bluuebunny/crossref_metadata_2025 huggingface dataset:
236 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATOR REGULATORS IN ASSOCIATION WITH BMP15 ON BOVINE EMBRYO DEVELOPMENT IN VITRO
Authors: Machado M. F. , Nogueira M. F. G. , Gilchrist R. B. , Sutton-McDowall M. L. , Mottershead D. G. , White M. A. , Thompson J. G.
Published: December 2014

BMP15 is a promising peptide to improve oocyte competence; also, addition of cyclic adenosine monophosphate modulator (cAMP) regulators prevents spontaneous maturation in vitro and promotes embryo development. We aimed to assess embryo development after prematuration [pre-in vitro maturation (IVM)] with IBMX and Forskolin (FSK) and maturation in the presence or absence of a purified pro mature region of BMP15. Immature cumulus-oocyte complexes (COC) were cultured in vitroMat (IVF Vet Solutions, Adelaide, Australia) plus 4 mg mL–1 fatty acid free-BSA and rhFSH (0.1 IU mL–1), then divided into the following treatment groups: 1) spontaneous IVM: 24 h of IVM; 2) spontaneous IVM + BMP15: 24 h of IVM in the presence of BMP15 (100 ng mL–1); 3) Pre 2 h: pretreatment with IBMX (500 µM; Sigma-Aldrich) and FSK (100 µM; Sigma-Aldrich) for 2 h following 24 h maturation; and 4) Pre 2 h + BMP15: pretreatment with IBMX and FSK for 2 h following 24 h maturation in the presence of BMP15 (100 ng mL–1). After maturation, oocytes were inseminated and zygotes were cultured for 5 days in VitroCleave (IVF Vet Solutions, Adelaide, Australia) and transferred into VitroBlast (IVF Vet Solutions, Adelaide, Australia) until blastocyst assessment (Days 7 and 8). Zona-intact embryos were retrieved to assess differential staining of trophectoderm and inner cell mass. Data were transformed into a logarithm and analysed by 1-way ANOVA and post hoc least significant difference using SigmaStat software (SPSS Inc., San Jose, CA, USA; P < 0.05). There was no difference among groups on cleavage rates or blastocyst rates at Day 7; however, both Pre 2 h treatments increase hatched blastocyst rates at Day 8 of embryo development (Table 1). Supplementation with BMP15 increased total blastocyst rates at Day 8, regardless of pretreatment with IBMX+FSK (Table 1). Our data demonstrate that embryos from oocytes matured in the presence of BMP15 or pretreated with IBMX+FSK increase trophectoderm and total cell numbers; however, no differences were observed for inner cell mass. We conclude that Pre 2 h treatment or BMP15 increase embryo development; however, no effect of cAMP regulators in association with BMP15 on embryo development was observed.
Table 1.Embryo development
Supported by FAPESP (project numbers: 2012/1073-8; 2013/12960-9; 2013/05083-1; 2012/50533-2).
25 DEVELOPMENTAL COMPETENCE OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED BY TRANSFECTED OR NONTRANSFECTED FIBROBLASTS TRANSFER TO ENUCLEATED OOCYTES DERIVED FROM OVUM PICK-UP AND ABATTOIR OVARIES
Authors: Yang C. , Shang J. , Zheng H. , Chen M. , Huang F. , Li C. , Yang B. , Liang X.
Published: December 2014

The objective of this study was to explore whether fibroblasts transfection and the source of oocytes – ovum pick-up (OPU) versus abattoir ovaries – affected the in vitro and in vivo developmental competence of somatic cell nuclear transferred (SCNT) embryos in buffalo. To this aim, the serum-starved ear fibroblasts were fused into enucleated oocytes derived from abattoir ovaries (Group 1) and OPU (Group 2). Furthermore, the enucleated buffalo oocytes derived from abattoir ovaries were also fused with pEGFP-N1 transfected ear fibroblasts, and the cloned embryos were enhanced green fluorescent protein (EGFP)-positive confirmed by fluorescence microscopy (Group 3). The reconstructed embryos cultured in Groups 1 to 3 were 262, 83, 120, respectively (5 replicates); and the data were analysed by one-way ANOVA (SPSS Inc., Chicago, IL, USA). As a result, the cleavage rate in Group 3 was significantly higher than that in Group 1 (75.0% v. 54.3%; P < 0.01), and the total blastocyst rate of reconstructed embryos in Group 3 (27.3%) was significantly higher than that in Group 1 (17.4%; P < 0.01) and Group 2 (24.4%; P < 0.05). The SCNT blastocysts were vitrified with 20% ethylene glycol + 20% dimethylsulfoxide + 0.5 M sucrose; the cryosurvival rates of SCNT blastocysts in the 3 groups were not different from each other (90.0%, 94.7%, 92.3%). Following culture, the cryosurvived blastocysts were transferred into synchronized local and crossbred buffaloes, with each recipient receiving 1 or 2 embryos. The pregnancy rates after transferring embryos derived from Groups 1 to 3 were not different from each other, and were 18.75% (3/16), 33.33% (4/12), and 26.67% (4/15), respectively. These results indicate that the oocytes derived from OPU can be enucleated as recipient cytoplasm and transfected fibroblast can be adopted as nuclei donor without decreasing the SCNT efficiency in buffalo.This research was supported by grants from the National Natural Science Foundation of China (31160456) and the Natural Science Foundations of China under Grant No. 0991011, No. 2011GXSFB018045).
254 IN VITRO BLASTOCYST PRODUCTION FROM PREPUBERTAL GOAT OOCYTES ACCORDING TO SEASON
Authors: Catalá M. G. , Roura M. , Hammami S. , Rodriguez M. , Izquierdo D. , Paramio M. T.
Published: December 2014

In our laboratory, we have been working for more than 20 years on in vitro embryo production from prepubertal goat oocytes. We have observed significant differences on blastocyst production according to the season of year. The present study is aimed to asses these differences in summer, autumn, winter, and spring. Oocytes with more than 3 compact cumulus layers (cumulus-oocyte complex, COC) and homogeneous cytoplasm were obtained after slicing prepubertal goat ovaries (45-day-old Murciano-Granadina rearing animals) recovered from slaughterhouse. Groups of 30 COC were matured in 100-µL drops of TCM199 medium (10% FBS) for 24 h with 5% CO2 and humidified atmosphere. Fresh buck semen (pool of 3 males) was selected by swim-up and capacitated with 0.05 mg mL–1 of heparin for 45 min. The IVF was performed by transferring 20 oocytes to 100-μL drops of Tyrode's medium with 4 × 106 capacitated sperm with 5% CO2 and humidity. After 17 h of IVF, a sample of oocytes was fixed in ethanol:acetic (3 : 1) for staining (1% lacmoid) to assess nuclear stage (Table 1). At 20 h post-insemination (pi), groups of 10 presumptive zygotes were transferred to 10-μL drops of SOF medium (10% FBS) under paraffin oil in a humidified atmosphere with 90% N2 for 8 days. Cleavage rate was evaluated at 48 h pi, and blastocyst rate was recorded at Day 8 pi (Table 1). Table 1 shows a decrease in normal pronuclear formation during winter (43%) and spring (47%). However, winter (16%) and spring (16%) produced significantly more blastocysts than summer (10%) and autumn (5%). Similar results were found by Sousa et al. (2014 Theriogenology 82, 1149–1162) in adult goats producing significantly higher blastocysts in the anoestrus season. We hypothesise that prepubertal goat oocytes in winter and spring may have a better cytoplasmic quality, producing more blastocyst even though the penetration rate is significantly lower than in autumn. In conclusion, under in vitro conditions and using fresh semen, the rate of penetration, cleavage, and blastocysts in prepubertal goats is influenced by season. Further studies in oocyte and sperm quality should be done to clarify this result.

Table 1.Nuclear stage of prepubertal goat oocytes according to season (3 replicates; top) and in vitro blastocyst production of prepubertal goat oocytes according to season at Day 8 postinsemination (7 replicates; bottom)1
265 TESTICULAR DEGENERATION AFFECTED PLASMA, ACROSOMAL AND MITOCHONDRIAL MEMBRANE INTEGRITY, AND DNA FRAGMENTATION IN RAM SPERMATOZOA
Authors: Bianchi Rodrigues Alves M. , Furugen Cesar de Andrade A. , Paes de Arruda R. , Batissaco L. , Lançoni R. , Andrade Torres M. , Mouro Ravagnani G. , Florez-Rodriguez S. A. , Marcelle Martins de Oliveira B. , Vellone V. , Carla Carvalho Celeghini E.
Published: December 2014

Testicular degeneration, an important cause of male infertility, adversely affects sperm motility and morphology. However, few studies describe effects on integrity of plasma and acrosomal membranes, mitochondrial membrane potential, and DNA fragmentation; therefore, they were evaluated in the present study. Testicular degeneration was induced in 17 White Dorper rams (scrotal insulation for 72 h). Semen was collected (artificial vagina) twice before insulation and twice thereafter (15-day intervals between post-insulation collections). Sperm motility and morphology were analysed by SCA software (Sperm Class Analyser®, MICROPTIC®, Barcelona, Spain) and differential interference contrast microscopy (DIC, model 80i, Nikon, Tokyo, Japan), respectively. Membrane integrity and potential were assessed with fluorescent probes: Hoescht 33342, propidium iodide, FITC-PSA, and JC-1 (Celeghini et al. 2010 Arq. Bras. Med. Vet. Zootec. 62, 536–543) and imaged with fluorescence microscopy (Nikon Model 80i, Nikon, Tokyo, Japan). Fragmentation of DNA was evaluated with a Halomax® kit (Halotech® DNA, Madrid, Spain). Data were analysed with Statview software (Stat View 1998, SAS Institute Inc., Cary, NC, USA). Data obtained from the periods (before × after insulation) were evaluated by analysis of variance (ANOVA) and means were compared using Tukey's test. Total motility (before: 87.53 ± 1.21%; after: 46.53 ± 4.46%) and progressive motility (before: 58.64 ± 2.00%; after: 31.33 ± 3.82%) were reduced (P < 0.01) by scrotal insulation, as were sperm major defects (before: 10.64 ± 1.65%; after: 54.30 ± 3.67%) and total defects (before: 20.50 ± 2.40%; after: 63.85 ± 3.41%; P < 0.0001). Sperm with intact plasma and acrosomal membranes and high mitochondrial potential (PIAIH) decreased (P < 0.0001) after insulation. In that regard, 53.19 ± 2.20 and 28.48 ± 3.48% of sperm were classified as PIAIH before v. after insulation, respectively. Furthermore, plasma membrane integrity, acrosome membrane integrity, and high mitochondrial potential were assessed independently. The quantity of plasma membrane integrity cells (before: 62.01 ± 2.07%; after: 33.92 ± 3.94%), acrosome membrane integrity cells (before: 57.17 ± 2.30%; after: 31.47 ± 3.77%), and high mitochondrial potential cells (before: 85.72 ± 1.42%; after: 57.28 ± 3.12%) were also reduced (P < 0.0001) after insulation. Likewise, DNA integrity decreased (P = 0.002) from 98.87 ± 0.26% before insulation to 91.88 ± 2.6% afterward. In conclusion, sperm plasma and acrosomal membrane integrity, mitochondrial membrane potential, and DNA fragmentation were adversely affected by testicular degeneration in rams induced by scrotal insulation.Research was supported by FAPESP process 2012/00040-0 and 2011/16744-3.
267 EFFECT OF SEMINAL PLASMA IN LAMA GLAMA SPERM
Authors: Carretero M. , Fumuso F. , Miragaya M. , Herrera C. , Giuliano S.
Published: December 2014

In South American camelids, raw semen only presents sperm with oscillatory movements. Therefore, it is necessary to treat these cells to enable them to acquire progressive motility. The effects of raw seminal plasma (SP) on sperm movement patterns (oscillatory, progressive, and hyperactive) have apparently not yet been reported. The objective of this study was to determine effects of raw seminal plasma on sperm motility, viability, and acrosomal status in fresh llama semen. A total of 15 ejaculates were collected (electroejaculation) from 5 llamas (n = 5, r = 3). Each ejaculate was diluted 4 : 1 in 0.1% collagenase in HEPES-TALP (HT) medium and incubated 4 min at 37°C, with the objective of separating spermatozoa from SP. Immediately after incubation, each ejaculate was divided into 2 and centrifuged for 8 min at 800 × g. Pellets were resuspended in either HT or raw SP and maintained at 37°C until evaluation (at 0, 1.5, and 3 h). Sperm motility was evaluated using a phase contrast microscope and a warm stage. Propidium iodide and carboxyfluorescein diacetate were used for assessing membrane integrity (viability). Acrosomal status was evaluated with the Coomassie blue stain. A split-plot design was used with treatment as a factor, with 2 levels (HT and SP) and time as the other factor, with 3 levels (0, 1.5, and 3 h), and blocked by males. There was no significant interaction between treatments (HT and SP) and times (0, 1.5, and 3 h) for each of the seminal characteristics evaluated. Progressive sperm motility was observed after collagenase treatment in all samples. Progressive motility disappeared immediately after the addition of raw SP and showed only oscillatory movements. In contrast, samples incubated in HT maintained progressive motility and became hyperactive. There were no differences (P > 0.05) in total motility of sperm incubated in HT among incubation times (0 h: 30.8 ± 18.9%; 1.5 h: 26.5 ± 11.5%; and 3 h: 21.5 ± 13.5%). However, in samples incubated with SP, a decrease (P < 0.05) in total sperm motility was detected after 3 h of incubation (0 h: 16.5 ± 12.6%, 3 h: 2.3 ± 3.2%). Sperm viability was not different (P > 0.05) between treatments (HT and SP); samples incubated in HT retained 78.4% of the initial viability (32.8/41.8, 3 h/0 h), and samples incubated in SP retained 69.7% of their initial viability (24.4/35.0, 3 h/0 h). The percentage of spermatozoa with intact acrosomes was not different (P > 0.05) between treatments (HT and SP); however, the percentage of sperm with intact acrosomes decreased after 3 h of incubation in both samples (HT and SP). Due to the presence of a high percentage of progressive and hyperactive motile sperm in samples incubated in HT and their absence in samples incubated in SP, we concluded that raw seminal plasma preserved oscillatory sperm motility. Further studies are needed to understand the effects of SP on South American camelid spermatozoa.
277 LIPID ACCUMULATION DURING BOVINE OOCYTES Gyr/HOLSTEIN MATURATION COLLECTED BY OPU IN SPOM SYSTEM
Authors: Leal G. R. , Monteiro C. A. S. , Saraiva H. F. R. A. , Camargo A. J. R. , Vasconcelos C. O. P. , Rodrigues A. L. R. , Nogueira A. G. , Serapião R. V. , Oliveira C. S.
Published: December 2014

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr/Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Oocyte quality is crucial for IVP, and lipid accumulation is a detrimental factor. The aim of this study was to assess the effect of SPOM maturation system (Albuz et al. 2010 Hum. Reprod. 25) on lipid accumulation in bovine oocytes. Oocytes obtained by ovum pick-up (OPU) from heifers without ovarian stimulation were transferred to control media (TCM 199 + sodium pyruvate, ITS, penicillin-streptomycin, BSA, FSH, oestrogen, and hCG) or SPOM (2 h pre-IVM: TCM 199 hepes + sodium pyruvate, ITS, penicillin-streptomycin, BSA, IBMX, and forskolin; followed by 28 h IVM: control media + cilostamide) in 5% CO2 atmosphere at 38°C. Oocytes were collected after 20, 24, and 28 h (groups: C20, C24, C28; S20, S24, S28) and evaluated for nuclear maturation using HOECHST (experiment 1, n = 301, 35–62 per group) and lipid content using Oil Red (experiment 2, n = 163, 14–42 per group). Oocytes from 4 replicates were fixed with PFA and stored at 4°C. For Oil Red, all structures were stained and evaluated at the same day. After being washed in 50% ethanol, oocytes were incubated in 0.2% Oil Red O solution for 10 min. Stained area fraction in each oocyte cytoplasm was measured using ImageJ (NIH). Nuclear maturation analysis was performed by Chi-squared test and Lipid analysis by Kruskal-Wallis and Dunn's post-test (P = 0.05). Distinct superscript letters indicate statistical difference between groups. In experiment 1, we detected a reduction in the percentage of matured (MII) oocytes only in S20 group (C20 = 64.28%a, C24 = 74.28%a, C28 = 60.46%a, S20 = 25.8%b, S24 = 59, 01%a, S28 = 74.13%a). In experiment 2, we detected an increase in lipid content in both control and SPOM groups from 20 to 28 h of IVM (S20 = 5.72a ± 4.41, S24 = 15.95bd ± 7.41, S28 = 37.46c ± 8.68, C20 = 8.65a ± 2.58, C24 = 12.14ad ± 8.08, C28 = 18.50b ± 8.09). For SPOM groups, the lipid content increased 2.78-fold from S20 to S24, and 6.54-fold from S20 to S28. In the control group, only in C28 wasa lipid increase detected, 2.13-fold higher than C20 content. Comparing control and SPOM groups at each time point, S28 displayed a 2.02-fold increase in lipid content compared to C28. We concluded that despite SPOM system being effective in maintaining meiotic arrest until 20 h of IVM and forskolin being present during pre-IVM in SPOM groups, Gyr/Holstein crossbreed oocytes obtained by OPU presented a significant increase in lipid content when matured in SPOM system, even higher than observed for the control group. During the last 8 h of IVM we observed a huge lipid accumulation in both systems, suggesting this might be a crucial period.
296 IMPACT OF CO-CULTURING CUMULUS-ENCLOSED PORCINE OOCYTES WITH DENUDED OOCYTES DURING IN VITRO MATURATION IN A DEFINED MEDIUM ON CUMULUS EXPANSION AND OOCYTE MATURATION
Authors: Appeltant R. , Somfai T. , Nakai M. , Bodo S. , Maes D. , Kikuchi K. , Van Soom A.
Published: December 2014

Recent research has revealed that oocyte-secreted factors (OSF) affect cumulus expansion and play important roles during maturation and embryo development of mammalian oocytes. The use of denuded oocytes (DO) as supplements during in vitro maturation (IVM) in a nondefined medium improved developmental competence of cumulus-enclosed porcine oocytes (COC; Gomez et al. 2012 Zygote 20, 135–145). We investigated the effect of DO on cumulus expansion and nuclear maturation of COC in pigs during IVM using a defined medium. If the DO exert a positive influence on IVM, the defined medium can then be analysed for the presence of OSF. Immature COC were collected in the slaughterhouse from prepubertal gilts. To obtain DO, some COC were completely denuded by pipetting through a narrow-bore glass pipette. The COC used as a source for DO fulfilled the same morphological criteria as the COC used for IVM. The IVM medium was porcine oocyte medium (POM; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213) with hormone supplementations applied only during the first 20 h of the IVM period. The COC were fixed to the bottom of 35-mm plastic Petri dishes in 3 × 3 grids by Cell-Tak (BD Bioscience, Bedford, MA, USA) in 100-µL droplets POM covered by paraffin oil. Culture droplets (each including 1 COC grid) were supplemented with (DO+ group, n = 179) or without 16 DO (DO– group, n = 143). After 20 h of IVM, the medium was replaced with a preincubated hormone-free POM and oocytes were cultured for an additional 28 h. At 0, 20, and 48 h of IVM, images of each grid were taken at the same magnification. The size of each COC was measured as a 2-dimensional area in pixels by analysing images with ImageJ software. Relative cumulus expansion was calculated at 20 and 48 h of IVM on the basis of the initial COC size at 0 h, which was assigned as 1. At 48 h of IVM, the COC were denuded and examined for oocyte maturation by orcein staining. The experiment was replicated 5 times. Cumulus expansion ratios at 20 and 48 h of IVM were compared between the DO+ and DO– groups by ANOVA. Maturation rates were compared between the DO+ and DO– groups by binary logistic regression. No difference in cumulus expansion between DO– and DO+ could be observed at 20 h (1.83 ± 0.04 and 1.75 ± 0.03, respectively) and 48 h (1.41 ± 0.03 and 1.47 ± 0.02, respectively) of IVM. Nuclear maturation rates of COC in DO– and DO+ groups did not differ significantly (39.0 ± 5.4 and 32.9 ± 8.8%, respectively). In conclusion, addition of DO to the defined IVM medium did not affect the cumulus expansion and oocyte maturation of follicular porcine COC. Further research is needed to assess the effects of DO during IVM on subsequent fertilization. If DO prove to be beneficial for fertilization, the nature of the OSF will be investigated.This study was supported by FCWO of UGent and by FWO-Flanders (grant number FWO11/ASP/276).
298 CYCLIC AMP AND CYCLIC GMP CONTENTS IN PORCINE OOCYTE–CUMULUS COMPLEXES, DENUDED OOCYTES, AND CUMULUS CELLS DERIVED FROM SMALL AND MIDDLE FOLLICLES DURING IN VITRO MATURATION
Authors: Okudaira Y. , Funahashi H.
Published: December 2014

Drastic changes in intracellular cAMP and cGMP levels play a critical role in the regulation of meiotic resumption. The objective of this study was to compare cAMP and cGMP contents in cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell masses (CC) derived from small (SF) and middle follicles (MF) during in vitro maturation (IVM). The COC were aspirated from SF (1–3 mm in diameter) or MF (3–6 mm in diameter) of prepubertal gilt ovaries. The COC were cultured in modified porcine oocyte medium (mPOM) with eCG, hCG, and dibutyryl cAMP for 20 h and then in fresh mPOM without those supplements for 4 h in an atmosphere of 5% CO2 in air at 39°C. At 0, 10, 20, and 24 h of IVM, COC, DO, and CC were collected. The DO were prepared by removal of cumulus cells and zona pellucida. The CC were prepared by puncturing ooplasm by using 18-gauge needle. Intracellular contents of cAMP and cGMP were determined by direct enzyme immunoassay kits. Statistical analyses of 3 to 7 replicated data were performed by ANOVA. There were no significant differences in contents of cAMP and cGMP between DO from SF and MF in all observation points (P > 0.05). Cyclic AMP contents in COC and CC derived from MF were higher than those from SF at 20 h of IVM (MF 33.0 ± 0.5 fmol/COC v. SF 28.4 ± 1.0 fmol/COC, MF 20.9 ± 0.9 fmol/CC v. SF 14.6 ± 0.8 fmol/CC; P < 0.05), whereas there were no significant differences between origins of those (SF v. MF, P > 0.05) at 0, 10, and 24 h of IVM. Furthermore, although cAMP content in CC from MF was not significantly different between 10 and 20 h of IVM (25.4 ± 1.7 and 20.9 ± 0.9 fmol/CC, respectively; P > 0.05), the content in CC from SF significantly decreased between 10 and 20 h (23.1 ± 1.2 , and 14.6 ± 0.8 fmol/CC, respectively; P < 0.05). At 0 and 10 h of IVM, cGMP contents in COC and CC from MF were significantly higher than those from SF (0 h: 81.8 ± 4.5 fmol/COC from MF v. 41.7 ± 10.6 fmol/COC from SF and 82.7 ± 7.5 fmol/CC from MF v. 10.7 ± 2.7 fmol/CC from SF; 10 h: 64.8 ± 8.4 fmol/COC from MF v. 24.8 ± 8.2 fmol/COC from SF, 49.3 ± 14.9 fmol/CC from MF v. 13.5 ± 4.8 fmol/CC from SF; P < 0.05). However, there were no significant differences in cGMP contents in COC and CC between the origins (MF v. SF) at 20 and 24 h of IVM (P > 0.05). From these results, we conclude that cAMP and cGMP contents in cumulus cells are significantly differences between the origins (MF v. SF) during IVM.
30 POSTMORTEM FINDINGS IN CLONED AND TRANSGENIC PIGLETS DEAD BEFORE WEANING
Authors: Schmidt M. , Winther K. D. , Callesen H.
Published: December 2014

Around 50% of cloned and transgenic piglets are lost during the first month after birth (Reprod. Fertil. Develop. 26, 124), and one reason is malformations of vital organs. The aim of the present study was to describe the distribution of malformations in piglets (until weaning, i.e. age <Day 28) born after transfer to Large White (LW) recipients of cloned embryos. Donor cells were fibroblasts either from LW (non-transgenic) or from Yucatan or Göttingen (made transgenic with 1 of 7 genes related to different human diseases). Handmade cloning was used to produce embryos that, after 5–6 days in vitro culture, were transferred to 202 LW sows 4 days after natural heat. Abortion occurred in 29 sows, and 6.5 ± 0.4 piglets per litter (from 1 to 22) were delivered from 116 sows (46 litters with LW piglets, 40 with Göttingen, 30 with Yucatan). In 78 of these litters (67%), autopsies were performed on 55 ± 4% of piglets stillborn or dead before weaning. Data were analysed by Fisher's Exact test with P < 0.05 as significance level. Malformations were found in 1 to 12 piglets per litter, with a higher malformation rate in transgenic Göttingen and Yucatan piglets (35% and 46% of all born, respectively) than in non-transgenic LW (17%). Many piglets showed 2 (24%) or more (6%) malformations. Some malformations seemed to be related to breed and/or transgene (see Table 1 for most frequent malformations); for example, heart malformations were most frequent in Yucatan litters independent of the transgene, whereas gall bladder and limb malformations were more frequent in Göttingen and in various litters with the same transgene. These results show that pig cloning results in a considerable loss of piglets, and that a majority of these can be related to various malformations. Use of transgenic cells for cloning only adds to this problem. Some malformations are related to the specific breed in use, but the general finding is that the problem is related to the cloning technique as such. However, because approximately half of the cloned piglets still survive, perhaps with unknown minor malformations, use of pigs as a model for human diseases is still realistic. But choice of breed and possible improvements of the transgenic technologies for this kind of work should be considered carefully.

Table 1.Malformations in transgenic and nontransgenic cloned piglets dead before weaning
300 GANGLIOSIDE IMPROVES MEIOTIC MATURATION AND PREIMPLANTATION DEVELOPMENT OF PORCINE OOCYTES
Authors: Kim J.-W. , Chae S.-K. , Lee Y.-H. , Ahn J.-H. , Do G.-Y. , Park H. , Koo D.-B.
Published: December 2014

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptor (EGFR). These compounds are localised in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role of gangliosides in porcine embryos is not yet clearly understood. Therefore, in this study, the relationship between ganglioside (GD1a) and EGFR activation was investigated during meiotic maturation and further development in porcine oocytes. Porcine oocytes were cultured in the NCSU-23 medium supplemented with or without EGF and GD1a for 44 h. Assessment of meiotic maturation was performed by using aceto-orcein staining method. Also, protein levels in matured oocytes were evaluated by Western blot analysis. After fertilization, presumptive zygotes were cultured in the PZM-3 medium for 6 days. All experiments were repeated more than 3 times. Data were analysed by using Student's t-test and Duncan's multiple range test. The proportion of GV arrested oocytes at 22 h was significantly different between the EGF + GD1a-treated and untreated group (41.6 ± 1.5 v. 25.0 ± 0.0%; P < 0.05). After completion of meiotic maturation (44 h), the proportion of MII was significantly different between the EGF + GD1a-treated and untreated group (89.9 ± 3.6 v. 57.4 ± 5.3%; P < 0.05). Also, expression of EGFR protein in matured porcine oocytes was increased in the presence of EGF and GD1a. After IVF, the percentage of penetrated oocytes was significantly higher in the EGF + GD1a-treated group (89.1 ± 2.3%), resulting in higher than normal pronucleus formation (2PN of 43.1 ± 5.2%). Finally, in result, differences in preimplantation developmental potential were detected between the oocytes that were matured with or without EGF and GD1a (50.4 ± 6.1 v. 27.2 ± 2.7%; P < 0.05). These results suggest that GD1a improves the developmental competence of embryos via enhanced meiotic maturation of porcine oocytes by EGFR activation.
31 PRODUCTION EFFICIENCY OF GENE KNOCKOUT PIGS USING GENOME EDITING AND SOMATIC CELL CLONING
Authors: Matsunari H. , Watanabe M. , Nakano K. , Uchikura A. , Asano Y. , Hatae S. , Takeishi T. , Umeyama K. , Nagaya M. , Miyagawa S. , Hanazono Y. , Nakauchi H. , Nagashima H.
Published: December 2014

Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5–83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN).

Table 1.Production efficiency of gene knockout cloned pigs using genome editing

This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, JST, CREST, Meiji University International Institute for Bio-Resource Research (MUIIBR), and JSPS KAKENHI Grant Number 26870630.
310 COBALAMIN SUPPLEMENTATION DURING IN VITRO MATURATION IMPROVES PREIMPLANTATION DEVELOPMENT OF SHEEP EMBRYOS
Authors: Zacchini F. , Toschi P. , Loi P. , Ptak G. E.
Published: December 2014

Oocyte maturation process includes the establishment of proper epigenetics marks, fundamental to ensure successful pregnancy. Epigenetic maturation of the oocyte depends on one-carbon-metabolism (OCM), whose key elements (cobalamin, folate) are crucial cofactors for the transfer of methyl groups onto chromatin. Commercially available IVM-media only partially supports oocyte metabolic requirements, and thus may negatively affect epigenetic maturation. Of relevance, cobalamin, one of the OCM cofactors normally present in follicular fluid, is missing in IVM-media. We investigated if cobalamin supplementation of IVM media affects sheep oocyte developmental competence in term of subsequent embryo development, epigenetic pattern, and fetal survival. Briefly, sheep oocytes were isolated from ovaries and divided into 2 groups: an untreated control (in vitro CTR group) and oocytes in vitro matured in medium containing cobalamin at 200 p.m. (Cobalamin group). Following maturation, MII oocytes were in vitro fertilized and cultured until blastocyst stage. A cohort of blastocysts was surgically transferred to recipient ewes and then conceptuses were collected at 20 days of pregnancy. Naturally mated conceptuses were also collected (in vivo CTR group). In vitro-matured oocytes and -derived blastocysts were analysed (i) by immunofluorescence anti-5-methylcitydine and (ii) by qRT-PCR for the expression of a panel of genes (MATb, ACHY, CBS, MTHFR, DNMT1) involved in OCM pathway. Immunofluorescence results were analysed by Image J software. Decimal variables were analysed using the Mann-Whitney test, while variables were expressed as percentage with the Fisher exact test. Cobalamin exposure during IVM significantly increased (i) cleavage rate (cobalamin 130/220 v. in vitro CTR 134/191, P < 0.02) following in vitro fertilization and (ii) global DNA methylation in blastocyst-stage embryos (P < 0.05). Then, qRT-PCR analysis of a select panel of OCM genes following IVM supplemented with cobalamin revealed a downregulation of MATb, ACHY, and DNMT1 in cobalamin-treated oocytes v. in vitro CTR group (P < 0.05), while no differences were observed at blastocyst stage. Finally, we found that the survival rate at 20 days of pregnancy was comparable in in vitro-produced (IVP) embryos from cobalamin and in vitro CTR oocytes, but reduced compared to in vivo CTR (cobalamin 60%, in vitro CTR 72%, and in vivo CTR 100%). Altogether, our results showed that cobalamin supplementation in IVM medium positively affects competence of oocytes and methylation profile at the blastocyst stage, presumably through the OCM pathway. Moreover, postimplantation survival of IVP conceptus, derived from untreated and treated oocytes, was reduced compared to naturally mated ones. Further investigation will clarify whether cobalamin supplementation influences fetal and placental development of IVP conceptus.
311 TEMPERATURE DURING OVERNIGHT HOLDING IN MEIOSIS INHIBITOR-FREE MEDIUM AFFECTS CHROMATIN CONFIGURATION AND MEIOTIC RESUMPTION IN EQUINE OOCYTES
Authors: Martino N. A. , Dell'Aquila M. E. , Uranio M. F. , Lampignano R. , Lacalandra G. M. , Hinrichs K.
Published: December 2014

Immature equine oocytes may be held overnight in an Earle's/Hanks' M199-based medium in the absence of meiotic inhibitors (EH medium) to schedule the onset of in vitro maturation. Holding in EH has been shown not to affect meiotic or developmental competence of equine oocytes (Choi et al. 2006 Theriogenology 66, 955–963). However, no studies have been performed to identify the mode by which this medium suppresses meiosis. We hypothesised that holding temperature may affect oocyte meiotic arrest. The effect of 3 holding temperatures (25, 30, 38°C) on chromatin status was investigated after Hoechst 33258 staining (Hinrichs et al. 2005 Biol. Reprod. 72, 1142–1150). Oocytes were recovered by scraping of follicles from slaughterhouse-derived ovaries. Data were analysed by Chi-squared test and one-way ANOVA followed by Dunn's or Holm-Sidak Multiple Comparison methods. A level of P < 0.05 was considered significant. There were no significant differences in chromatin configuration between oocytes held overnight at 25°C (25°C-held) and controls (immediately-fixed oocytes); the proportion of oocytes showing meiotic resumption was 1/27, 4% and 0/26, 0%, respectively (not significant, NS). In contrast, holding at higher temperature significantly increased meiosis resumption (14/38, 37% and 14/28, 50%, at 30 and 38°C, respectively; P < 0.01) and reduced the proportion of oocytes showing the most meiotically-competent germinal-vesicle (GV) configuration (condensed chromatin, CC; 24 to 29% v. 65 to 70% for control and 25°C-held, respectively; P < 0.05). Based on these results, a subsequent experiment was performed in which oocyte meiotic stage and mitochondrial (mt) potential of 25°C-held (n = 29) and control (n = 36) oocytes was evaluated. Nuclear chromatin, mt activity (MitoTracker orange), intracellular reactive oxygen species (ROS) levels (2′,7′-dichlorodihydrofluorescein diacetate, DCDHFDA), and mt/ROS colocalization (Pearson's coefficient) were analysed by epifluoscence and confocal microscopy (Martino et al. 2012 Fertil. Steril. 97, 720–728). Meiotic arrest after EH treatment at 25°C was confirmed (0/29, 0% v. 5/36, 14% for meiotic resumption in 25°C-held and controls, respectively; NS). At any GV stage, 25°C-held treatment had no effect on mt activity, ROS levels, or mt/ROS colocalization. For example, in CC oocytes, values for control and 25°C-held, respectively, were: MitoTracker, 547.8 ± 499.5 v. 722.9 ± 390.3; DCF fluorescence intensity, 278.5 ± 179.3 v. 378 ± 185, and mt/ROS colocalization, 0.5 ± 0.1 v. 0.5 ± 0.2; these were not significantly different (NS). In conclusion, EH holding at 25°C maintains meiotic arrest, viability, and mt potential of equine oocytes.
313 PRE-IN VITRO MATURATION WITH CYCLIC AMP AND CYCLIC GMP MODULATORS IMPROVES DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES
Authors: Santiquet N. W. , Greene A. F. , Schoolcraft W. B. , Krisher R. L.
Published: December 2014

In vitro maturation (IVM) of cumulus-oocyte complexes (COC) results in oocytes with reduced quality and is still not as efficient as in vivo maturation in most species. One hypothesis that could explain the low developmental competence of oocytes following IVM is that the oocytes resume meiosis too quickly after being retrieved from the follicles. Studies in mice and bovine have shown that a short period of prematuration in the presence of cAMP modulators, before IVM, enhances oocyte developmental competence. Moreover, other studies have recently demonstrated that cGMP is also a crucial molecule involved in meiotic resumption. Here, our objective was to examine the effect of a cGMP modulator in combination with a cAMP modulator during a short period of prematuration on mouse oocyte nuclear maturation and subsequent embryo development following IVF. The COC were collected (6 replicates) from 2-month-old outbred CF1 mice 48 h after PMSG (5 IU) injection in the presence (pre-IVM) or absence (control) of cGMP and cAMP modulators. Pre-IVM COC (n = 184) were then placed in prematuration medium that also contained these cGMP and cAMP modulators. After 2 h, pre-IVM COC were washed and transferred to our in-house prepared, completely defined IVM medium (Paczkowski et al. 2014 Reprod.) for the remaining 16 h of culture; 10 oocytes per 50 µL drop under oil, at 37°C in 7.5% CO2 and 6.5% O2 due to the increased altitude at our location. Control COC (n = 161) were matured in the same IVM medium under identical conditions for 18 h, without prematuration. After IVM, oocytes were fixed for assessment of nuclear maturation, or fertilized and cultured in vitro and subsequent development (96 and 112 h) was recorded (Paczkowski et al. 2014 Reprod.). Results were analysed by ANOVA. A short 2-h prematuration period in the presence of cGMP and cAMP modulators had no impact on oocyte nuclear maturation to metaphase II after IVM or on embryo cleavage after IVF. However, pre-IVM treatment improved the developmental competence of the oocyte, as demonstrated by increased embryo development. More (P < 0.02) blastocysts (96 h of culture) and hatched blastocysts (112 h of culture) developed in the pre-IVM treatment compared to control (31.0 ± 3.4 v. 19.9 ± 3.2%; 31.5 ± 3.4 v. 19.9 ± 3.2%, respectively). In conclusion, a combination of cGMP and cAMP modulators during oocyte collection and a subsequent short pre-IVM improves oocyte developmental competence and could therefore be a potential tool to improve embryo yield following IVM.
319 APPROACHES TO IMPROVE INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS AND MAXIMIZE THE USE OF SEX-SORTED SPERM IN BOVINE
Authors: Canel N. G. , Bevacqua R. J. , Hiriart M. I. , Chavez Rabelo N. , Almeida Camargo L. S. , Salamone D. F.
Published: December 2014

Intracytoplasmic sperm injection (ICSI) mediated transgenesis is an effective tool for transgenic animal production. However, ICSI in cattle remains inefficient. In this work, we assayed approaches to improve egfp expressing blastocysts production by ICSI: the sperm pretreatment with heparin and l-glutathione (Hep-GSH), the use of sex-sorted sperm (SS), the refrozen/thawing of SS sperm, and the combination of these. Quality of ICSI blastocysts was analysed by studying the expression of 4 genes, and the rates of DNA fragmentation. Cumulus-oocyte complexes from slaughtered cow ovaries were in vitro-matured for 21 h. Nonsorted (NS) and sex-sorted (SS) frozen straws were thawed. Some of them were incubated with 80 μM Hep-15 mM GSH for 20 h (Hep-GSH+). The Hep-GSH-control group was not pretreated. Semen samples were co-incubated with 50 ng µL–1 of pCX-EGFP for 5 min before ICSI. Moreover, the SS sperm that are usually discarded after ICSI were cryopreserved and used for ICSI after a second thawing (ICSI SS refrozen). The ICSI NS, sham, and diploid parthenogenetic (Diplo PA) controls were included. Oocytes were activated with 5 µM ionomycin for 4 min, TCM-199 for 3 h (except for diploid PA), and 1.9 mM DMAP for 3 h. Cleavage and blastocyst/egfp expression rates were evaluated on Days 2 and 7 post-ICSI, respectively. Results are shown in Table 1. Relative expression of HMGN1, GLUT5, AQP3, and OCT4 genes from ICSI NS Hep-GSH+ and IVF blastocysts were compared by qPCR. Data were analysed by the pair-wise fixed reallocation randomisation test. None of the 4 genes showed differences between groups. The DNA fragmented nucleus index/blastocyst cell numbers were determined by TUNEL assay, not showing differences between groups (Kruskal–Wallis test, P ≤ 0.05). Means ± s.d. were 29 ± 17/91 ± 27 for ICSI Hep-GSH+; 27 ± 15/63 ± 34 for ICSI Hep-GSH–; 28 ± 17/68 ± 17 for ICSI SS, 28 ± 13/75 ± 24 for ICSI SS refrozen; and 21 ± 13/105 ± 59 for IVF SS control. The Hep-GSH pretreatment can increase blastocyst and transgene expressing blastocysts rates after TM-ICSI, except when SS semen is used. Interestingly, the use of SS sperm for ICSI can be maximized by cryopreservation and reuse of discarded sperm cells. The parameters analysed in this work indicate that the proposed approaches do not affect blastocyst quality. Therefore, Hep-GSH pretreatment of NS sperm and refrozen SS sperm could be applied for TM-ICSI in bovine for the production of transgenic animals.

Table 1.In vitro development and egfp expression of ICSI embryos fertilized with nonsorted (NS) and sex-sorted (SS) sperm pretreated with Hep-GSH, refrozen, or both
328 NONVIRAL REPROGRAMMING OF mCHERRY-EXPRESSING PORCINE FIBROBLASTS INTO INDUCED PLURIPOTENT STEM CELLS BY piggyBac TRANSPOSONS
Authors: Kumar D. , Talluri T. R. , Kues W. A.
Published: December 2014

The generation of induced pluripotent stem (iPS) cells is a promising approach for innovative cell therapies, as well as for animal biotechnology. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of tumorigenicity due to the preferential integration into active genes. The domestic pig is an attractive large animal model for preclinical testing of safety and efficacy of cell-based therapies. Porcine organs are similar in size and physiology to their human counterparts, and a suitable model for cardiovascular disease, muscular dystrophies, atherosclerosis, wound repair, diabetes, and ophthalmological diseases. Therefore, the present study was carried out to derive porcine iPS cells from transgenic fetuses systemically expressing mCherry (Garrels et al. 2011 PLOS ONE 6) through a nonviral piggyBac transposon. The piggyBac transposon system has several advantages: (i) piggyBac has no bias to integrate in expressed gene-like lenti- or retroviral vectors, (ii) the cargo capacity is >100 kb, (iii) seamless removal is possible, and (iv) the production of transposon plasmid is cost-efficient and does not require S2 safety cabinets. Porcine fetal fibroblasts isolated from CAGGS-mCherry founder porcine line fetuses (passage 2), were co-electroporated with a PB transposon carrying a multigene cassette consisting of human cDNA for OCT4, SOX2, KLF4, c-MYc, NANOG, and LIN28 separated by self-cleaving 2A peptide sequences, driven by a CAGGS promoter and a helper plasmid expressing the pCMV-PB transposase. On Day 6 postelectroporation, morphology of fibroblasts started change to round structure, and on Day 9 loose aggregates of cells developed. Putative iPS cell colonies were cultured, propagated, and characterised through morphology and expression of pluripotency markers, such as AP, OCT4, SSEA-1, and SSEA-4, through immunostaining. Further, various stemness genes, including OCT4, SOX2, NANOG, and UTF, were detected by porcine-specific primers through endpoint RT-PCR. In vitro differentiation potential was assessed by embryoid body (EB) formation. The formed EB exhibited the expression of mCherry in their cells and expressed differentiation markers, such as NESTIN, TUJI, GATA4 and AFP. To test their tumorigenic potential, 1 × 106 iPS cells were injected under the skin of nude mice. An mCherry-positive tumour was recovered 6 weeks later. Presently the tumour is being prepared for histological analysis. This study indicates that piggyBac transposon containing 6 transcription factors is able to reprogram porcine fetal fibroblasts into iPS cells. These cells could be cultured and maintained in vitro for a prolonged period, exhibit characteristics of stem cells, and offer a potential source for future blastocyst complementation experiments.
332 DERIVATION OF BOVINE-INDUCED PLURIPOTENT STEM CELLS BY piggyBac-MEDIATED REPROGRAMMING
Authors: Rao T. T. , Dharmendra K. , Silke G. , Garrels W. , Niemann H. , Debowski K. , Behr R. , Kues W. A.
Published: December 2014

Induced pluripotent stem (iPS) cells are a seminal breakthrough in stem cell research and are promising for the development of advanced regenerative therapies and farm animal biotechnology. Considering the potential of this technology for both basic and clinical research, it is tempting to extend this research to important livestock species, such as cattle, in which authentic embryonic stem cell lines are yet not available. The first attempts to produce iPS cells from livestock species were made using retro- and lentiviral vectors, which are associated with an increased risk of insertional mutagenesis and which are not removable after reprogramming. Here, we describe a nonviral method for the derivation of bovine iPS cells, employing a piggyBac (PB) transposon system. The reprogramming PB transposon encodes the primate cDNA of 6 core reprogramming factors, OCT4, SOX2, KLF4, MYC, LIN28, and NANOG, separated by self-cleaving 2A peptide sequences and driven by the chimeric CAGGS promoter. The derived bovine iPS line expressed typical endogenous genes (OCT4, SOX2, c-MYC, KLF4, NANOG, REX1, and ALP) by RT-PCR and OCT-4 as well as SSEA-1 and 4 pluripotency-related markers by immunostaining, and it exhibited silencing of exogenous reprograming factors. Moreover, the iPS line showed long-term proliferation (until the 40th passage) under feeder-free culture conditions, differentiated into derivatives of the 3 germ layers in vitro, and formed teratomas (4/6) after subcutaneous injection into immunodeficient nude mice. These results are a major step towards the derivation of authentic bovine iPS cells, and thus facilitate the genetic modifications of the bovine genome.