bluuebunny/crossref_metadata_2025 huggingface dataset:
207 GANGLIOSIDE IMPROVES THE MEIOTIC MATURATION AND ANTI-APOPTOTIC EFFECTS DURING IN VITRO MATURATION OF PORCINE OOCYTES
Authors: Kim J.-W. , Park H.-J. , Chae S.-K. , Ahn J.-H. , Do G.-Y. , Park J.-Y. , Yang S.-G. , Koo D.-B.
Published: December 2015

Gangliosides are sialic-acid-glycosphingolipids expressed in almost all vertebrate cells. These play a role in regulating cell differentiation and signal molecules of cellular signaling, including interactions with molecules of signal transduction pathways, such as receptors tyrosine kinases (RTK). Gangliosides perform critical functions through interactions with proteins as receptors in cell‐cell recognition, such as the epidermal growth factor (EGF) receptor (EGFR). Recent studies investigated functional a link between gangliosides-derived RTK signaling and embryo development until blastocyst in mice. However, there is no report on the directly relationship of GM3 or GD1a and oocyte maturation in pigs. Our studies have verified an interaction mechanism between EGF receptor signaling and gangliosides (GM3 and/or GD1a) in porcine oocyte during meiotic maturation and further development. The GM3 expression increased in mouse oocytes that were in vitro-matured (IVM) under apoptotic conditions. To induce the apoptotic conditions of oocytes, we used H2O2. We first confirmed that the expression of GM3 in oocytes of H2O2-treated groups (1 mM) was gradually increased compared with those of the control group (P < 0.05). We investigated the anti-apoptotic effect of GM3 as the inhibition apoptosis in porcine oocyte IVM by treatment of GM3 (5 or 10 μM) after pretreatment with H2O2. Next EGFR protein levels and meiotic maturation were investigated by Western blot analysis and acetic-orcerin staining, respectively, of IVM oocytes. The proportion of germinal vesicle arrested oocytes at 22 h was significantly increased (P < 0.05; 41.6 ± 1.5% v. 25.0 ± 0.0% in the untreated group) in EGF (10 ng mL–1) + GD1a (10 μM) treated group. To confirm the completion of meiotic maturation, we investigated the proportion of metaphase II (MII) oocytes at 44 h. The MII oocytes were increased (P < 0.05; 89.9 ± 3.6% v. 57.4 ± 5.3% in the untreated group) in oocytes cultured with EGF+GD1a. Interestingly, p-EGFR protein levels of matured oocytes were significantly increased (P < 0.05) by the EGF+GD1a treatment in the maturation process. In addition, EGF+GD1a treatment increased the proportion of normal pronucleus formation (2PN of 43.1 ± 5.2%) and increased (P < 0.05; 50.4 ± 6.1% v. 27.2 ± 2.7% in the untreated group) pre-implantation developmental potential until blastocyst. These results suggest that EGF+GD1a treatment improved the developmental competence of embryos via enhanced meiotic maturation of porcine oocytes by inducing EGFR activation. We confirmed that GM3 has anti-apoptotic effects during porcine oocyte maturation periods. Furthermore, these data will be helpful for better understanding the receptor mechanisms during oocyte maturation in pigs.
209 MATURATION KINETICS AFTER HOLDING EQUINE OOCYTES IN EMBRYO HOLDING MEDIUM
Authors: Dini P. , Bogado O. , Smits K. , VanSoom A. , Daels P.
Published: December 2015

It has been reported that immature, equine oocytes can be maintained in meiotic arrest at 24°C. To evaluate a commercial equine embryo holding medium for storage of equine oocyte at 24°C and to determine the effect of holding on maturation kinetics, cumulus‐oocyte complexes (COC) were recovered from slaughtered mares and placed in Syngro® Embryo Holding Solution at 22–25°C for 18–20 h (OH Group) or placed directly in DMEM-F12-based in vitro maturation (IVM) conditions (D-Mat Group) at 5% CO2 in air at 38.5°C. Maturation rate (metaphase II percentage; MII%) was assessed (presence of polar body under stereomicroscope) after denudation at 22, 24, and 28 h. After assessment, the denuded oocytes that were considered immature were placed back in IVM, reassessed at 24 and 28 h, and MII% was compared with that of oocytes remaining uninterrupted in IVM for 24 and 28 h. One-way ANOVA was used to compare dependent variable in different groups using PROC ANOVA (SAS, version 9.2, SAS Institute, Cary, NC, USA). A random selection of mature oocytes from both groups were fertilised using intracytoplasmic sperm injection (ICSI). A total of 250 injected oocytes were cultured in DMEM-F12 with 10% FCS. Blastocyst rates in OH and D-mat groups were similar (7.1% v. 6.3%). At 22 h, significantly more oocytes reached the MII stage in the OH group than in the D-Mat group, but MII% was similar in both groups at 24 and 28 h (Table 1). Denuded, immature oocytes reached similar maturation rate as the undenuded oocytes in the same group. Our data suggest that oocytes can be held in Syngro® Embryo Holding Solution at 22–25°C for 18–20 h without compromising oocyte developmental competence. Overnight holding of oocytes accelerates maturation with similar maturation rate at 22, 24, and 28 h of IVM in the OH group. Denudation of immature oocytes after 22 h of IVM and returning the denuded oocytes to IVM does not affect the progression of maturation. In subsequent experiment, overnight held oocytes were fixed and stained (Hoechst 33342) and MII% was evaluated after 20, 22, and 28 h of IVM. Chromatin configuration confirmed that stored oocytes reach the MII stage at 22 h. Maturation rates were significantly lower at 20 h, suggesting that 22 h of IVM is required for stored oocytes.

Table 1.Maturation rates (% in MII stage) at 22, 24, and 28 h of IVM for equine oocytes held in Syngro® Embryo Holding Medium before IVM (OH) and oocytes placed directly in IVM (D-Mat)
Thanks to I. Lemahieu and P. Van Damme. Study was supported by the Special Research Fund at UGent.
21 GENERATION OF GGTA1 AND CMAH BIALLELIC KNOCKOUT PORCINE FIBROBLAST CELL LINES BY TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE AND CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9
Authors: Kang J.-D. , Ryu S.-M. , Zhu H.-Y. , Jin L. , Li W.-X. , Cui C.-D. , Kim J.-S. , Yin X.-J.
Published: December 2015

The use of porcine organs for pig-to-human transplantation is considered the most promising solution to overcome the growing shortage of human grafts for all transplantation. Unfortunately, human antibodies recognise Gala-1, 3-gal (Gal), and N-glycolylneuraminic acid (Neu5Gc) epitopes on the surface of pig cells, causing hyperacute rejection. Transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are 2 powerful tools for producing gene knockouts with high efficiency. Here we generate α-1, 3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene knockout porcine fibroblasts cell lines with TALEN and CRISPR/Cas9. Porcine fibroblasts were treated with TALEN and CRISPR/Cas9 designed against the region coding for the catalytic core of GGTA1 and CMAH. A magnetic separation of these cells was used in somatic cell NT. The cloned fetuses and piglets were characterised by T7E1, fluorescent PCR, and DNA sequencing. Nineteen fetuses were harvested using TALEN; 4 of them were GGTA1 gene mutated, and 11 were CMAH gene mutated. The DNA sequence analysis confirmed 5 of 11 are biallelic disruptions of the CMAH gene. For the CRISPR/Cas9, 6 piglets were obtained, and DNA sequence analysis confirmed 1 of 6 is biallelic disruptions of the GGTA1 and 2 of 6 are mono-allelic disruptions of the CMAH gene. We produced several fetuses or piglets via gene editing technology (TALEN and CRISPR/Cas9) and somatic cell NT technology. Furthermore, GGTA1 and CMAH gene knockout porcine fetal fibroblast cell lines derived from the fetus individuals were successfully established. However, expression of α-Gal and Neu5Gc epitopes is needed to be further examined by flow cytometry and confocal microscopy. Our study will provide the possibility of making GGTA1 and CMAH knockout pigs in the future.
212 EFFECT OF CANINE OVIDUCT CELLS AND CUMULUS CELLS CO-CULTURE ON IN VITRO MATURATION OF PORCINE OOCYTES AND EMBRYO DEVELOPMENT
Authors: Lee S. H. , Oh H. J. , Kim G. A. , Kim M. J. , Choi Y. B. , Kwang Jo Y. , Nugraha Setyawan E. , Lee B. C.
Published: December 2015

In oestrus stage, canine oocytes surrounded by cumulus cells undergo maturation in oviduct for 3 days after ovulation. We hypothesised that canine cumulus cells (cCC) and canine oviduct cells (cOC) in oestrus stage might affect the maturation of oocyte and embryo development. Therefore, the present study was aimed to compare the effects of cCC and cOC co-culture system on oocyte in vitro maturation and embryo in vitro development. cCC were separated from cumulus‐oocyte complex (COC) in ovary from bitches in oestrus phase. cOC were collected from oviduct flushing of bitches in oestrus phase. Both cCC and cOC were cultured and cryopreserved until use for co-culture. In the first experiment, the effect of co-culture using cCC and cOC on porcine oocyte in vitro maturation (IVM) were investigated. The porcine COC were randomly cultured in different co-culture groups as follows: 1) co-culturing with cCC for 42 h, 2) co-culturing with cOC for 42 h, and 3) culturing in absence of cCC or cOC. After IVM, extrusion of the first polar body was observed under a microscope. In the second experiment, the matured oocytes with the first polar body derived from each group were activated with electrical stimulus. Parthenotes were cultured in porcine zygote medium-5 (PZM-5) for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The embryo developmental competence was estimated by assessing the in vitro development under microscope. The third experiment was to evaluate the reactive oxygen species (ROS) levels in each supernatant medium obtained from cCC and cOC co-culture group after IVM using a OxiselectTM ROS ELISA Assay kit. Last, analysis of genes (MAPK1/3, SMAD2/3, GDF9 and BMP15) expression in cCC and cOC co-cultured with porcine COC using real-time PCR is in progress. As results, IVM rate of cOC group (91.19 ± 0.45%) was significantly higher than that of cCC and control group (86.50 ± 0.61% and 79.81 ± 0.82%; P < 0.05). Also, cOC groups expressed the highest efficiency in cleavage rate, blastocyst formation rate, and the total cell number in blastocyst (P < 0.05). In ROS levels, cOC group (555 ± 7.77 nM) were significantly lower than cCC and control groups (596.8 ± 8.52 nM and 657.8 ± 11.34 nM). The present study demonstrated that co-culture with cOC improved the in vitro oocyte maturation and the in vitro development rate of porcine embryos. The ROS level decreased in cOC co-culture would have beneficial influence on oocytes maturation. For further study, we will investigate the relation between gene expression related to oocyte maturation and the co-culture results.
This research was supported by a global PhD Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), RDA (#PJ010928032015), IPET (#311011–05–4-SB010, #311062–04–3-SB010), Research Institute for Veterinary Science, and the BK21 plus program.
214 PRE-IMPLANTATION EMBRYO SEX DETERMINATION BY AMPLIFICATION OF AMELOGENIN GENE IN WATER BUFFALOES (BUBALUS BUBALIS)
Authors: Shang J.-H. , Yang C.-Y. , Zheng H.-Y. , Qin J. , Gu Y.-P. , Liu H.-Z.
Published: December 2015

Sex-specific sequence variability of the amelogenin (AMEL) gene has been observed in a variety of mammalian species. AMEL is a multi-copy gene expressed in abundance in the development of mammalian tooth enamel matrix. Its homologous genes are located on the X- or Y-chromosome, respectively. In our study, a pair of primers was designed to allow amplification of a single fragment of 312 bp from buffalo cows. Because the gene is slightly differently encoded on the Y-chromosome, the same primers were expected to produce 2 fragments of 312 and 249 bp from bulls. The primers were successfully applied to single, in vitro-produced Day-6 to Day-8 water buffalo blastocysts by direct lysis of the whole blastocysts, followed by PCR. The amplification on presumed female and male embryos consistently displayed the same sex-specific pattern. Of 100 blastocysts, 43 were determined to be females, 51 males, and 6 unknown (samples missed). We concluded that PCR amplification of the AMEL gene can be used for early embryo sex identification of water buffalo.
This work was supported by the National Natural Science Foundation of China under Grant No. 31160456 and the Guangxi Natural Science Foundation of China under Grants No. 2013GXNSFAA019075 and 2014GXNSFAA118116.
224 CHARACTERIZATION OF EQUINE ENDOMETRIAL-DERIVED MESENCHYMAL STROMAL CELLS
Authors: Rink E. , French H. , Watson E. , Aurich C. , Donadeu F. X.
Published: December 2015

Equine mesenchymal stromal cells (MSC) are mainly harvested from bone marrow and adipose tissue, requiring surgical procedures. Although human endometrium is known to harbor mesenchymal precursor cells, the presence of MSC in equine endometrium, a dynamic tissue, has not been investigated. This study reports for the first time the culture and characterisation of MSC from equine endometrium compared with equine bone marrow (BM)-derived MSC. Samples of equine endometrium (n = 6) and BM (n = 3) were collected postmortem. Endometrial tissue was digested using a dissociation medium containing collagenase I and DNase type I, and CD227 (mucin-1)-bound magnetic beads were utilised to separate epithelial (CD227+) from stromal (CD227–) cell fractions. Red blood cells from BM samples were excluded using a density gradient. All cell fractions were cultured in DMEM/F-12 containing 10% fetal bovine serum. After expansion, colony-forming unit (CFU) assay at passage 2, trilineage differentiation (adipogenic, chondrogenic, osteogenic), and flow cytometry analysis at passage 3/4 were performed for CD227– fractions and BM-MSC. Descriptive statistical analysis and 2-tailed t-test was performed with IBM SPSS Statistics 22 (SPSS Inc./IBM, Chicago, IL, USA). Both isolated cell fractions were plastic adherent and grew well under standard MSC culture conditions, although endometrial CD227– cells attached quicker to culture plasticware than did BM-MSC. The CFU assay at passage 2 showed no significant difference in cloning efficiency (CE) between BM-MSC (20.78 ± 2.86%) and CD227– (24.89 ± 3.04%) cell lines (P = 0.36). Flow cytometry showed the expression of MSC markers (CD29, CD44, CD90, CD105) and perivascular markers (CD146, NG2) but almost no expression of haematopoietic markers (CD34, CD45) in both cell lines (Table 1). No statistically relevant difference was seen except for the higher expression of NG2 in BM-MSC (P = 0.054). Trilineage differentiation was successfully induced in both cell lines. In conclusion, we showed the presence of putative MSC in equine endometrium. We successfully isolated and cultured these cells, which display comparable characteristics in MSC criteria as well-established BM-derived MSC. These endometrial-derived MSC may provide a convenient source for veterinary regenerative therapies in equine reproduction.

Table 1.Flow cytometry marker expression (mean ± standard error) at passage 4
236 SUPEROVULATION IN SHEEP WITH REDUCED INJECTION FSH/eCG PROTOCOLS FOLLOWED BY TRANSFER OF FRESH OR VITRIFIED EMBRYOS
Authors: Fry R. C. , Mapletroft R. , Bo G. A. , Izzo M. M. , Humphris M. A.
Published: December 2015

The aim of this experiment was to compare a single FSH/eCG, or double FSH-0.5% hyaluronan/eCG injection protocol with a multiple FSH/eCG injection protocol on the superovulatory response and embryo production in sheep. In addition, the effect of vitrification of these embryos on the pregnancy rate following embryo transfer was evaluated. Eighty Dohne Merino ewes received an 8-day CIDR-S device (0.33 g P4; Zoetis, Florham Park, NJ, USA) plus 10 mg of FSH i.m. (Folltropin-V; Vetoquinol, Lavaltrie, QC, Canada) and 400 IU of eCG i.m. (Pregnecol; Vetoquinol) in 3 treatment groups. Group 1 (n = 21) received a single 10-mg FSH injection in saline and 400 IU of eCG in saline 2.5 days before CIDR withdrawal. Group 2 (n = 23) received 6.7 mg of FSH in hyaluronan (MAP-5, 50 mg; Vetoquinol) and 400 IU of eCG in saline 2.5 days before CIDR withdrawal and 3.3 mg of FSH in hyaluronan 0.5 days before CIDR withdrawal. Group 3 (n = 36) received 7 injections (am, pm) of FSH in saline (2.5, 2.0, 1.5, 1.5, 1.0, 1.0, 0.5 mg) starting 2.5 days before CIDR withdrawal and 400 IU of eCG in saline at the first injection. Ewes were inseminated with semen collected from 1 of 5 rams at 36 to 40 h after CIDR withdrawal. Donor ewes were slaughtered 6 days after AI (Day 0), CL were counted and ova/embryos were collected. Viable embryos were transferred in singles into Day 6 synchronised recipients as either fresh (n = 128) or following vitrification/thawing using the CVM (CryoLogic, Blackburn, VIC, Australia; n = 97; Fry et al. 2005 Reprod. Fertil. Dev. 17, 243). Pregnancy was diagnosed by ultrasound scanning on Day 45. Data for CL and transferable embryo were analysed by the Kruskall-Wallis test and differences between groups determined by the Dunn test. Data for pregnancy rates was compared by chi-squared analysis. The mean number of CL in Groups 2 and 3 were both significantly higher than that in Group 1 (12.3 and 12.0 v. 8.5; P < 0.05). Similarly, the total number of embryos/ova recovered in Groups 2 and 3 were significantly higher than for Group 1 (8.3 and 7.0 v. 5.0; P < 0.05). Group 3 produced more viable embryos than either Group 2 or Group 1 (4.6 v. 2.7 and 2.1; P < 0.05); however, data were skewed by the extensive use of semen from one ram in Group 2 that had a low fertilization rate (28%). The transfer of vitrified/thawed embryos resulted a nonsignificant (P > 0.05) 10% decrease in pregnancy rate compared with fresh embryos (66% v. 76%). In conclusion, the administration of the sustained release FSH-MAP-5 in a 2-injection protocol in sheep was as effective as a multiple FSH injection protocol in inducing an ovarian response but more research is required to elucidate the effect of FSH-MAP-5 on embryo quality. The successful vitrification of sheep embryos provides a promising technique for the storage and transport of embryos in large-scale sheep embryo transfer programs.
253 THE MARE MODEL FOR LUTEINIZED UNRUPTURED FOLLICLE SYNDROME: INTRAFOLLICULAR ENDOCRINE MILIEU
Authors: Gastal E. L. , Bashir S. T. , Gastal M. O. , de Tarso S. G. S. , Tazawa S. P. , Cuervo-Arango J. , Baerwald A. R. , Hales D. B.
Published: December 2015

Mechanisms underlying the development of luteinized unruptured follicle (LUF) syndrome in mares were studied to provide a model for human anovulation. The aims of the present study were (1) to determine the effect of recombinant equine LH (reLH) and prostaglandin F2α (PGF2α) on LUF formation (Experiment 1), (2) to identify an optimal dose of COX-2 inhibitor (flunixin-meglumine; FM) required to experimentally induce LUF (Experiment 2), and (3) to evaluate the intrafollicular endocrine milieu in induced LUF (Experiment 3). In Experiment 1, mares (n = 30) were treated with reLH from Days 7 to 15 (Day 0 = ovulation), with PGF2α on Day 7 or in combination. In Experiment 2 (n = 18 mares), FM at doses of 2.0 or 3.0 mg kg–1 every 12 h and hCG (1500 IU) were administered after a follicle ≥32 mm was detected. In Experiment 3 (n = 23 mares), FM at a dose of 2.0 mg kg–1 every 12 h plus hCG was used to induce LUF and investigate the intrafollicular endocrine milieu. Shapiro-Wilk tests were used for analysing data for normal distribution, and data not normally distributed were transformed to ranks before any further analyses. For sequential data, mixed model repeated-measures ANOVA were used. For single-point data, Student’s t-tests were used. No LUF were induced in mares treated with reLH or PGF2α, in combination or separately. However, LUF were induced in 100% of mares using FM at the dose rate of 2.0 and 3.0 mg kg–1 of body weight in combination with 1500 IU of hCG. Intrafollicular PGF2α metabolite (PGFM), PGF2α, and prostaglandin E2 (PGE2) were lower, and the ratio of PGE2 : PGF2α was higher in induced LUF. Higher levels of intrafollicular growth hormone, cortisol, oestradiol, testosterone, and total primary sex steroids were observed in the induced LUF group; however, LH, prolactin, vascular endothelial growth factor-A, and nitric oxide did not differ between the control and induced LUF groups. In conclusion, COX-2 inhibitors used in conjunction with hCG can be used to pharmacologically induce LUF with 100% success in mares. This study revealed part of the intrafollicular endocrine milieu and the association of prostaglandins in LUF formation. We postulated that LUF result from decreased intrafollicular prostaglandin concentrations or altered prostaglandin synthesis, as indicated by disparity in PGE2 : PGF2α ratio. Increased intrafollicular oestradiol, testosterone, cortisol, and growth hormone were associated with LUF formation; however, further studies are necessary to ascertain the cause-effect relationship. The effect of LH on LUF formation remains unclear. This study further encourages the use of intrafollicular v. systemic biomarkers for evaluating ovulatory disorders. Finally, results from this study support the use of the mare as a model for investigating the poorly understood LUF syndrome in women.
26 PILOT STUDY: YOLK SAC VEGF EXPRESSION IN BOVINE EMBRYOS FROM REPRODUCTIVE TECHNIQUES
Authors: Mess A. M. , Carreira A. C. O. , Marinovic de Oliveira C. , Fratini P. , Favaron P. O. , Barreto R. S. N. , Meirelles F. V. , Miglino M. A.
Published: December 2015

The bovine yolk sac morphology and function are vulnerable to alterations by reproductive techniques. We hypothesised that the early haematopoiesis, vasculogenesis, and angiogenesis of yolk sac affect embryo nutrition and placenta differentiation. A pilot study on bovine yolk sac vascularization was done using gestations with 25, 30, 35, and 40 days of pregnancy, produced by IVF (n = 10) and NT of somatic cell (NT, n = 5). Samples from an abattoir were used as controls (n = 9). All pregnancies were conduced with zebuine cross-breed. We examined yolk sac gross and fine morphology. Also immunohistochemistry and qPCR were done for VEGF-A and receptors (VEGFR-1/Flt-1 and VEGFR-2/KDR). Morphological aspects, including vascularization, of yolk sac from control and IVF groups were tightly similar, although IVF is discretely precocious. However, NT group at Day 30 had low vascularization with thin endothelium, and large intercellular spaces in the endodermal layer, when compared with control and IVF. At Day 35 to 40, NT group improved vascularization and decreased intercellular spaces when compared with early NT samples, remaining less developed than control and IVF. Protein of VEGF-A and receptors (VEGFR-1/Flt-1 and VEGFR-2/KDR) were detected in all groups and ages. The VEGF-A was particularly present in cytoplasm of endodermal, endothelial cells, and in yolk sac blood islands. Furthermore, VEGFR-1 and VEGFR-2 were restricted to endodermal cells. At mRNA level, VEGF-A was highly expressed at Day 25 for IVF (P < 0.05) and at Day 30 for NT (P < 0.05). In addition, VEGFR-1 was highly expressed at Day 30 for IVF group (P < 0.05). For VEGFR-2 high averages were observed at Days 25 and 40 for IVF (P < 0.05). The high expression of VEGF-A and receptors in comparison with control may be due to the precocious yolk sac development in this group. The increase of VEGF-A at Day 30 in NT group is related to the moment of yolk sac vascularization development. In conclusion, yolk sac development is affected by reproductive techniques that alter the vascularization pattern, decreasing embryo nutrition and organogenesis.
27 AN IMPROVED LARGE ANIMAL MODEL FOR THE STUDY OF ADULT STEM CELLS
Authors: Simpson S. , Gonzalez L. , Chung J. , Blikslager A. , Magness S. , Piedrahita J.
Published: December 2015

Murine models for the study of adult stem cell populations have broadened the understanding of previously uncharacterized stem cell niches. The development of murine reporter lines for the leucine-rich repeat-containing G-protein-coupled receptor-5 (Lgr5) has highlighted the importance of this gene as a stem cell marker in the stomach, intestine, hair follicle, liver, and kidney in mice. These models however have significant limitations in terms of translational applications because of anatomical and physiological differences between humans and mice. In order to overcome these limitations, we have sought to develop a porcine LGR5 reporter line. We report the generation of a porcine stem cell reporter line using the combination of transcription activator-like effector nucleases and somatic cell NT. Transcription activator-like effector nuclease-mediated homologous recombination was used to drive the integration of an internal ribosome entry site green fluorescent protein fusion into the 3′ untranslated region of the LGR5 locus in porcine fetal fibroblast cells. Multiple cell lines were developed and screened for the proper integration event. Upon confirmation of proper integration by genomic DNA sequencing, these lines were used as donors for somatic cell NT. Transfer of the somatic cell NT reconstructed embryos to a surrogate gilt resulted in 3 live births, and the establishment of a founder line of LGR5-green fluorescent protein reporter pigs. We have begun to characterise these lines, having observed fluorescent labelling of putative stem cell populations in the intestinal crypts and hair follicles from these animals. Many of these observations parallel the expression patterns observed in similar murine models. We have confirmed the fluorescent reporter signal by immunohistochemistry using an anti-green fluorescent protein antibody, and are working towards colocalization studies using anti-LGR5 antibodies and RNA in situ hybridization, as well as the characterisation of additional stem cell populations in the pig. The development of this line of transgenic pigs represents significant progress toward the study of adult stem cells, their progenitors, and the stem cell niche, using a large animal model with an anatomy, physiology, and ability to recapitulate human disease that overcomes the current limitations of rodent models.
Funding was provided by NIH R21OD019738.
29 THE NOVEL CRYOPROTECTIVE AGENT CARBOXYLATED ε-POLY-L-LYSINE IS EFFECTIVE FOR VITRIFICATION OF PRE-IMPLANTATION MOUSE EMBRYOS AT THE DIFFERENT STAGES
Authors: Ito J. , Kawasaki Y. , Shibao Y. , Matsumura K. , Hyon S.-H. , Kashiwazaki N.
Published: December 2015

Embryo cryopreservation is one of the important technologies in various research and clinical areas. Very recently, carboxylated ε-poly-L-lysine (COOH-PLL) has been developed as a new cryoprotective regent, and we previously demonstrated that combined treatment of COOH-PLL with ethylene glycol (EG), which is a commonly used cryoprotective regent, is effective for vitrification of unfertilized mouse oocytes and pronuclear embryos. In this study, we examined different concentration of COOH-PLL and tried to vitrify the embryos at the different stages beyond the 2-cell stage using the same protocol. Embryos at the pronuclear, 2-cell, 4-cell, 8-cell, morulae, and blastocyst stages were treated with 7.5% (vol/vol) EG and 7.5% (vol/vol) COOH-PLL for 3 min and then vitrified with 15% EG (vol/vol) and 15% (vol/vol) COOH-PLL (E15P15) for 1 min by the Cryotop. The embryos except for pronuclear embryos showed low rates of survival, blastocyst, and hatched blastocysts. To improve the survival and developmental ability of the vitrified embryos, blastocysts were vitrified with different concentrations of COOH-PLL. At the results, the survival and developmental rates of vitrified blastocyst in E20P10 group were higher (98.0 ± 2.5% and 93.3 ± 4.0%) than those of the E30, E25P5, and E15P15 groups (P < 0.05). When 2-cell, 4-cell, and 8-cell embryos or morulae were vitrified with E20P10, survival, blastocyst, and hatched blastocyst rates were dramatically improved (more than 86.0%). Taken together, vitrification with 20% COOH-PLL and 10% EG is available for vitrification of mouse pre-implantation embryos at the different stages.
34 EFFECTS OF L-CARNITINE AND trans-10,cis-12 CONJUGATED LINOLEIC ACID SUPPLEMENTATION DURING MATURATION ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO
Authors: Block J. , Zolini A. M. , Carrascal-Triana E. , Ruiz A. , Hansen P. J. , Torres C. A. A.
Published: December 2015

The objective of the present study was to determine the effect of supplementation of maturation media with L-carnitine and trans-10,cis-12 conjugated linoleic acid (CLA) on embryo development and survival following cryopreservation. Immature bovine cumulus-oocyte complexes (n = 1796) were harvested from abattoir-derived ovaries and randomly assigned in a 2 × 2 factorial design to be matured in maturation medium [TCM-199 with Earle salts supplemented with 10% (vol/vol) bovine steer serum, 2 μg mL–1 oestradiol 17-β, 20 μg mL–1 bovine FSH, 22 μg mL–1 sodium pyruvate, 50 μg mL–1 gentamicin sulfate, and 1 mM glutamax®] supplemented with or without 100 mM CLA and with or without 3.03 mM L-carnitine for 22 to 24 h at 38.5°C in a humidified atmosphere of 5% CO2. The proportion of oocytes that cleaved was determined on Day 3 after insemination, and the proportion of oocytes developing to the blastocyst and advanced blastocysts stages (expanded, hatching, and hatched) was assessed on Day 7. Blastocyst and expanded blastocyst stage embryos (n = 270) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 5 times. There was no effect of supplementation of maturation medium with either CLA or L-carnitine on the proportion of oocytes that cleaved at Day 3 or that developed to the blastocyst and advanced blastocyst stages at Day 7 after insemination. There was no interaction between CLA and L-carnitine affecting cleavage rate or embryo development. Supplementation of maturation medium with L-carnitine did not affect post-thaw re-expansion or hatching rates. In contrast, treatment with CLA during maturation reduced (P < 0.05) post-thaw re-expansion (24 h: 75.2 ± 3.8% v. 60.3 ± 4.1%; 48 h: 82.0 ± 3.4% v. 64.9 ± 4.0%; 72 h: 78.9 ± 3.6% v. 65.9 ± 4.0%, respectively) and hatching (24 h: 33.7 ± 4.2% v. 23.5 ± 3.6%; 48 h: 61.1 ± 4.3% v. 44.0 ± 4.2%; 72 h: 62.6 ± 4.3% v. 50.2 ± 4.2%, respectively) rates at all time points. There was no interaction between CLA and L-carnitine affecting post-thaw viability. In conclusion, supplementation of maturation medium with L-carnitine did not affect embryo development or post-thaw viability. Although addition of CLA during maturation did not affect embryo development, post-thaw cryotolerance was reduced following CLA supplementation. There was no beneficial effect of supplementing maturation medium with both CLA and L-carnitine on embryo development or post-thaw cryosurvival.
45 ACROSOME REACTION AND HETEROLOGOUS ZONA BINDING ASSAY OF FROZEN STALLION SPERM AFTER HYPERACTIVATION
Authors: Lagares M. A. , Martins H. S. , Souza M. R. , Penna C. F. A. M. , Leme F. O. P. , Silva G. C. , Cortes S. F. , Stahlberg R.
Published: December 2015

During cryopreservation and due to the large portion of seminal plasma removal, there is a decrease in equine sperm antioxidant protection. Lactoferrin and catalase in seminal plasma play an antioxidant role. The fertilizing ability of equine sperm has been analysed in vitro using sperm-zona binding assays with heterologous oocytes. The results have been correlated with in vivo fertility by means of acrosome reaction (AR) and the number of attached sperm to the zona pellucida (ZP). The aim of the present work was to estimate the potential fertilizing ability of stallion sperm frozen with INRA82 extender (Battelier et al. 1997) with lactoferrin and catalase, and after hyperactivation with procaine and calcium ionophore A-23187 (Ca-I) by determining the AR rate and number of attached sperm to the bovine ZP. Semen from 6 stallions was frozen with 3 extenders: (T1) control, INRA 82; (T2) T1 + 500 μg mL–1 lactoferrin; and (T3) T1 + 200 IU mL–1 catalase. After semen thawing, the sperm were selected by swim-up and distributed in 3 aliquots according to the hyperactivation treatments: (H1) control, after thawing; (H2) capacitating Whitten’s medium + 5 mM procaine chloride; and (H3) capacitating Whitten’s medium + 5 μM Ca-I. To the zona binding assays, bovine oocytes derived from abattoir ovaries were incubated at 38.5°C with 5% CO2 (1 h), and 5 oocytes were poured into each treatment droplet under mineral oil. Sperm were stained with Hoechst 33342 dye (35 μg mL–1), and after 2 h co-culture, the number of sperm attached to the ZP was determined with epi-fluorescent microscopy. The rate of sperm AR was determined after freezing-thawing (control) and hyperactivation treatments with propidium iodide and fluorescein isothiocyanate/peanut agglutinin dies with a flow cytometer. The green fluorescent (peanut agglutinin+) and not red stained (propidium iodide) sperm were considered acrosome reacted. Means of ZP attached sperm and percentage of AR sperm were analysed by ANOVA and Tukey test. A probability of P < 0.05 was considered significant. The mean of ZP attached sperm (4.2 ± 3.5) and AR sperm rate (4.4 ± 3.7%) did not differ among the extenders (P > 0.05). The rate of sperm AR after hyperactivation with procaine (5.2 ± 2.4%) did not differ to the Ca-I (6.1 ± 3.7%); however, they were higher than the spontaneous AR rate (1.1 ± 0.5%, P < 0.05). Lower number of ZP attached sperm was observed by the Ca-I induced hyperactivation protocol (1.9 ± 2.1) compared with the procaine (5.9 ± 3.7; P < 0.05), although they did not differ to the control (3.3 ± 2.7). In conclusion stallion frozen sperm were better hyperactivated with procaine than with Ca-I, and therefore, it is a more suitable sperm hyperactivation inductor to study equine IVF protocols with frozen semen.
Acknowledgments are extended to CAPES, Brazil, for the financial support.
47 EFFECT OF L-CARNITINE TREATMENT DURING OOCYTE MATURATION ON THE POST-THAW DEVELOPMENT OF PORCINE EMBRYOS VITRIFIED AT THE PRONUCLEAR STAGE
Authors: Grupen C. G. , Somfai T. , Kikuchi K.
Published: December 2015

The extreme cryo-sensitivity of porcine oocytes and embryos is attributed to their endemically high content of cytoplasmic lipid droplets. In attempts to improve the cryo-tolerance of porcine embryos, various strategies have been used to reduce the amount of lipid droplets present in the cytoplasm before vitrification. Recently, the cryo-tolerance of bovine oocytes vitrified at the metaphase II stage was improved by supplementing in vitro maturation (IVM) medium with l-carnitine (LC), a stimulator of lipid metabolism (Chakitisakul et al. 2013 Theriogenology 79, 590–598). The objective of this study was to determine the effect of supplementing IVM medium with LC on the post-thaw development of porcine embryos vitrified at the pronuclear stage. Oocytes recovered from the ovaries of prepubertal gilts were matured in modified porcine oocyte medium supplemented with 0 (control) or 12 mM LC during the final 22 h of IVM. Following IVF, presumptive zygotes were cultured in porcine zygote medium-3. At the pronuclear stage, cohorts of embryos from each group were either vitrified using a solid surface vitrification procedure (Somfai et al. 2009 Biol. Reprod. 80, 42–49) or cultured for 7 d without being vitrified. Vitrified zygotes were subsequently warmed and cultured for 7 d. The rates of cleavage, blastocyst formation, and hatching were recorded, and all blastocysts were stained to determine the total cell numbers. Three replicates were performed. Proportional data were arcsine transformed and subjected to ANOVA, and cell number data were analysed by t-test. The post-thaw survival rates of the embryos that were vitrified did not differ between the groups (control: 95.7%; LC: 97.1%; P > 0.05). There were no significant effects of LC treatment or vitrification on the rates of cleavage, blastocyst formation, and hatching (Table 1). Vitrified embryos derived from LC-treated oocytes produced blastocysts with fewer cells than vitrified embryos derived from untreated oocytes (Table 1; P < 0.05). In contrast to previous findings in other species, the results indicate that supplementing IVM medium with LC did not enhance the post-thaw development of porcine embryos vitrified at the pronuclear stage.
Table 1.Effect of l-carnitine (LC) treatment and vitrification on porcine embryo development

C. Grupen was supported by an OECD Fellowship.
49 EFFECT OF ADDING Trolox C AND ASCORBIC ACID TO RAM SPERM BEFORE CRYOPRESERVATION ON THE MOTILITY AND BINDING CAPABILITY
Authors: Costa J. , Lima W. , Moraes E. , Sousa P. , Ramon L. , Lima D. , Coelho V.
Published: December 2015

Different antioxidants have been tested to improve sperm quality, but distinct and consistent beneficial effects are lacking. The objective of this study was to evaluate whether the addition of different concentrations of the antioxidant Trolox C and ascorbic acid before cryopreservation could improve the binding of sperm to chicken egg perivitelline membrane (PM) after cryopreservation. Three ejaculates of ram were split and diluted with Tris egg yolk diluent to a final concentration of 200 × 106 cells mL–1, and after, the ejaculates were divided into 5 tubes. Each tube received one of the following antioxidants: control, no antioxidant; 200 μM of Trolox C; 300 μM of Trolox C; 0.05% of ascorbic acid; and 0.25% of ascorbic acid. The samples were cooled to 5°C/2 h, packaged into 0.5-mL straws, and frozen in static LN vapor for 15 min before being plunged into LN. Straws were thawed (37°C/30 s). The motility was determined using computer-assisted semen analysis. For PM binding test, PM was put in tubes with 1 mL of TALP and inseminated with 50 000 sperm. The PM and sperm were incubated for 90 min at 37°C in an atmosphere of 5% CO2 in air, and 20 min before the end of the incubation time, 10 μL of Hoechst 33342 was added in each treatment. After each PM was washed 5 times in TALP, placed under the coverslip on a slide, and evaluated by fluorescence microscopy at 400×. Spermatozoa were counted in 6 random fields of each piece of PM. Percentage data were transformed using arcsine prior analysis. Treatment differences were determine by analysis of variance and Tukey test. The total and progressive motility of sperm treated with 0.25% ascorbic acid Trolox C was higher (64.5 and 45%) than 100 μM of Trolox C (61.9 and 42.6%) and 200 μM of Trolox C (64.3 and 46.7%) and control (59.8 and 39.6%; P < 0.05), respectively. The binding test was higher when using 0.25% of ascorbic acid (155.73 cells; P < 0.05) compared with other treatments. Addition of 0.25% ascorbic acid to ram sperm before cryopreservation improved cell cryosurvival rates.
51 OPTIMIZATION OF VITRIFICATION PARAMETERS FOR RHESUS MACAQUE BLASTOCYSTS
Authors: Ramsey C. , Hanna C. , Hennebold J.
Published: December 2015

The cryobiological characteristics of rhesus macaque embryos are not well defined, and only a few studies report limited success rates after recovery from slow-freezing cryopreservation. Vitrification is the method currently in use by many infertility clinics with 85 to 96% embryos surviving post-thaw. However, success of thawing rhesus macaque embryos after vitrification is not well documented. Therefore, multiple vitrification parameters were evaluated on rhesus expanded blastocysts by comparing culture medium, vitrification method, and embryo integrity. Embryos (n = 204) were produced by standard IVF and cultured in either Global (single-step; LifeGlobal Group, Guilford, CT, USA) or Quinns Advantage (sequential; SAGE, Trumbull, CT, USA) in a humidified tri-gas mixture (6% CO2, 5% O2, 89% N2) at 37°C until blastocyst expansion was observed after 7 to 9 days of culture. Embryos were then vitrified in straws using the Global Fast Freeze Vitrification protocol or in cryotops using the Kitazato method (VT 601-Top-Kitazato, Japan). Additional embryos were biopsied with an objective-mounted laser to remove ~10 to 20 trophectoderm cells before vitrification with Global Fast Freeze in straws. Embryos were thawed at 30°C and cultured at 37°C for an additional 48 h or until arrested. Morphology was graded as good (re-expanded), poor (shrunken cytoplasm), or lysed, following thaw, and success of good or hatched embryos was analysed by Fisher’s exact test with P < 0.05. Because our goal was to provide good quality embryos for embryo transfer, the ability to hatch was our standard for determining success. There was no significant difference in embryo quality between culture medium, vitrification method, or embryo integrity. Overall, 90% of the embryos were scored as morphologically good regardless of culture medium based on re-expansion post-thaw <24 h (55/61) but had a relatively low hatching rate of 25% (15/61), demonstrating that not all re-expanded embryos will go on to achieve the hatched state. Initial trials vitrifying with the Global protocol were associated with decreased recovery rates as many embryos were lost. Cryotops were able to increase our recovery rate to 100% (n = 22) but with only 5% surviving post-thaw (1/22). Decreasing the amount of media used to recover embryos from straws enabled a re-evaluation of the cryotop technique with an improved recovery of 92% (60/65). Biopsied embryos tended to have a higher hatch rate (48%; 10/21) compared with intact embryos frozen at the same stage (26%; 9/35). The trophectoderm biopsy likely allows for better permeation of the cryoprotectant before vitrification in embryos due to a collapsed blastocoel. Taken together, these data suggest that vitrification is a viable method of preserving embryos of genetic value and those that undergo manipulation, including trophectoderm.
56 THE EFFECTS OF SERUM LEVEL OF ANDROGENS, LUTEINIZING HORMONE, AND INSULIN-LIKE GROWTH FACTOR 1 IN EARLY FOLLICULAR PHASE ON OVARIAN FOLLICULAR GROWTH PARAMETERS AND PREGNANCY RATE
Authors: Raoofi Z. , Hosseini F. , Pegah Parvar S. , Paniz Parvar S.
Published: December 2015

Many studies have showed the role of androgens on ovarian follicular maturation. The present study investigated the effect of serum concentration of androgens, LH, and insulin-like growth factor 1 (IGF1) in the early follicular phase on the results of the ovulation induction (I/O) and intrauterine insemination (IUI) cycles. This cross-sectional study was done in the infertility clinic of Akbar Abadi hospital. Fifty-nine infertile patients who were not affected by polycystic ovary syndrome (PCO) and were candidates for induction ovulation or intrauterine insemination were chosen at random. The serum concentrations of androgens (free testosterone, dihydroepiandrosterone, and androstenedione), LH, and IGF1 were measured on the third day of menstruation. Sonographic monitoring was started from the ninth day of the cycle. The ampule of hCG was injected when there was at least one follicle with the diameter of 18 mm. The relationships of concentration of androgens, LH, and IGF1 with follicular growth parameters and pregnancy rate were analysed. There was no statistically significant link between the number and diameter of follicles with concentration of free testosterone, dihydroepiandrosterone, androstenedione, IGF1, and LH. There was no statistically significant link between the number of follicles in the ovaries and concentrations of testosterone (P = 0.090 and r = 0.223), dihydroepiandrosterone (P = 0.642 and r = 0.062), androstenedione (P = 0.526 and r = 0.084), IGF1 (P = 0.470 and r = 0.096), and LH (P = 0.446 and r = 0.102). There was no statistically significant link between the mean follicular diameter and concentration of testosterone (P = 0.822 and r = 0.03), dihydroepiandrosterone (P = 0.733 and r = 0.045), androstenedione (P = 0.526 and r = 0.084), IGF1 (P = 0.799 and r = 0.034), and LH (P = 0.626 and r = 0.065). Beta human chorionic gonadotropin (β-hCG) was positive in 11 patients (18.6%) and negative in 48 patients (81.4%). Serum concentrations of androgen profile, LH, and IGF1 in the positive (β-hCG) group were not significantly different in comparison with the negative β-hCG group. It seems that in women who were not affected by PCO, concentrations of free testosterone, dihydroepiandrosterone, androstenedione, IGF1, and LH in the early follicular phase were not related to follicular growth parameters and pregnancy rate.
6 THE EFFECTS OF DEPLETING DONOR CELL MITOCHONDRIAL DNA ON CATTLE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER
Authors: Srirattana K. , St. John J. C.
Published: December 2015

Although somatic cell nuclear transfer (SCNT) is a valuable tool for producing animals for agricultural and research purposes, the resultant mixing of mitochondrial DNA (mtDNA) from the donor cell and recipient oocyte (heteroplasmy) affects embryo development and offspring survival and health. The aim of this study was to determine the effects of depleting donor cells of their mtDNA before SCNT on embryo development. mtDNA was depleted from cattle fibroblasts using 2′,3′-dideoxycytidine. mtDNA copy number in cells depleted for 30 days (0.85 ± 0.05) was significantly decreased when compared with nondepleted cells (150.12 ± 29.90; P < 0.0001, ANOVA). Moreover, mtDNA copy number in depleted cells could not be replenished after depletion for 30 days. Depleted cells and nondepleted cells were used as donor cells for SCNT. Somatic cell nuclear transfer embryos were produced by electrofusion of a single donor cell with an enucleated cow oocyte. Reconstructed oocytes were chemically activated and cultured for 7 days (nontreated embryos). Another cohort of embryos was treated with Trichostatin A (TSA), to enhance reprogramming, by activating reconstructed oocytes and culturing them in the presence of 50 nM TSA for up to 10 h. The embryos were then cultured in the absence of TSA. In nontreated groups, the fusion rates of depleted cells (78.0 ± 0.8%) were significantly lower than those of nondepleted cells (92.1 ± 1.4%; P < 0.05). No positive effect on fusion rates was found after TSA treatment. The blastocyst rate for SCNT embryos derived from depleted cells (18.7 ± 4.9%) was significantly lower than the nondepleted group (32.5 ± 3.1%; P < 0.05). Trichostatin A treatment increased blastocyst rates for SCNT embryos derived from depleted cells (32.5 ± 5.3%) to levels equivalent to those of nondepleted cells but did not have any beneficial effect on SCNT embryos derived from nondepleted cells. We have analysed blastocysts for the presence of donor cell mtDNA by high resolution melting analysis. Four out of 10 SCNT blastocysts derived from nondepleted cells were heteroplasmic, whereas others had no donor cell mtDNA. However, all 10 analysed SCNT blastocysts derived from depleted cells were homoplasmic as they harboured only oocyte mtDNA. From RNA sequencing results, TSA treatment of SCNT blastocysts derived from depleted cells increased the expression of key developmental transcription regulators and decreased expression of the mtDNA-specific replication factors, which is essential for embryo development. In conclusion, homoplasmic SCNT embryos were successfully produced by using mtDNA depleted donor cells. Trichostatin A treatment enhanced nuclear reprogramming efficiency in SCNT embryos derived from depleted cells.
This work was supported by MitoStock Pty. Ltd., Australia.