bluuebunny/crossref_metadata_2025 huggingface dataset:
4 PRODUCTION OF 11β-HYDROXYSTEROID DEHYDROGENASE TYPE 1 (11β-HSD1) OVER-EXPRESSED PIGS FOR THE STUDY OF METABOLIC SYNDROME DISEASE
Authors: Jeon Y. , Kim Y. K. , Yoon J. D. , Cai L. , Hwang S. U. , Kim E. , Lee S. , Jeung E. B. , Hyun S. H.
Published: December 2013

Metabolic syndrome shows a complication at risk for cardiovascular disease and diabetes. With the high prevalence of obesity globally, the incidence of metabolic syndrome is increased. However, the basic mechanisms of metabolic syndrome are not completely known yet. Therefore, we attempted to develop large-animal model for the study of metabolic syndrome disease. Some studies have shown that constitutive overexpression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in mice leads to metabolic syndrome. Therefore, we tried to produce the 11β-HSD1 gene overexpressed pig by somatic cell nuclear transfer (SCNT). First, we established 11β-HSD1 overexpressed cells for the preparation of the transgenic (TG) donor cells. Porcine fetal fibroblasts were transfected with cytomegalovirus vector that included the porcine 11β-HSD1 gene. The 11β-HSD1-TG cell was injected into the enucleated ooplasm to produce 11β-HSD1-TG cloned embryos. In total, 833 TG porcine SCNT embryos (TG-SCNT embryos) were made. Of these, 155 TG-SCNT embryos were cultured in procine zygote medium-3 to evaluate the in vitro developmental potential of TG-SCNT embryos. Among these porcine SCNT-TG embryos, 109 embryos (70.3%) were cleaved at 48 h. On Day 7, 31 transgenic porcine SCNT embryos (20.0%) developed to the blastocyst stage. The remaining 678 TG-SCNT embryos were transferred to 6 surrogates (average 113 embryos per surrogate). On 25 days after embryo transfer, 2 surrogates were diagnosed as pregnant (pregnancy rate, 33.3%). On Day 114, we obtained 9 live piglets and 3 stillborn piglets from 2 surrogates. By PCR analysis, we confirmed that 1 live piglet and 2 stillborn piglets were integrated with 11β-HSD1 gene. We succeeded to obtain TG piglets at sixth trials, and for the re-cloning by SCNT, a stable cell line transfected with the 11β-HSD1 gene was established from a TG cloned piglet. This study presents new possibilities for large-animal model development for the study of metabolic syndrome disease.
This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.
53 COMPARISON OF 4 DIFFERENT CRYOPROTECTANTS ON FREEZING NGUNI BULL SPERMATOZOA EVALUATED BY COMPUTER-AIDED SPERM ANALYSIS
Authors: Seshoka M. M. , Mphaphathi M. L. , Nedambale T. L.
Published: December 2013

Cryopreservation has been reported to damage approximately 40 to 50% of viable spermatozoa in bulls. It is critical to evaluate frozen-thawed spermatozoa with computer-aided sperm analysis (CASA) and find a suitable cryoprotectant for Nguni semen. The study was conducted to compare cryo-effectiveness of glycerol (GLY), dimethyl sulfoxide (DMSO), propanediol (PND), and ethylene glycol (EG) cryoprotectants at 12% concentrations during freezing of Nguni bull semen. Semen was collected from 18 stud Nguni bulls of proven fertility with the use of an electro-ejaculator. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories. Semen samples were pooled and sperm motility rate was evaluated using CASA. Semen was then diluted (1 : 2; vol : vol) with egg-yolk citrate extender supplemented with either 12% GLY, DMSO, PND, or EG. Semen samples were equilibrated for 4 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled rate programmable freezer using a customized freezing curve (from 5 to –5°C at 0.008°C min–1 and from –3 to – 130°C at 6°C min–1). Following thawing of semen, artificial insemination was conducted on 104 oestrus-synchronised Nguni cows. The IVF was also conducted on 120 oocytes to check the cleavage rate. Data were analysed using ANOVA. There was a significant difference (P < 0.05) between raw total sperm motility (94.70 ± 2.63) and frozen-thawed sperm total motility with GLY (77.80 ± 11.03), EG (20.35 ± 11.86), DMSO (15.68 ± 10.14), and PND (11.19 ± 11.27) groups. The pregnancy rate following artificial insemination was 75.9% and a total of 86.6% oocytes had cleaved after fertilization with frozen (12% GLY)/thawed semen. In conclusion, cryopreservation process reduced sperm motility and velocity rates, regardless of cryoprotectant. Egg-yolk citrate extender supplemented with 12% glycerol had recorded the highest post-thaw sperm motility rate.
54 EFFECT OF THAWING TEMPERATURE ON POST-THAW SURVIVAL OF BOAR SPERM
Authors: Athurupana R. , Funahashi H.
Published: December 2013

The survival of cryopreserved sperm is strongly influenced by the warming rate. Aims of the study were to examine the time taken by the extender inside the straw to reach 39°C and to evaluate the effect of thawing temperature on post-thaw survival of boar sperm. The sperm samples were diluted in egg yolk-based freezing extender containing trehalose (100 mM) and 0.25% Equex STM™. The samples were cryopreserved using the straw-freezing procedure (Buranaamnuay et al. 2009 Reprod. Domest. Anim. 44, 69–73). To examine the change of extender temperature inside the straw, sealed 0.5-mL straws with the extender were immersed in water (40, 60, or 80°C) after freezing and the temperature inside the straw was measured using a 2-channel digital record thermometer (TNA-140, Tasco Japan Co. Ltd., Osaka, Japan). After frozen sperms were thawed at different temperatures for the time taken by the extender inside the straw to reach 39°C, the sperms were analysed for motility, viability, and acrosome integrity. Statistical analysis from 5 replicated trials was performed by ANOVA. Average time taken to reach 39°C in 40, 60, and 80°C water was 36, 13, and 9 s, respectively (n = 8). There were 2 phases of the rates of change of temperature (RCT) inside the straw: rapid at the beginning of the course of thawing (–196 to approximately 0°C) and relatively slower at the liquid phase. The RCT was very rapid during the first 2 s, with a mean rate of 73.3, 81.3, and 86.9°C s–1 in 40, 60, and 80°C water, respectively. The RCT was not significantly different among the 3 groups between 2 and 6 s after the start of warming, whereas the RCT was significantly higher in 80°C water compared with 60 and 40°C from the next second (P < 0.01). Starting from 12th second, RCT was significantly higher in 60°C water compared with 40°C water (P < 0.01). When frozen sperms were thawed at 40, 60, or 80°C for 36, 13, or 9 s, respectively, however, there were no significant differences in post- thaw parameters (motility and viability) of sperms thawed in different temperatures. The motility of sperms thawed at 40, 60, and 80°C water was 41.2, 42.5, and 30.0%, whereas the viability was 42.3, 47.6, and 42.6%, respectively. The percentage of sperms with intact acrosomes was 54.8, 64.2, and 68.0%, respectively. There was a trend of increased acrosome integrity when the sperms were thawed at higher temperature (P = 0.06). In conclusion, this preliminary result suggests that rapid thawing may affect acrosome integrity rather than the motility and viability of cryopreserved sperm. However, additional observations are necessary to reach further conclusions.
56 COMPARISON OF 4 DIFFERENT DILUENT AGENTS ON CRYOPRESERVATION OF SEMEN FROM UNIMPROVED INDIGENOUS SOUTH AFRICAN GOATS
Authors: Matshaba B. , Mphaphathi M. L. , Schwalbach L. M. , Greyling P. C. , Nedambale T. L.
Published: December 2013

The cryopreservation process is one of the most crucial factors that affect the post-thaw quality of frozen goat semen. The aim of this study was to compare 4 different semen diluents (Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®). A total of 5 indigenous bucks were used as semen donors. Semen was evaluated for macroscopic and microscopic characteristics. Semen was pooled and diluted randomly in each of the 4 different extenders. Semen samples were equilibrated for 3 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled-rate programmable freezer using a customized freezing curve (from 5 to –5°C at 4°C min–1, –5 to –110°C at 25°C min–1, and then from –110 to –140°C at 35°C min–1). Data was analysed with the aid of the statistical program GenStat®, using a complete randomised design. Following fresh semen dilution with Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®, the sperm progressive motility percentage was 41.5 ± 4, 43.5 ± 7, 48.6 ± 5, and 50.2 ± 7%, respectively (P > 0.05). In addition, the live normal sperm percentage following fresh semen dilution was 59.2 ± 5, 49.2 ± 6, 57.8 ± 5, and 54.4 ± 3% for Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®, respectively (P > 0.05). After thawing semen samples frozen either with Tris-BSA, Tris-yolk, Bioxcell®, or Ovixcell, the sperm progressive motility percentage was 5.3 ± 2, 1.6 ± 1, 10.6 ± 2, and 13.3 ± 3%, respectively (P < 0.05). Furthermore, the live normal sperm percentage following thawing was 19.1 ± 3, 14.3 ± 3, 39.6 ± 6, and 37.1 ± 3% for Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®, respectively. In conclusion, sperm progressive motility percentage was reduced following the freezing-thawing process, irrespective of the type of extender used. The Bioxcell® and Ovixcell® extenders showed to be better for South African indigenous goat semen cryopreservation purposes.
6 GENE EXPRESSION ANALYSIS OF IN VIVO- AND IN VITRO-MATURED PORCINE METAPHASE II OOCYTES
Authors: Cox L. , Saunders G. , Stevens J. , Isom S. C.
Published: December 2013

In vitro-matured (IVM) oocytes lack the same developmental competence as oocytes that are matured in vivo (IVV), yet no compelling explanation for this discrepancy has been provided at the molecular level. The aim of this study was to quantify and compare mRNA levels in IVM and IVV oocytes for genes from a wide variety of functional gene categories, including RNA degradation, pluripotency, epigenome modification, oocyte-specific, and apoptosis. Quantitative real-time PCR (qPCR) was used to evaluate the relative gene expression levels of 70 genes in each of 33 individual IVM oocytes from 4 different collection days and 29 individual IVV oocytes from 4 different donor animals. The qPCR data were analysed using ANOVA and significance was assigned at P < 0.05. After a multiple testing correction was applied, relative transcript abundances for 32 of the 70 genes tested were found to be significantly different (q < 0.05) between the IVM and IVV oocytes. Of these significantly different genes, 23 were higher in the IVM oocytes and only 9 were higher in the IVV oocytes. The 32 significantly differentially expressed genes were then evaluated in relation to their corresponding functional gene categories. Of particular interest, transcripts for 7/14 RNA degradation-related genes (CNOT3, DCP1A, DDX6, LSM1, PABPN1, PABPN1L, PARN) and 3/9 oocyte specific genes (BMP15, YBX2, H1FOO) were significantly more abundant in the IVM oocytes. In contrast, transcripts for 4/8 epigenetic related transcripts (ASH2l, DNMT1, EHMT2, EZH2), 2/2 apoptosis related genes (BCL2, XIAP), and 1/4 pluripotency factors (LIN28) were significantly more abundant in the IVV oocytes. Gene set enrichment analysis confirmed that, within the context of this experimental design, RNA degradation and chromatin remodelling pathways are significantly perturbed in IVM oocytes. We conclude that in vitro maturation has profound effects on transcript populations of metaphase-II oocytes, with most transcripts being higher in IVM oocytes. We expect that this data will lead to a better understanding of how we can improve the quality of oocytes that are matured in vitro as well as provide information to help to identify markers that could be indicative of oocyte quality.
61 TRANSGENIC HUNTINGTON'S DISEASE MONKEY SPERM HAS A LOWER CRYOTOLERANCE
Authors: Moran S. P. , Chi T. , Prucha M. S. , Engelhardt H. R. , Yuksel A. , Chan A. W. S.
Published: December 2013

Cryopreservation is an important tool routinely used for preserving sperm for artificial reproductive technologies (ART), as well as genetic preservation of unique animal models. The cryopreservation process is harsh and detrimental to the fragile gametes, and damage to the sperm is not only known, but inevitable. This study presents new data in which sperm from 3 transgenic Huntington's disease (HD) monkeys (rhesus macaques) are compared with 3 wild-type (WT) rhesus sperm donors. Currently, there are no data comparing HD versus WT sperm viability and cryotolerance in humans. The goal of this study was to investigate differences between fresh and frozen semen by quantitative analysis on sperm viability based on (1) motility, (2) membrane integrity, and (3) acrosome integrity. Sperm motility was determined by visual evaluation. Membrane and acrosome integrity were assessed simultaneously by Hoechst 33342, propidium iodide (PI), and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) triple staining. Sperm viability analysis was divided into 3 groups: (1) fresh HD versus fresh WT, (2) fresh versus cryopreserved-thawed WT, (3) and fresh HD versus cryopreserved-thawed HD sperm. Interestingly, fresh HD sperm had a lower percentage of membrane-damaged cells (38.57 ± 3.15) compared with WT (49.67 ± 3.56; P < 0.03). However, after cryopreservation and subsequent thawing, HD sperm had a significantly higher percentage increase in damaged membranes than WT sperm (27.91 ± 2.93 v. 8.27 ± 8.28; P < 0.001), respectively. No significant difference in acrosome damage between groups was identified in either fresh or cryopreserved sperm populations. Motility significantly declined in both cryopreserved populations [HD: 89.7 to 43.4% (P < 0.001) and WT: 90.0 to 45.6% (P < 0.001)]. There was no significant difference between either freeze-thawed group. These data illustrate that HD sperm have a lower cryotolerance than WT sperm. Our findings suggest that the optimization of the HD sperm cryopreservation method and investigation on biochemical differences (e.g. membrane lipid composition) are necessary to improve post-thaw survival. This in turn is important for the establishment of a sperm cryobank and future derivation of a unique animal model such as HD monkey. Our study also suggests that HD monkey could be a useful model for optimizing cryopreservation method for HD patients.
63 THE EFFECT OF A MODIFIED CRYOPRESERVATION METHOD ON THE VIABILITY OF FROZEN–THAWED PRIMORDIAL GERM CELL ON THE KOREAN NATIVE CHICKEN (Ogye)
Authors: Kim H. , Kim D. H. , Han J. Y. , Choi S. B. , Ko Y.-G. , Do Y. J. , Seong H.-H.
Published: December 2013

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus, conservation of genetic material in chickens was attempted by preserving primordial germ cells (PGC) in LN2. This study established a method for preserving chicken PGC that enables long-term storage in LN2 for preservation of species. The purpose of this study was to clarify the effects of fetal bovine serum (FBS) or chicken serum (CS) treatment on the viability of cryopreserved PGC in the Korean native chicken (Ogye). Primordial germ cells were separated from a germinal gonad using a fine glass micropipette under a microscope and were suspended in a freezing medium containing freezing and protecting agents [e.g. dimethyl sulfoxide (DMSO) and ethylene glycol (EG)]. The PGC were then purified using the magnetic-activated cell sorting (MACS) method. The viability of the PGC in both groups was determined by the trypan blue exclusion method. The values of the 0, 5, 10, and 15% DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39, and 48.36%, respectively. The viability of PGC after freeze-thawing was significantly higher for the 10% EG plus FBS treatment than for the 10% EG plus CS treatment (P < 0.05; 64.36 v. 50.66%). This study established a method for preserving chicken PGC that enables systematic storage and labelling of cryopreserved PGC in LN2 at a germplasm repository and ease of entry into a database. In the future, the importance of this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germ line chimeras.
7 OESTRAL RESPONSE AND FERTILITY IN DAIRY COWS TREATED WITH PROGESTERONE FOR 6 DAYS PRIOR TO OESTRUS SYNCHRONIZATION WITH PROSTAGLANDIN F2α
Authors: Orozco M. , Gutierrez C. G. , Lopez R. , Aguilar C. , Roque C. , Hernandez-Ceron J.
Published: December 2013

The present study tested whether the increase in serum progesterone concentrations for 6 days before oestrus synchronization with prostaglandin F2α (PGF2) increases oestral response, pregnancy rate, and reduces the incidence of twin births in dairy cattle. Seven hundred and eighty-three first-service dairy cows were synchronized with 2 injections of PGF2 14 days apart, starting on Day 35 postpartum. Six days before the second PGF2 injection, cows were assigned to 2 groups: P4 and control. The P4 group (n = 387) received a progesterone-releasing intravaginal device and an intramuscular injection of 500 mg of progesterone. The control group (n = 396) did not receive the progesterone-releasing intravaginal device or progesterone injection. Cows were inseminated 12 h after exhibiting oestrus. Pregnancy diagnosis was performed 40 and 45 days after insemination by rectal palpation and the cumulative pregnancy rate on Day 90 postpartum was determined retrospectively through the individual records. The response to the progesterone treatment varied according to the parity of the cows (P < 0.05). Thus, progesterone administration improved the percentage of cows detected in oestrus in multiparous [192/255 (75.2%) v. 161/267 (60.2%)] but not in primiparous [93/132 (70.4%) v. 90/129 (69.7%)] cows. Progesterone treatment increased pregnancy rate in multiparous [53/192 (27.6%) v. 27/161 (16.7%)] but not in primiparous cows [25/93 (26.8%) v. 29/90 (32.2%)]. Likewise, progesterone administration increased the cumulative percentage of pregnant cows on day 90 postpartum in multiparous [149/255 (58.4%) v. 129/267 (48.3%)] but not in primiparous cows [77/132 (58.3%) v. 82/129 (63.5%)]. The incidence of twin births was lower (P = 0.07) in cows treated with progesterone [1/74 (1%)] than in the control group [4/53 (7%)]. It is concluded that progesterone administration before oestrus synchronization with PGF2 in first service dairy cows improves oestral response, increases pregnancy rate, and percentage of pregnant animals on Day 90 postpartum in multiparous cows, but not in primiparous cows, and decreases the incidence of twin births.
This research was supported by PAPIIT-UNAM research grant (IN219811-3).
70 PROTEIN EXPRESSION OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR DURING BOVINE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT
Authors: Poppicht F. , Stinshoff H. , Wrenzycki C.
Published: December 2013

Insulin-like growth factor 1 (IGF1) is essential for regulating physiological processes such as growth and development of fetal and placental tissues (Bauer et al. 1998, Fowden 2003). During early embryonic development, IGF1 plays an important role, as it leads to a reduction of apoptosis and decreases early embryonic mortality (Block et al. 2007). The signal transduction of IGF1 is carried out by its specific binding to the membrane-embedded insulin-like growth factor 1 receptor (IGF1R). The expression of IGF1R is a potential quality marker of in vitro produced embryos (Liu et al. 1997, Yaseen et al. 2001). Thus far, analysis of the relative amount of specific transcripts is the method of choice to study bovine pre-implantation embryos, as information on protein expression is scarce. Therefore, it is of great interest to analyse protein expression and to determine if and to which extent these results differ from results obtained in previous mRNA expression analyses. In the present study, a total of 4800 cumulus-oocyte-complexes were deployed in 60 in vitro produced runs. The cleavage rates averaged 57.4 ± 7.3% and blastocyst rates were 27.6 ± 7.5% at Day 8 of culture. Embryos at the blastocyst stage were frozen and stored at –80°C for further experiments. The protein expression of the IGF1R during early embryonic development was investigated by Western blot analysis testing 8 different antibodies. Seven of these antibodies were commercially available and mainly not tested in the bovine species. Only 1 of these antibodies resulted in a weak signal for the IGF1R protein in bovine blastocysts. Therefore, a specific peptide antibody against 2 peptide sequences of the α unit of the bovine IGF1R was produced. The analysis of the IGF1R protein with this antibody resulted in the determination of a signal in a pool of 100 blastocysts, which was weaker than in the positive control (20 μg of bovine liver protein extract). The detection of the IGF1R protein localization was possible in all different stages of embryonic development from the zygote to the expanded blastocyst using immunfluorescence staining with the specific peptide antibody. The IGF1R protein was mainly expressed in the plasma membrane of single blastomeres and also weakly in the cytoplasm. As the early bovine embryo expresses IGF1R throughout all stages, the main function of IGF1 in embryonic development needs to be further elucidated.
We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).
72 EFFECTS OF A PRECONCEPTIONAL AND GESTATIONAL MULTI-VITAMIN-MINERAL-OMEGA3 SUPPLEMENTATION ON FETOPLACENTAL DEVELOPMENT IN A RABBIT MODEL
Authors: Mourier E. , Tarrade A. , Ralliard-Rousseau D. , Larcher T. , Albert J.-P. , Aubrière M.-C. , Dahirel M. , Richard C. , Lévy R. , Chavatte-Palmer P.
Published: December 2013

The developmental origins of health and disease concept stipulates that nutritional imbalance in the preconceptional and gestational periods induces risks for the development and long-term health of the individual. In industrialized countries, most women take nutritional supplements before and during pregnancy, regardless of their diet. Potentially harmful embryonic and fetal effects of an excess multi-micronutrient supplementation, however, are not known. The objective of this study was to assess, using a rabbit model, the effects of a multi-vitamin-mineral-omega 3 supplementation (Gestarelle® G) administered in slight excess during preconceptional and gestational periods. Twenty-seven New Zealand does were used. Supplementation contained vitamins (B1, B2, B5, B6, B8, B9, B12, C, E), minerals (magnesium, iron, copper, iodine), and n-3 polyunsaturated fatty acids (n-3 PUFA; namely DHA and EPA) in the form of oral capsules administered daily from 10 days before mating until the end of pregnancy. Group 1X posology allometrically matched that used in humans (n = 10), representing an excess of 6 to 50% of rabbit needs. Group 3X rabbits received 3 times the recommended posology (n = 9; 18 to 150% of rabbit needs), whereas the control group received capsules containing only excipient (n = 8). Embryo and fetoplacental development was monitored by transabdominal two- and three-dimensional ultrasound and Doppler on Days 5, 7, 14, 21, and 28 with 5 conceptuses per doe examined each time. Does were killed on Day 28 (term = 31 days), 24 h after the last capsule administration. Maternal and fetal blood, as well as placenta and fetal organs, were collected. Data were analysed by ANOVA and Kruskall-Wallis as appropriate. The n-3 PUFA plasma concentrations increased in the 3X does (P < 0.01). Fetal plasma concentrations were increased only in males in the 1X group (P < 0.001) and both in females and males in the 3X group (P < 1e–15). No statistical differences were observed between treatments for any of the ultrasound or Doppler analyses. In total, 208 fetuses were collected with no difference in litter size, sex ratio, nor biometric measurements between groups. No structural anomaly was observed by histological analysis of fetal organs. In conclusion, multi-vitamin-mineral-omega 3 supplementation administered during the preconceptional and gestational periods in a rabbit model in the present study and within the measured parameters had no deleterious effect for dam and conceptuses at 1 and 3 times the recommended posology. Further work is on-going to study the effects on fetal brain development. Further studies are needed to evaluate putative post-natal effects.
This study was funded by IPRAD Santé Laboratories, Paris, France.
73 INTRAUTERINE GROWTH RESTRICTION AFTER BETWEEN-BREED EMBRYO TRANSFER IS ASSOCIATED WITH STRONG ALTERATIONS IN PLACENTAL STRUCTURE AND FUNCTION IN HORSES
Authors: Peugnet P. , Valentino S. , Tarrade A. , Wimel L. , Reigner F. , Serteyn D. , Chavatte-Palmer P.
Published: December 2013

In equids, placentation is diffuse and the nutrient supply to the fetus is determined by uterine size, which is correlated with maternal size. The size of the mare affects fetal development as shown by embryo transfer (ET) between Ponies and Thoroughbreds. In turn, insulin sensitivity in the newborn foal and subsequent postnatal growth rate are affected. We enhanced or restricted fetal growth through ET using Pony (P), Saddlebred (S), and Draft (D) horses and investigated placental morphology, structure, and function at term. Control pregnancies of P-P (n = 21), S-S (n = 28), and D-D (n = 8) were obtained by AI. Enhanced and restricted pregnancies were obtained by transferring P (P-D, n = 6) and S embryos (S-D, n = 8) into D mares or S embryos into P mares (S-P, n = 6), respectively. Placental weight and surface were recorded at delivery. Samples were collected for stereology and RT-qPCR analysis of expression of genes involved in placental growth, vascularization, and nutrient transport. Housekeeping genes were RPL32, SCAMP3, and B2M. Data were analysed by Kruskal-Wallis followed by Dunn's post hoc test. Placental weight and surface were increased in S-S and in D-D compared with P-P, whereas S-S and D-D were not different. No histological changes were observed among controls, but most genes had their expression decreased in P-P compared with S-S and D-D. The P-D foals had a 57% increased birthweight with heavier and larger placentas than P-P foals. The S-D foals were similar to both S-S and D-D in terms of birthweight and placental weight and surface. No major modifications in placental histology or transcript levels were observed in both enhanced groups. In contrast, S-P foals had a 37% decreased birthweight with lighter and smaller placentas compared with S-S and S-D foals. There was no gross histological difference between S-P and S-S but the microcotyledonary surface density was higher in S-P compared with S-D. Moreover, the expression of IGF2, IGF2R, SLC2A1, and eNOS was decreased in S-P compared with S-S. There was no difference in gene expression between S-P and P-P. In conclusion, intrauterine growth restriction led to marked changes in placental morphology, histology, and gene expression. The increased microcotyledonary surface density suggests a lengthening of villi, which could increase feto-maternal contact surface as a compensatory mechanism for the restricted uterine capacity. Surprisingly, placental adaptation to the restricted intrauterine environment in S-P induced gene profiles resembling that of control P, whereas no difference was observed in enhanced pregnancies.
81 EFFECT OF IN VITRO CULTURE SYSTEM MODIFICATION USING CR1aa MEDIUM ON EMBRYO DEVELOPMENT AND PREGNANCY RATE IN CATTLE
Authors: Singina G. , Taradajnic T. , Taradajnic N. , Zinovieva N.
Published: December 2013

The culture of in vitro matured and fertilized oocytes is a critical step of in vitro production of bovine embryos. Generally, oocytes are co-incubated with sperm in TALP medium containing different additions and then zygotes are transferred to a medium with another composition. At the same time the effect of the medium alteration on the development of early embryos is unknown. Continual adjustment of fertilized oocytes to the changing culture environment may result in a reduction of their developmental potential. The aim of the present study was to compare effects of two different culture systems on the embryo development and subsequent pregnancy rate in cattle. Slaughterhouse-derived cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. Frozen/thawed sperm from different Russian Black Pied bulls were prepared in Sperm-TALP medium by swim-up procedure. In vitro matured oocytes were co-incubated for 18 h with prepared sperm in the modified Fert-TALP medium containing 10 μg mL–1 heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine), and 0.1% MEM nonessential amino acids. The embryo culture was carried out using 2 systems. A total of 340 presumptive zygotes were incubated in CR1aa medium (Rosenkrans et al. 1994 J. Anim. Sci. 72, 434–437) up to Day 5 post-insemination (System 1) and a total of 442 presumptive zygotes were incubated for 24 h in a fresh Fert-TALP medium without PHE and heparin and then cleaved embryos were transferred to CR1aa medium and incubated until Day 5 post-insemination (System 2). Thereupon, the embryos were transferred to a fresh CR1aa medium supplemented with 5% FCS and cultured for 3 or 5 days. The embryo development was evaluated at Days 2, 8, and 10 for cleavage and blastocyst formation and hatching rates, respectively. A portion of blastocysts (of Grade 1 according to IETS classification) obtained at Day 8 were immediately transferred to recipients or were frozen in 1.5 M ethylene glycol and stored in liquid nitrogen until transplantation. The embryo development data (from 6–8 replicates) were analysed by ANOVA and the embryo transplantation data were analysed using the chi-squared test. The cleavage rates did not differ among Systems 1 and 2 and were 63.6–65.7%. On the other hand, the significant differences between culture Systems 1 and 2 were detected in rates of blastocysts (21.9 ± 1.4 v. 28.8 ± 2.8; P < 0.05) and hatched blastocysts (7.2 ± 1.2 v. 12.3 ± 1.6; P < 0.05). The pregnancy rate for frozen embryos was also higher (but not significantly) in System 2 than in System 1 [26.3% (9/34) v. 16.7% (2/12)], whereas for fresh embryos the similar values of the pregnancy rate were observed [on average 42.9% (6/14)]. Thus the additional 24-h culture of zygotes in Fert-TALP medium favourably affects bovine embryo development in vitro.
88 EFFECTS OF REMOVAL OF CUMULUS CELLS AND pb1 PRESENCE OF IN VITRO-MATURED BUFFALO OOCYTES PRIOR TO IVF ON CLEAVAGE RATE AND SUBSEQUENT EMBRYO DEVELOPMENT
Authors: Shang J.-H. , Zheng H.-Y. , Yang C.-Y. , Huang F.-X. , Yang B.-Z. , Liang X.-W.
Published: December 2013

The efficiency of oocyte maturation and embryo production in vitro in buffalo is relatively poor when compared with that in cattle. The percentage of oocytes selected by pb1 (the 1st polar body) presence for somatic cell nuclear transfer (SCNT) ranged from 50 to 70% in our laboratory, which meant that 30 to 50% oocytes have been abandoned. The present study was designed to identify the effect of cumulus cells removal and pb1 presence or absence before the IVF of matured buffalo oocytes on cleavage rate and subsequent embryo development and to try to reuse those oocytes without pb1 for embryo in vitro production. In vitro-matured oocytes enclosed with cumulus cells were randomly selected and denuded mechanically, then the denuded oocytes (DO) were divided into 3 groups by non-selection (pb1 ± ), selection of pb1 presence (pb1+) and absence (pb1–). Intact cumulus–oocyte complexes (COC, control) and pb1 ± , pb1+, and pb1– DO (treatments) were inseminated with motile buffalo sperm in Tyrode's medium for 24 h. The presumed zygotes were washed 3 times and transferred into 50-μL droplets of IVC medium (TCM 199 + 10% fetal bovine serum) and co-cultured with buffalo cumulus cells monolayer for more than 10 days to evaluate the developmental ability of embryos. Cleavage rate (CR) and blastocyst rate (BR) were assessed at 48 h and 240 h after fertilization (0 h). The results indicated that CR and BR for COC (61.69 ± 9.22% and 34.07 ± 7.61%) and pb1+ (66.59 ± 15.50% and 35.96 ± 10.87%) were significantly higher (P < 0.01) than those for pb1 ±  (49.11 ± 6.83% and 21.88 ± 8.17%) and pb1– (35.09 ± 9.17% and 13.16 ± 5.38%). In addition, there was a significant difference (P < 0.05) in the CR and BR between pb1 ±  and pb1– but no difference (P > 0.05) between COCs and pb1+ DO. These data show that removal of cumulus cells before IVF significantly reduces the overall developmental competence to cleavage and blastocyst stage and this negative effects mainly caused by the immature oocytes (indicated by the absence of pb1), but there was no effect on mature oocytes (presence of pb1). However, the oocytes without pb1 can still be used for in vitro embryo production even with lower efficiency when compared with intact COC.
This research was supported by grants from the National Natural Science Foundation of China (31160456), the Natural Science Foundation of Guangxi, China (2011GXSFB018045, 2013GXNSFAA019075).
89 CONCENTRATION OF ZINC IN FLUID FOLLICULAR OF FEMALE HORSES
Authors: Parra-Forero L. Y. , Vela-Correa G. , Cano-Flores O. , Mendoza G. , Pulido S. , Guevara J. A. , Gongora A. , Garcia-Contreras A. C.
Published: December 2013

The follicular fluid manage and keeps the nutrition of the oocyte and granulosa cell, which are develop inside this liquid. As well this fluid contains growth hormones, growth factors, and some micro-minerals as zinc (Zn). Resources report that Zn could avoid the earlier meiotic detention of the gametes, strengthen the structure of the chromatin due to the discovery of the Zn fingers, while also decrease the oxidative damage and inhibit the electrodes transportation by connecting to the Cytochrome b. The goals of this research were to determine the Zn concentration in the follicular fluid of female horses and define suitable concentration and supplementation in an embryonic culture. The first step of this research was to obtain follicular fluid from 23 female horses; these were classified by follicular size: F1 <1 cm, F2 between 1 and 2 cm, F3 >2 cm. The Zn was measured by spectrophotometry atomic absorption (μg mL–1). The use of the statistics program SAS version 9.3 (SAS Institute Inc., Cary, NC, USA) with a complementary random model, with factorial adjustments. Significance was established when P < 0.001. Obtaining significant differences on the Zn concentration on different follicles (P < 0001). Group F1 0.68 ± 0.15 μg mL–1, F2 1.42 ± 0.17 μg mL–1, and F3 1.8 ± 0,32. Table 1 shows the results of the volume and total concentration. Results indicate that Zn concentration varies with size and maturity of the follicle. Therefore, Zn required to be supplemented could be different as a function of the follicular development; further studies are required to evaluate the embryonic response to Zn addition in different maturation media and to correlate with survival rates.

Table 1.Concentration of zinc in fluid follicular of female horses
90 CHARACTERIZATION OF A PRIMARY CULTURE OF OVIDUCTAL CELLS IN THE BITCH
Authors: Gibson C. , Reynaud K. , Thoumire S. , Grimard B. , Saint-Dizier M.
Published: December 2013

The oviduct is of particular importance in canine reproduction as it supports oocyte meiosis resumption, sperm capacitation and storage, fertilization and embryo development to the morula/blastocyst stage for 8 to 10 days post-ovulation. A long-time co-culture with oviducal cells could be employed to improve the yield of reproductive biotechnologies in this species, but no characterisation of canine oviduct cells in vitro has been reported to date. The objectives of this study were to (1) evaluate the viability and proportion of epithelial/fibroblast cells in a primary culture of canine oviducal cells collected around ovulation; (2) study the responsiveness of the cultured cells to steroids. Beagle bitches (n = 9) were ovariectomized between Day –1 and Day +1 around ovulation, and their oviducts were sectioned at the ampulla-isthmus junction. Mucosal cells (including stromal and epithelial cells) were collected by squeezing from the ampulla and isthmus sections and cultured separately at a concentration of 5 × 105 cells/well in 500 μL of M199 + 10% FCS at 39°C for 9 days. At Days 3 and 6, 1 × 106 cells were stimulated with 17β-oestradiol (E2, 20 pg mL–1) or progesterone (P4, 20 ng mL–1) for 6 h. At Days 3, 6, and 9 of culture, the viability of the cells was evaluated using the Live/Dead kit (Invitrogen, Carlsbad, CA, USA), and proportions of fibroblast and epithelial ciliated cells were evaluated by immuno-cytochemistry using anti-vimentin and anti-tubulin antibodies, respectively. At Days 0, 3 and 6, the total RNA was extracted from cells and mRNA levels of the oviduct-specific glycoprotein (OVGP, synthesised by nonciliated epithelial cells), E2 (ERα, ERβ) and P4 (PR) receptors were evaluated by RT-qPCR. The effects of the day of culture and of steroid exposure on mRNA levels were analysed by ANOVA followed by a Tukey test. Cell confluence was observed around Day 6 of culture and more than 90% of cells survived during the 9-day culture period. From Day 3 to Day 9, the proportion of vimentin-positive (fibroblast) cells was greater than 68% in both ampulla and isthmus cells. In contrast, the proportion of epithelial ciliated cells was low at Day 3 (9% in ampulla, 12% in isthmus) and null at Days 6 and 9 in both regions. The mRNA levels of OVGP, ER, and PR decreased significantly after 3 days of culture, and then remained stable in both ampulla and isthmus cells (P < 0.001). The steroid exposure had no effect on gene expression, except for ERα mRNA levels at Day 3, which was increased by E2 and reduced by P4 (P < 0.05). In conclusion, the method of collection did not allow us to collect a high proportion of epithelial oviducal cells. However, the relatively stable gene expression of PR and ER during the culture period provides us with a useful tool to study the steroid regulation of canine oviduct mucosal cell functions.
96 EFFECT OF TWO DIFFERENT EMBRYO TRANSPORTERS ON DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS
Authors: Zhang M. , Sui L. , Li Y. , Chen Z. , Zhang Y. , Liu T. , Xu J. , Zhang X. , Zhang Y.
Published: December 2013

In the present study, we investigated two embryo transport methods, including a commercial cell transporter and a self-made, simple embryo transporter, for the pre-implantation development of porcine parthenogenetic embryos. The cleaved embryos were randomly distributed between the two types of embryo transport methods and were conserved in vitro for 2, 3, and 4 h. Embryo development efficiency testing and blastocyst differential staining were utilized to assess embryo developmental quality. There were no significant differences in embryo early development efficiency between the commercial cell transporter group, self-made embryo transporter group, and control group. The blastocyst hatch rate (7.75 ± 2.96%) in the self-made simple embryo transport method maintained for 3 h was significantly higher compared to the other groups (P < 0.05). The results (Table 1) showed that blastocyst differential staining showed that the ratio of inner cell mass (ICM) to total cells in both the 2-h-transport group and 3-h-transport group from the self-made simple embryo transport method and the 4-h-transport group from the commercial cell transporter were significantly higher than other groups (P < 0.05).The self-made simple embryo transporter and commercial cell transporter are both effective for transport and conservation of embryos for 3 h.

Table 1.Effect of different modes of transport and transit time on embryo development1
99 EFFECT OF SEASON AND WEIGHT GAIN ON PREGNANCY RATES OF EMBRYO RECIPIENTS RAISED UNDER PASTURE CONDITIONS
Authors: Fernandes C. A. C. , Ribeiro J. R. , Palhao M. P. , Figueiredo A. C. S. , Viana J. H. M. , Costa D. S. , K. N. Arashiro E.
Published: December 2013

In most Brazilian farms, the management of embryo recipients is done exclusively under grazing systems. Thus, the seasonal variation of grass availability can affect the energy balance and reduce weight gain, and consequently affects the rate of pregnancy. The aim of the present study was to evaluate the effect of the seasonal variation of daily weight gain (DWG) on the pregnancy rate of crossbred heifers (Bos taurus × Bos indicus). The study was performed in the southwest region of Minas Gerais State, Brazil, during one year. Two periods were evaluated: dry (April to September, average precipitation of 470 mm and low grass availability) and rainy (October to March, average precipitation of 1930 mm and high grass availability) seasons. The recipients were kept in a grazing system (Brachiaria brizantha) with water and minerals available ad libitum. Embryos were produced in vitro by the same laboratory, using oocytes and sex-sorted semen from the Gir breed (Bos indicus). A total of 404 fresh embryos (N = 484, 45.4% during dry and 54.5% during rainy season) were transferred by the same technician on Day 7 days of the oestrous cycle – synchronized with prostaglandin – of the recipients, which were weighted at the day of embryo transfer and 23 to 25 days later, at the diagnostic of pregnancy. Animals were retrospectively allocated into 4 groups according to the DWG observed between embryo transfer and pregnancy diagnosis: G1 (N = 132) up to 150 g; G2 (N = 132) between 151 and 250 g; G3 (N = 119) between 251 and 350 g; and G4 (N = 99) greater than 350 g of DWG. The pregnancy rates were compared among DWG groups and between seasons by chi-squared. The logistic regression model was performed using the PROC GENMOD to test the model, including the effects of DWG, season, and the interaction (DWG × season). During the dry season, there was change in frequency distribution of the heifers among DWG groups, with lower frequency (P < 0.05) of heifers in high (G3 and G4) when compared with low (G1 and G2) DWG groups (33.2, 31.4, 21.8, and 13.6% for G1, G2, G3, and G4, respectively). Altogether, pregnancy rate was not different between dry and rainy seasons (42.3 v. 45.8%, respectively; P > 0.05). When compared to G1, pregnancy rate was higher (P < 0.05) for recipients with daily weight gain greater than 250 g day–1 (G3 and G4) (35.6, 40.6, 52.1, and 51.0% for G1, G2, G3, and G4, respectively). Only the effect of the DWG was significant in logistic regression, and the odds rate for each gram of DWG was 0.0039, which means that pregnancy rate improved 1.17 times for recipients with 300 g of DWG. In conclusion, the DWG after embryo transfer, but not the season itself, affect pregnancy rate of in vitro-produced embryo recipients.
Biotran, FAPEMIG, and CnPQ are acknowledged.
Do Part–Whole Relations Produce Facilitation in the Picture–Word Interference Task?
Authors: Sailor Kevin , Brooks Patricia J.
Published: December 2013

Three experiments used the picture–word interference task to evaluate competing models of lexical access in spoken word production. Both the presence of a part–whole relation and association between the target and the interfering word were manipulated. Part terms associated with targets produced facilitation at early stimulus onset asynchronies (SOAs; –300 ms in Experiment 1, –300 and –150 ms in Experiment 3), but not at SOA 0 ms. Otherwise, part terms tended to produce interference, with unassociated part terms producing a significant semantic interference effect (SIE) at SOA of 0 ms in Experiment 1, and a similar trend in Experiment 3. Experiment 2 replicated the materials and procedure of Costa, Alario, and Caramazza (2005, Experiment 2. On the categorical nature of the semantic interference effect in the picture–word interference paradigm. Psychonomic Bulletin & Review, 12(1), 125–131), yet failed to find any semantic facilitation at SOA 0 ms. We propose that these findings are consistent with lexical competition accounts of SIE but difficult to explain in terms of the plausibility of the interfering words as responses to the target.
Amplification and thrifty single-molecule sequencing of recurrent somatic structural variations
Authors: Patel Anand , Schwab Richard , Liu Yu-Tsueng , Bafna Vineet
Published: December 2013

Deletion of tumor-suppressor genes as well as other genomic rearrangements pervade cancer genomes across numerous types of solid tumor and hematologic malignancies. However, even for a specific rearrangement, the breakpoints may vary between individuals, such as the recurrent CDKN2A deletion. Characterizing the exact breakpoints for structural variants (SVs) is useful for designating patient-specific tumor biomarkers. We propose AmBre (Amplification of Breakpoints), a method to target SV breakpoints occurring in samples composed of heterogeneous tumor and germline DNA. Additionally, AmBre validates SVs called by whole-exome/genome sequencing and hybridization arrays. AmBre involves a PCR-based approach to amplify the DNA segment containing an SV's breakpoint and then confirms breakpoints using sequencing by Pacific Biosciences RS. To amplify breakpoints with PCR, primers tiling specified target regions are carefully selected with a simulated annealing algorithm to minimize off-target amplification and maximize efficiency at capturing all possible breakpoints within the target regions. To confirm correct amplification and obtain breakpoints, PCR amplicons are combined without barcoding and simultaneously long-read sequenced using a single SMRT cell. Our algorithm efficiently separates reads based on breakpoints. Each read group supporting the same breakpoint corresponds with an amplicon and a consensus amplicon sequence is called. AmBre was used to discover CDKN2A deletion breakpoints in cancer cell lines: A549, CEM, Detroit562, MOLT4, MCF7, and T98G. Also, we successfully assayed RUNX1–RUNX1T1 reciprocal translocations by finding both breakpoints in the Kasumi-1 cell line. AmBre successfully targets SVs where DNA harboring the breakpoints are present in 1:1000 mixtures.
TIGRA: A targeted iterative graph routing assembler for breakpoint assembly
Authors: Chen Ken , Chen Lei , Fan Xian , Wallis John , Ding Li , Weinstock George
Published: December 2013

Recent progress in next-generation sequencing has greatly facilitated our study of genomic structural variation. Unlike single nucleotide variants and small indels, many structural variants have not been completely characterized at nucleotide resolution. Deriving the complete sequences underlying such breakpoints is crucial for not only accurate discovery, but also for the functional characterization of altered alleles. However, our current ability to determine such breakpoint sequences is limited because of challenges in aligning and assembling short reads. To address this issue, we developed a targeted iterative graph routing assembler, TIGRA, which implements a set of novel data analysis routines to achieve effective breakpoint assembly from next-generation sequencing data. In our assessment using data from the 1000 Genomes Project, TIGRA was able to accurately assemble the majority of deletion and mobile element insertion breakpoints, with a substantively better success rate and accuracy than other algorithms. TIGRA has been applied in the 1000 Genomes Project and other projects and is freely available for academic use.