bluuebunny/crossref_metadata_2025 huggingface dataset:
120 SUPPLEMENTATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR DURING IN VITRO MATURATION OF PORCINE IMMATURE CUMULUS - OOCYTE COMPLEXES AND SUBSEQUENT DEVELOPMENTAL COMPETENCE AFTER PARTHENOGENESIS AND SOMATIC CELL NUCLEAR TRANSFER
Authors: Biswas D. , Jeon Y.-B. , Kim G.-H. , Jeung E.-B. , Hyun S. H.
Published: August 2011

In the present study, pig cumulus–oocyte complexes were cultured in medium supplemented with different concentrations (0, 5, 50, and 500 ng mL–1) of vascular endothelial growth factor (VEGF), and then the maturation and intracellular glutathione (GSH) concentration of oocytes were examined. In addition, the development of oocytes matured with different concentrations of VEGF after parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT) was observed. Although the maturation rate of oocytes was not affected by VEGF concentrations (81.13 ± 2.61%, 83.93 ± 1.97%, 82.14 ± 4.03%, 75.24 ± 2.68%, respectively), the intracellular GSH concentrations of oocytes matured with 5 and 50 ng mL–1 VEGF were significantly higher (12.68 ± 0.08, 12.33 ± 0.53 pMol/oocyte, respectively) than those of oocytes matured with 0 or 500 ng mL–1 VEGF (10.19 ± 0.66, 10.54 ± 0.54 pMol/oocyte, respectively). The blastocyst formation rates after PA of oocytes matured with 5 and 50 ng mL–1 VEGF were significantly higher (58.99 ± 4.70% and 54.00 ± 1.09%, respectively) than that of oocytes matured with 0 or 500 ng mL–1 VEGF (30.15 ± 4.52%, 34.79 ± 4.01%, respectively). Total cell number of PA blastocyst after oocytes matured with 5 and 50 ng mL–1 VEGF was significantly higher (83.21 ± 4.89, 78.16 ± 6.15, respectively) than that of control and 500 ng mL–1 VEGF (56.91 ± 4.78, 55.93 ± 3.89, respectively). Similarly, the blastocyst formation rate after SCNT of oocytes matured with 5 ng mL–1 VEGF was significantly higher (14.54 ± 1.42%) than that of oocytes matured without VEGF (7.95 ± 1.44%). Total cell number of SCNT blastocyst after oocytes matured with 5 ng mL–1 VEGF was significantly higher (67.83 ± 6.56) than control (48.09 ± 5.36). Fully cumulus cell expansion was significantly higher in the 5 ng mL–1 VEGF treated group (85.37 ± 0.73%) compared with the control (58.89 ± 0.88%). In conclusion, adding 5 ng mL–1 VEGF during IVM improved the developmental potential of PA and SCNT in porcine embryos by increasing the intracellular GSH level.

This work was supported by a grant (#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea.
121 DEVELOPMENTAL CHANGES IN THERMOPROTECTIVE ACTIONS OF INSULIN-LIKE GROWTH FACTOR-1 ON THE PREIMPLANTATION BOVINE EMBRYO
Authors: Bonilla A. Q. S. , Oliveira L. J. , Ozawa M. , Newsom E. M. , Lucy M. C. , Hansen P. J.
Published: August 2011

Insulin-like growth factor-1 (IGF1) is an important endocrine signal for regulation of early embryonic development. It increases the proportion of preimplantation embryos becoming blastocysts, alters blastocyst gene expression, improves resistance of embryos to various stresses and can enhance survival of embryos after transfer to recipients. The present study had 2 objectives. The first was to determine whether the thermoprotective actions of IGF1 on the preimplantation bovine embryo were developmentally regulated, with the 2-cell embryo being refractory to IGF1. The second was to determine the molecular basis for the improved competence of embryos treated with IGF1 to establish pregnancy after transfer to heat-stressed recipients. Heat shock at 41°C decreased (P < 0.005) the percentage of 2-cell embryos becoming a blastocyst at day 8 (39.5 v. 21% for 38.5 and 41°C, respectively), and treatment of embryos with 100 ng mL–1 IGF1 did not provide thermoprotection to 2-cell embryos heat shocked at 41°C (21 v. 21% for control and IGF1-treated embryos, respectively). Heat shock at 41°C had no effect on blastocyst development of day 5 embryos. However, exposure to 42°C reduced (P < 0.001) blastocyst development of day 5 embryos (87 v. 47.6% for 38.5 and 42°C, respectively). Furthermore, treatment of embryos with 100 ng mL–1 IGF1 reduced (P = 0.05) the effect of heat shock at 42°C on day 5 embryos (48 v. 66% control and IGF1-treated embryos, respectively). Failure of IGF1 to alter 2-cell embryo survival after heat shock was not associated with reduced expression of genes involved in IGF1 signaling (IGF1R, RAF1, PI3K, and MAPK), as shown by quantitative real-time RT-PCR assay, or in amounts of immunoreactive IGF1R protein. Treatment with IGF1 had little effect on the transcriptome at the blastocyst stage, with a total of 102 differentially expressed genes identified. Among the differentially expressed genes were several involved in apoptosis, protection against free radicals, and development. Changes in gene expression are consistent with IGF1 acting to induce an anti-apoptotic state and inhibit neurulation. In conclusion, thermoprotective actions of IGF1 are developmentally regulated. Failure of IGF1 to protect the 2-cell embryo from heat shock could reflect the fact that these embryos are maximally sensitive to damage caused by heat shock or reflect the quiescence of the embryonic genome at this early stage in development. Changes in gene expression at the blastocyst stage induced by IGF1 could contribute to the increased survival of IGF1-treated embryos when transferred during periods of heat stress.

Support: USDA NRI 2007-35203-18070 and 2009-65203-05732.
122 EXTENSIVE ALTERATIONS IN THE QUANTITATIVE PROTEOME PROFILE DURING EARLY EMBRYONIC DEVELOPMENT
Authors: Demant M. , Fröhlich T. , Wolf E. , Arnold G. J.
Published: August 2011

Early embryonic development is morphologically well characterised, but there is limited knowledge available about the underlying molecular processes. Several studies have addressed early bovine embryonic development on the transcriptome level, but to our best knowledge, no comprehensive approach on the proteome level has been performed so far, although early embryos contain a huge number of proteins originating from the oocyte protein pool. Due to strong similarities in estric cycle (non-seasonal polyestric), kinetics of early embryo development, and singleton pregnancy, the bovine system reflects crucial parameters of the human reproductive system. Being interested in the dynamics of protein expression profiles during early embryogenesis, we here present a differential proteomic analysis comparing morulae and blastocysts. Protein lysates of embryos were analysed using the ultrasensitive 2D DIGE saturation approach, which facilitates quantitative 2D gel analysis from total protein amounts of <0.5 μg. Six biologically independent replicates of morulae and blastocysts, containing 25 embryos each, were prepared and analysed. Quantitative evaluation of spot signals was performed using DeCyder 6.5 software. Applying the FDR correction feature and a P-value of <0.01, 60 differentially abundant protein spots differing in their abundance ratios between morulae and blastocysts up to a factor of 12 were detected. Spots already identified by LC-MS/MS represent proteins involved in metabolic processes and cell redox homeostasis. Among several oxidoreductases decreasing during ongoing development, 3 isoforms of Peroxiredoxin-2 have been detected, which were not described previously. To characterize which proteins in early embryonic stages are contributed by the maternal part of the embryo, we generated a comprehensive qualitative proteomic dataset of bovine oocytes using a nano-LC-MS/MS approach on an Orbitrap XL mass spectrometer. From 900 oocytes, 1221 proteins were identified. For 1045 proteins, existence on the protein level was experimentally proved for the first time in the bovine system.
125 EFFECT OF GLYCERALDEHYDE-3-PHOSPHATE DURING BOVINE IN VITRO EMBRYO CULTURE
Authors: Rubessa M. , Di Francesco S. , Suárez Novoa M. V. , Boccia L. , Longobardi V. , De Blasi M. , Gasparrini B.
Published: August 2011

Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183–1192; Takahashi and First 1992 Theriogenology 37, 963–978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from culture media. Glyceraldehyde-3-phosphate (G3P), a glucose-derived high-energy compound, is the end product of the energy-consuming phase of glycolysis that enters the pay-off phase of the pathway characterised by a net gain of energy. The aim of this study was to determine whether G3P is a valid energy source for supporting in vitro embryo development in cattle. Abattoir-derived oocytes (n = 832, over 4 replicates) were matured in vitro in TCM-199 with 15% bovine serum (BS), 0.5 μg mL–1 FSH, 5 μg mL–1 LH, 0.8 mM L-glutamine, and 50 mg mL–1 gentamicin. Mature COC were fertilized in Tyrode’s modified medium, with 30 mg mL–1 heparin, 30 mM penicillamine, 15 mM hypotaurine, 0.15 mM epinephrine, and 1% BS. Both IVM and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF containing either 1.5 mM glucose (control group) or G3P at 3 different concentrations (0.125, 0.5, and 1.5 mM). It is worth specifying that in the 3 G3P-supplemented groups small amounts of glucose were left (0.15 mM) because it is known that a complete removal would affect embryo development by interfering with ribose synthesis through the pentose–phosphate pathway. In vitro culture was carried out at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2 in air for 7 days, when the percentages of tight morulae-blastocysts (TMBL) and superior quality blastocysts (BL) were recorded. Differences in embryo yields among groups were analysed by chi-square test. Supplementation of IVC medium with 1.5 mM G3P reduced (P < 0.01) TMBL (5.0%) and BL (5.0%) rates compared with all other groups, indicating a toxic effect. However, when G3P was added at lower concentrations, no differences in TMBL (37.3 and 26.1, respectively, with 0.125 and 0.5 mM G3P) and in BL rates (35.3 and 25.5%, respectively, with 0.125 and 0.5 mM G3P) were observed compared with the control (32.7% TMBL and 31.4% BL, respectively). Within G3P-treated groups, the higher embryo yields were recorded with 0.125 mM compared with 0.5 mM (P < 0.05) and 1.5 mM (P < 0.01). Interestingly, embryos produced with G3P at the lower concentrations (0.125 and 0.5 mM) seemed to show a faster development compared with the control. In conclusion, these results demonstrated that G3P is a valid energy source for bovine preimplantation embryos and, hence, that G3P supplementation of IVC medium may be a suitable option for reducing glucose concentration in the media. However, further studies are needed to investigate lower concentrations of G3P and to better evaluate embryo viability.
127 EFFECT OF DIETARY FATS ON EMBRYO QUALITY AND YIELD IN HEAT-STRESSED DAIRY CATTLE
Authors: Guzey Y. Z. , Onal A. G.
Published: August 2011

The thermal environment is a major factor that can negatively affect production and reproduction of dairy cattle (Kadzere et al. 2002 Livest. Prod. Sci. 77, 59–91). Heat stress (HS) at and immediately after the time of breeding may result in a lower conception rate (Epperson and Zalesky 2007; http://wrac.sdstate.edu/pubs/ansci/ExEx2018.pdf). High temperatures compromise endometrial function and alter its secretory activity, which may lead to termination of pregnancy (Wolfenson et al. 2000 Anim. Reprod. Sci. 60–61, 535–547). One of the strategies to reduce thermal environment stress is improved nutritional management schemes like increasing dietary fat (Umphrey et al. 2001 J. Dairy Sci. 84, 2680–2685). Our objective was to determine (define) effects of protected fats on ovulation rate, embryo yield, and quality in heat-stressed Holstein cattle in Turkey. Twenty Holstein cows were assigned to control and treatment groups (86.50 ± 8.0 and 83.17 ± 10.8 DIM, respectively). Intravaginal devices (PRID) were placed in all cows (Day 0) and were superstimulated with twice-daily treatments with FSH (5, 4, 3, and 1 mL, respectively) on days 9 to 12. Also cows were treated with PgF2α (Dynolytic, 2 mL) twice on day 11. On the night of day 12, PRID were removed and animals were artificially inseminated on days 13 and 14. On day 20, inseminated cows were palpated per rectum and flushed transcervically. Following uterine flushings, each cows received PgF2α (Dynolytic, 2 mL) to induce luteolysis of developing corpora lutea. Embryos were scored and quantified, and good-quality embryos were fixed to determine embryonic cell counts. Two-twelve cell embryo and blastocyst counts were statistically different between seasons (P < 0.01 and P < 0.05, respectively); only 2-12 cell embryos were different between groups (P < 0.01) and only 2-12 cell embryos were different (P < 0.05) for season × treatment interaction. In the cool season experiment, morula and expanded blastocyst embryos were higher; and in the hot season experiment, unfertilized egg and morula embryos were higher for treatment group than those of controls. Also in the hot season experiment, total embryo yield was higher than the control group. However, dietary fats reduced rectal temperatures in these experiments. According to these results, we can assume that high rectal temperatures caused changes in blood parameters and very early embryonic deaths occurred in the control group. Consequently, dietary fats added to dairy cattle rations in hot seasons can positively affect embryo quality and embryo yield in dairy cattle.
129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION
Authors: Kang J. T. , Atikuzzaman M. , Kwon D. K. , Park S. J. , Kim S. J. , Moon J. H. , Gomez M. N. , Koo O. J. , Jang G. , Lee B. C.
Published: August 2011

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality.

This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.
13 THE RELATIONSHIP BETWEEN EARLIER POSTPARTUM CYCLICITY AND SUBSEQUENT REPRODUCTIVE PERFORMANCE IN DAIRY COWS: A FIELD TRIAL
Authors: Kim I. H. , Lee H. C. , Kang H. G.
Published: August 2011

Earlier postpartum cyclicity based on hormonal analysis was related to the subsequent fertility, although it has not been clarified under field conditions. This field study examined the relationship between the detection of corpus luteum (CL) at the first post partum month and subsequent reproductive performance in dairy cows. Holstein dairy cows underwent ultrasonography (Sonoace 600 with 7.5 MHz linear-array transducer; Medison Co. Ltd., Korea) at 30 (±7) days postpartum to determine the existence of CL in ovaries and then divided into 2 groups based on the existence of CL; the CL group (n = 156) or the non-CL group (n = 281). At the same time of ultrasonography, all cows were scored for body condition. Cows received the normal herd reproductive management program including oestrous detection or synchronization, or synchronization of ovulation in the breeding period. Pregnancy was determined per rectum 60 days after artificial insemination by both ultrasonography and palpation. Reproductive performance data were collected for a minimum of 210 days postpartum. Reproduction data between the CL and non-CL groups were evaluated by t-test, chi-square test, or logistic regression using an SAS program (Version 9.1: SAS Institute, Inc., Cary, NC, USA). The proportion of cows with CL in ovaries at 30 (±7) days postpartum was 35.7% of all cows. The number of days to first insemination after calving was more delayed (P < 0.0001) in the non-CL group (100.1 ± 2.7 days) than in the CL group (80.9 ± 2.9 days), while the pregnancy rate to first insemination did not differ (P > 0.05) between the CL group (44.2%) and the non-CL group (45.9%). The CL group had higher pregnancy proportions (PP) within 60 (odds ratio, OR = 2.3; P = 0.0063), 90 (OR = 1.6; P = 0.04), and 120 days (OR = 1.5; P = 0.05) postpartum than the non-CL group; however, the PP within 150 or 210 days postpartum did not differ between the 2 groups (P > 0.05). Besides, logistic analysis revealed that cows with body condition score (BCS) ≥3.00 were more likely to have CL (OR = 1.8; P = 0.015) compared with cows with BCS ≤ 2.75, while primiparous cows were less likely to have CL (OR = 0.6; P = 0.0085) compared with multiparous cows. In addition, cows that underwent ultrasonography later than 30 days postpartum were more likely to have CL (OR = 1.6; P = 0.0192) compared with cows that underwent ultrasonography earlier. In conclusion, the earlier cyclicity based on the detection of CL using ultrasonography at the first postpartum month, which might be in part due to a higher BCS, predicts an enhanced reproductive performance in dairy cows, particularly during the early and early-mid lactations.

This work was supported by the research grant of the Chungbuk National University in 2010.
131 EFFECT OF BREED AND FROZEN - THAWED RAM SEMEN ON IN VITRO FERTILIZATION AND OVINE EMBRYONIC DEVELOPMENT
Authors: Lehloenya K. C. , Mahoete N. , Greyling J. P. C. , Nedambale T. L.
Published: August 2011

Ovine embryonic development was evaluated 8 days following in vitro fertilization, after using fresh or frozen–thawed Merino and indigenous (Pedi and Zulu) sheep semen. Semen used was collected twice weekly over a 3-month period with the aid of an electro-ejaculator. Following collection, semen samples were evaluated and semen with acceptable sperm motility and a percentage live sperm of 60% diluted with an egg yolk-based extender (Egg-Yolk Citrate). Semen samples were cryopreserved in straws with a programmable freezer to –130°C and then plunged into liquid nitrogen (–196°C) until used for IVF. Fresh and frozen–thawed semen was used to fertilize the matured oocytes in vitro. A total of 791 oocytes were fertilized using fresh semen and 802 oocytes fertilized using frozen–thawed semen. No significant differences were recorded between the fresh and frozen–thawed semen regarding the embryonic developmental stages. The performance of fresh and frozen–thawed semen followed the same trend, with the cleavage rate gradually declining with the progression in time and the embryonic developmental stage. The lowest developmental rate recorded was the occurrence of blastocyst formation, ranging between 0.4 ± 0.4% and 2.6 ± 1.0%. Regarding breed, no significant difference was observed from cleavage to the 2- to 4-cell stages. The use of fresh and frozen–thawed Zulu semen resulted in a significantly (P < 0.05) higher percentage of 8-cell development compared with the Pedi semen. However, the 8-cell embryonic stage recorded with the use of the Zulu ram semen (fresh and frozen–thawed), did not differ significantly from that of the Merino breed. No significant difference between the breeds regarding blastocyst formation was recorded. The overall cleavage rate, 2- to 4-cell, and blastocyst embryonic developmental stages following the use of fresh and frozen–thawed semen from the different rams were generally lower than those recorded by other researchers. The low blastocyst rates obtained warrant more research regarding the in vitro embryo production technique in order to improve the ovine blastocyst formation rate.

The study was funded by the University of the Free State and conducted at the Germplasm Conservation and Reproduction Biotechnologies ARC.
134 EFFECTS OF VITAMIN E AND VITAMIN C ON THE DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION, IN VITRO FERTILIZATION, AND NUCLEAR TRANSFER
Authors: Lu F. , Zhang Z. , Zhang S. , Li N. , Jiang J. , Shi D.
Published: August 2011

The purpose of this study was to explore the effects of vitamin E (VE) and vitamin C (VC) on the in vitro development of embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (NT) in buffalo (Bubalus bubalis). Buffalo oocytes obtained from ovaries at slaughter were matured in vitro for 22 to 24 h. After maturation, oocytes were separated to 3 groups: one group of oocytes was fertilized in vitro with buffalo sperm; one group of oocytes was parthenogenetically activated by exposing them to 5 μM ionomycin for 5 min and then cultured in 2 mM 6-DMAP for 3 h; the other group of oocytes was enucleated, and fibroblasts in DMEM + 10% FBS for 4 to 5 days were transferred into enucleated oocytes by electronic fusion (100 v mm–1, 15 μs, and 3 pulses). After fusion, the activation of reconstructed embryos was induced by exposure to 5 μM ionomycin for 5 min and then cultured in 2 mM 6-DMAP for 3 h. The embryos of PA, IVF, and NT were respectively cultured in the culture medium (CM) containing different concentrations of VE, VC, or VE + VC for 7 to 9 days to evaluate embryonic development. As a result, when the embryos were cultured in the CM with different concentrations of VE (0, 50, 100, 150, and 200 μM), the blastocyst development rate of the embryos derived from PA, IVF, and NT gradually rose with increasing concentrations of VE and reached the highest amount [PA: 32.9% (81/246); IVF: 21.4% (45/210); and NT: 21.1% (47/223)] in the group containing 150 μM of VE; it was significantly higher than that of other groups (P < 0.05). When the different concentrations of VC (0, 50, 100, 150, and 200 μM) were added to the CM, the blastocyst development rate of the embryos derived from PA, IVF, and NT also enhanced according to the increasing concentration of VC, and more embryos developed to blastocysts in the group containing 150 μM of VC [PA: 31.2% (72/231); IVF: 20.2% (43/213); NT: 19.8% (48/243)] than in the other groups (P < 0.05). Compared with the control group (0 μM), the blastocyst rate of PA and IVF, as well as NT embryos, cultured in the CM with 150 μM VE + 150 μM VC groups was significantly higher (P < 0.05), but there were no significant differences in the percentage of blastocysts among groups of the 150 μM VE, 150 μM VC, and 150 μM VE + 150 μM VC (P > 0.05). These results indicated that adding VE (150 μM), VC (150 μM), or VE (150 μM) + VC (150 μM) in the CM could efficiently enhance the developmental competence of buffalo embryos during in vitro culture.

This work was funded by China High Technology Development Program (2007AA100505), Guangxi Science Foundation (0718005-3A), Fok Ying Tung Education Foundation (111034).
135 EXPRESSION OF APOPTOSIS-RELATED GENES IN BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED THROUGH IN VITRO FERTILIZATION AND PARTHENOGENETIC ACTIVATION
Authors: Saw S. , Singh K. P. , Kaushik R. , Muzaffar M. , Chauhan M. S. , Manik R. S. , Singla S. K. , Palta P. , Singh M. K.
Published: August 2011

Apoptosis, a highly conserved evolutionary mechanism that allows an organism to tightly control cell numbers, tissue size, and protect itself from dangerous cells and unfavourable environments that threaten homeostasis, is generally directed by specific genes involved in the regulation of a series of pro-apoptotic (BAX) and anti-apoptotic (BCL-XL) proteins that are expressed during early development. All mammalian species show the highest level of spontaneous apoptotic processes at the blastocyst stage. These proteins prevent apoptosis by maintaining the cell survival by interfering with the release of cytochrome-C from mitochondria. In this study, immature oocytes were obtained from buffalo slaughterhouse ovaries and were subjected to in vitro maturation (IVM) in TCM-199 + 10% FBS + 5 μg mL–1 porcine FSH for 24 h in a CO2 incubator (5% CO2, 90 to 95% relative humidity) at 38.5°C. The mature oocytes were used for IVF, and the cleaved embryos were cultured for 8 days in culture medium (CR2 medium containing 0.6% BSA and 10% FBS) for production of embryos at different stages. The parthenotes were produced with exposure of 7% ethanol, 6-dimethyl aminopurine and cultured for 8 days in culture medium. The total RNA was isolated from oocytes and embryos and transcribed using Cell-to-cDNA-II (Ambion, Austin, TX, USA), according to manufacturer protocol. The PCR cycle included heating to 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60 (BAX) and 62°C (BCL) for 30 s, and 72°C for 45 s with a final extension at 72°C for 10 min. The amplified product of both genes were separated on agarose gel and densitometry data for band intensities were generated using AlphaDigiDocTM AD-1201 software under a WindowsTM environment and data analysed with the help of SYSTAT software. Relative abundance of BCL-XL transcripts in immature, mature oocytes and embryos produced through IVF (i.e. 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stage) were 25.33 ± 0.90, 12.67 ± 1.20, 37.67 ± 0.90, 30.67 ± 0.30, 23.67 ± 0.90, 18.33 ± 0.90, and 27.00 ± 1.20, respectively, whereas in parthenogenesis these values were 23.67 ± 0.88, 13.67 ± 1.20, 23.67 ± 1.20, 22.34 ± 0.88, 24.34 ± 0.88, 33.67 ± 0.88, and 45.34 ± 1.20, respectively. Relative abundance of BAX transcripts by IVF were 23.0 ± 0.60, 0.33 ± 0.10, 4.00 ± 0.60, 5.00 ± 0.60, 0.37 ± 0.06, 13.0 ± 0.66, and 56.7 ± 0.90; and by parthenonenesis were 22.3 ± 0.90, 0.13 ± 0.03, 13.67 ± 0.90, 14.0 ± 0.60, 15.33 ± 0.90, 64.67 ± 2.20, and 55.0 ± 2.10, respectively. In conclusion, the expression pattern of the apoptosis-related genes revealed that the incidence of apoptosis was significantly higher in IVF and parthenogenetically produced buffalo embryos at stages such as immature oocytes, morula, and blastocyst than the early cleavage stage embryos.
137 PHENAZINE ETHOSULFATE AND FETAL CALF SERUM EFFECTS ON THE DEVELOPMENT AND APOPTOSIS OF IN VITRO PRODUCED BOVINE EMBRYOS
Authors: Sudano M. J. , Paschoal D. M. , Rascado T. S. , Magalhães L. C. O. , Crocomo L. F. , Lima-Neto J. F. , Machado R. , Landim-Alvarenga F. C.
Published: August 2011

Phenazine ethosulfate (PES) is a metabolic regulator that inhibits fatty acid synthesis and favours the pentose-phosphate pathway. Supplementation of fetal calf serum (FCS) during culture has been correlated with the reduction of quality of in vitro produced bovine embryos (IVPE). The aim of the present study was to evaluate embryo development and apoptosis in blastocysts after the supplementation of PES and FCS in culture medium of IVPE. Oocytes (N = 4320) were matured and fertilized in vitro (Day 0). The zygotes (Bos indicus) were cultured in SOFaa medium with 4 concentrations of FCS (0, 2.5, 5, and 10%) and with the use or not of 0.3 μM PES from Day 4 (after 96 h of embryo culture). Embryo development was evaluated after 7 days of culture. Apoptosis in blastocysts (N = 60–80) was accessed through TUNEL reaction. Embryos (Bos indicus) recovered from superstimulated cows were used as in vivo control (n = 15). Data were analysed by ANOVA followed by LSD using PROC GLIMMIX (SAS; SAS Institute Inc., Cary, NC, USA) means ± SEM. Increasing FCS concentration in the culture media did not change cleavage (86.7 ± 1.7, 82.3 ± 1.6, 86.3 ± 1.4, 87.0 ± 1.5, P > 0.05) and augmented blastocyst production (30.5 ± 2.5a, 41.8 ± 2.4b, 40.5 ± 2.6b, 47.2 ± 2.8b, P < 0.05), respectively, for 0, 2.5, 5, and 10%. Additionally, increasing FCS concentration increased apoptosis in blastocysts (13.8 ± 1.2b, 19.1 ± 1.8b, 20.7 ± 1.9bc, 28.4 ± 2.3c, P < 0.05, respectively, for 0, 2.5, 5, and 10%). The addition of PES from Day 4 in the culture medium did not affect (P > 0.05) cleavage (87.0 ± 1.3 and 84.4 ± 1.3), blastocyst production (42.0 ± 2.8 and 43.0 ± 2.0), and apoptosis in blastocysts (20.7 ± 2.0b and 18.9 ± 2.1b), respectively, for control and PES Day 4 groups. Independent of FCS withdrawal or PES addition to culture medium, the in vivo control group presented the lowest apoptosis rate (6.3 ± 1.1a). Therefore, increasing FCS concentration augmented embryo development and reduced blastocyst quality. However, the addition of 2.5% of FCS in the culture medium increased the embryo development without the reduction of blastocyst quality. Moreover, the PES supplementation from Day 4 did not affect embryo development and blastocyst quality.

São Paulo Research Foundation – FAPESP.
139 TRICHOSTATIN A IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS PRODUCED BY A NEW ACTIVATION METHOD
Authors: Sui L. C. , Wang W. , Li Y. S. , Zhang Y. L. , Ji S. F. , Cao Z. B. , Chen J. W. , Gui T. , Zhang M. L. , Zhang X. R. , Zhang Y. H.
Published: August 2011

Recently, it has been reported that a new activation method, brief exposure to cycloheximide before electrical activation, could increase development rates and reduce cell death. In our study, we allocated reconstructed SCNT embryos into 3 groups: electrical activation followed by exposure to cycloheximide (10 μg mL–1) for 4 h (ELE+CHX); exposure to cycloheximide (10 μg mL–1) for 10 min followed by electrical activation (CHX+ELE); and electrical pulse treatment alone (ELE). We found the CHX+ELE (10 min) group had a similar blastocyst formation rate and total blastocyst number with the ELE+CHX (4 h) group, and both groups could increase in vitro development compared with the ELE group. Trichostatin A (TSA), an inhibitor of histone deacetylase, has been reported to potentially enhance cloning efficiency. We examine the effect of TSA on nuclear transfer embryos produced by the CHX+ELE activation method. The reconstructed embryos were treated with 50 nM TSA for 0 and 36 h. We found that 50 nM TSA for 36 h after activation had an increased blastocyst rate compared with the control (15.35 v. 8.84%; P < 0.05), but there was no difference in cleavage rate or in total blastocyst numbers. Our data demonstrate that TSA treatment could significantly improve pig nuclear transfer embryos produced by a new activation method.

S.L.C., W.W. equal contribution; Corresponding author ZH. X.R., ZH. Y.H.; Supported by NSFC (30700574), 863 (2008AA101003).
14 EFFECT OF PORCINE SEMINAL PLASMA AND EGG YOLK ON CHEMOTAXIS AND PHAGOCYTOSIS OF NEUTROPHILS DERIVED FROM PERIPHERAL BLOOD OF PIGS AND COWS
Authors: Li J.-C. , Funahashi H.
Published: August 2011

The aim of this study was to determine the effects of porcine seminal plasma (0 to 20%, vol/vol) and egg yolk (0 to 20%, vol/vol) on chemotaxis and phagocytosis of porcine and bovine polymorphonuclear neutrophil (PMN) in vitro. Chemotaxis was determined using a blind well chamber. The phagocytosis assay was performed according to (Matthijs et al. 2000 J. Reprod. Fertil. 120, 265–273) with modification in pigs and cows. The serum-stimulated chemotactic activity of PMN (porcine, 1126.1 ± 14.4 cells/mm2 and bovine, 1067.1 ± 9.5 cells/mm2) was reduced (n = 4; P < 0.05) in the presence of 5% pig seminal plasma (pig, 1009.3 ± 12.4 cells/mm2 and cow, 800.0 ± 17.3 cells/mm2). More than 5% (vol/vol) of pig seminal plasma significantly decreased the chemotaxis of PMN in pigs and cows. The presence of 1% and higher concentrations (vol/vol) of seminal plasma reduced (n = 4; P < 0.05) leukocyte phagocytosis in pigs (37.1 ± 1.4 v. 41.7 ± 1.0%) and cows (36.4 ± 1.6 v. 42.1 ± 1.1%) compared with controls, and higher concentrations decreased the phagocytotic activity of PMNs in a concentration-dependent manner (n = 4; P < 0.05). Interestingly, 20% egg yolk increased (n = 4; P < 0.05) chemotaxis of PMN in pigs (953.5 ± 11.6 v. 789.9 ± 13.1 cells/mm2) and cow (988.6 ± 14.6 v. 790.4 ± 19.4 cells/mm2) compared with controls. The egg yolk increased (n = 4; P < 0.05) phagocytosis ability of porcine PMN (35.3 ± 1.6 v. 24.9 ± 2.4%) compared with the control. However, 20% egg yolk did not affect phagocytotic ability of PMN in cows (15.1 ± 1.6 v. 15.8 ± 1.0%). These results demonstrated that porcine seminal plasma, regardless of whether the species was pigs or cows, reduced chemotactic and phagocytotic activities of PMN. However, egg yolk significantly increased chemotactic activity of PMN in pigs and cows, while the egg yolk increased phagocytosis of PMN in pigs, but not in cows. In addition, porcine seminal plasma and caffeine reduced the egg yolk-induced increase in chemotactic and phagocytotic activities of PMN in both species.

Supported by JSPS Grants-in-Aid (B20380154).
143 TREATMENT WITH VITAMINS A AND E IMPROVE OOCYTE QUALITY AND IN VITRO EMBRYO DEVELOPMENT IN BOS INDICUS COWS
Authors: Evangelista J. J. F. , Souza C. E. A. , Moraes M. E. A. , Moura A. A. A.
Published: August 2011

We assessed the impact of a single intra-muscular injection of vitamins A and E on oocyte quality and yield and early embryo development in Bos indicus cows. Twenty Bos indicus cows, of Gyr, Brahman, and Nellore breeds, weighing between 450 and 600 kg were subjected to ovum pick-up (OPU). Oocytes were collected in Dulbecco’s PBS (DBPS) containing heparin and antibiotics, counted, and morphologically classified. Viable oocytes were taken to the laboratory, in vitro matured for 24 h and in vitro fertilized using 25 million sperm mL–1. After 168 h of incubation (39°C, 5% CO2), viable embryos were counted and classified. Then, after 10 days, the cows received an intra-muscular injection of 1 000 000 UI of vitamin A and 1 g of vitamin E, and, after 12 days, were again subjected to the same procedure described above. Differences in oocyte yield and embryo development were analysed using paired t-test. The 40 OPUs from 20 cows yielded a total of 520 oocytes. Nellore and Brahman cows produced more embryos/cow (P < 0.01) compared with Gyr. After vitamin treatment, the cows produced more oocytes (n = 303; P < 0.01) compared with the previous OPU (n = 217), resulting in 95 more viable oocytes (31%). Brahman, Gyr, and Nellore cows yielded 11.2 ± 1.8, 8.5 ± 1.5, and 12.0 ± 2.6 oocytes before vitamin treatment, respectively. From those oocytes, 224 embryos were obtained, 89 before vitamin injection and 135 after treatment (P < 0.02), with 36 more embryos (40%) produced. Irrespective of breed, cows responded equally to vitamin injection. A single parenteral injection of vitamins A and E had a significant positive effect on oocyte yield after OPU and in vitro embryo development on Bos indicus cows. We suggest that this effect is probably due to the positive influence of retinoids on oocyte and embryo development.
145 EFFECT OF TRANSFER OF CATTLE ELONGATING EMBRYO TO A REPEAT BREEDER COW ON PREGNANCY RATE AND INCIDENCE OF A RETURN TO ESTRUS
Authors: Kimura K. , Matsuyama S. , Kojima T.
Published: August 2011

In cattle, a low pregnancy rate directly affects calf and milk production and brings about economic loss to livestock producers. The number of repeat breeder cows in herds decreases the overall pregnancy rate. Therefore, many studies have focused on improving the cows’ reproductive performance. On the other hand, it has been demonstrated that the major embryonic loss occurs in the first or second week of pregnancy, after which mother cows recognize the presence of their embryos via a signal protein secreted by the embryos, interferon-τ. Therefore, to bypass the period with a high incidence of pregnancy loss in cattle, we used elongating embryos for embryo transfer (ET) to repeat breeder cows. Elongating embryos on Day 14 postoestrus were used for the present study because embryos of this stage have the ability to produce interferon-τ and embryos of later stages are easily damaged when they are collected from the uterus by flushing. In this study, Japanese Black cows were superovulated and inseminated when they showed standing oestrus. Elongating embryos were collected by nonsurgical uterine flushing. Collected embryos that had evident embryonic discs and intact trophectoderms were used for ET. These embryos were transferred, using plastic catheters or ET guns to ipsilateral uterine horns, to the corpus luteum of synchronized crossbred (Japanese Black × Holstein-Friesian) recipient cows. Recipient cows were divided into 2 groups according to the previous reproductive history of each cow: reproductively normal cows that were pregnant with fewer than 3 times of AI or regular ET (using Day 7 embryos) after every parturition (n = 33) and repeat breeder (RB) cows (n = 42). Elongating embryos were transfer on Day 14 after oestrus, and all cows were monitored for signs of standing oestrus twice a day until Day 80. Pregnancy was diagnosed with ultrasonography every 10 days from Day 30 to 80 postoestrus. The data were analysed by Fisher’s exact test. The pregnancy rate of reproductively normal cows was 54.5%. However, in repeat breeder cows, the pregnancy rate was significantly decreased (2.4%; P < 0.01). When the repeat breeder cows did not become pregnant, transfer of the elongating embryos was repeated another 5 times continuously and the incidence of a return of oestrus was monitored daily. At the first ET period, the average interestrus interval in nonpregnant repeat breeder cows was 50 ± 7.2 days. As the ET period proceeded, the interestrus intervals were gradually decreased, and at a sixth ET period, it approximated the normal oestrus cycle length (22.8 ± 0.8). These results indicated that although transfer of elongating embryos could bypass the critical period of high incidence of embryo loss, it did not improve the pregnancy rate of repeat breeder cows. Moreover, it suggests that the uterine environment of repeat breeder cows was gradually deteriorating in a time-dependent manner.
151 PREGNANCY RATES AFTER A NONSTEROIDAL ANTI-INFLAMMATORY (FLUNIXIN MEGLUMINE) AND PROGESTERONE (P4) ADMINISTRATION TO MANGALARGA MARCHADOR RECIPIENT MARES
Authors: Rodrigues P. G. , Resende A. M. , Souza J. C. , Carvalho R. R. , Maculan R.
Published: August 2011

The aim was to evaluate whether pregnancy rates could be improved by randomly injecting recipient mares with flunixin meglumine (FM; Banamine®, Schering-Plough, São Paulo, Brazil) on the day of embryo transfer combined or not with long-acting progesterone (P4) in commercial embryo transfer programs. One hundred sixty-five Mangalarga Marchador recipient mares from the 2006–2007 breeding season of 4 different commercial programs were used. Ages ranged from 4 to 15 years old. In 86 mares, 1.1 mg kg–1 of BW of FM was given intravenously immediately after embryo transfer, and in 79 mares, only saline was injected. Additionally, 59 mares receiving saline were injected with 1 800.0 mg of progesterone at embryo transfer and every week thereafter until 110 days of gestation. Of the 86 receiving FM, 17 received the same progesterone protocol. Embryos were transferred nonsurgically in Ham’s F-10 or Encare medium. Pregnancy was checked by ultrasonography at 15, 30, and 60 days after transfer, and only a positive diagnosis on the latter was considered pregnancy in the statistical analysis. In a 2 × 2 factorial design (2 levels of FM and P4), pregnancy rates were analysed using the GENMOD procedure considering a binomial distribution (SAS®, Cary NC), and means were compared by orthogonal contrasts. Pregnancy rates were lower (P < 0.0001) in FM-treated (50.2 ± 5.3%) compared with control (88.2 ± 5.1%) mares and in P4-treated (85.9 ± 5.0%) compared with control (52.4 ± 5.5%) mares. An interaction effect between FM and P4 was not observed (P < 0.49). For mares receiving or not receiving FM, the addition of the P4 protocol lowered pregnancy rates. In mares not treated with FM, pregnancy rates were 81.4 ± 5.1% and 95.0 ± 8.8% for P4-treated and nontreated mares, respectively (P < 0.05). In FM-treated mares that did not receive P4, pregnancy rates were 76.8 ± 4.8% compared with 23.5 ± 9.6% of those treated with P4 (P < 0.0001). In conclusion, the indiscriminate use of progesterone and nonsteroidal anti-inflammatory drugs may actually decrease reproductive efficiency and even act synergistically to potentiate their effect. These results must be interpreted with caution, considering that the data are derived from field observations, although sources are from very reliable and well-controlled operations. Nevertheless, the authors consider it imperative to have a proper diagnosis before generalizing the use of medical tools to improve pregnancy rates in horses.

Financial support from CNPQ–Conselho Nacional de Desenvolvimento Científico e Tecnológico, CAPES–Coordenação de Aperfeiçoamento de Pessoal de nível Superior. Schering/Plough, JOFADEL, and ABCCMM–Associação Brasileira dos Criadores do Cavalo Mangalarga Marchador.
152 EFFECT OF EMBRYO TRANSFER AFTER ARTIFICIAL INSEMINATION ON THE CONCEPTION RATE IN DAIRY COWS UNDER HEAT STRESS IN SOUTHERN JAPAN
Authors: Tani M. , Tani C. , Tomokawa K. , Funakoshi D. , Sakatani M. , Takahashi M. , Kitahara G. , Kamimura S.
Published: August 2011

A serious decline in the reproductive performance of dairy cows occurs in southern Japan in the summer period, when the total number of hot days ≥35°C numbers more than 20 days annually. Previous reports have mentioned the effectiveness of embryo transfer (ET) at 7 days after AI (AI/ET) under heat-stressed conditions. In the present study, we investigated the effect of AI/ET on conception rate (CR) under heat-stressed conditions in the summer period. Artificial insemination was performed at 13 commercial dairies in this study from August through September in 2007 and 2008. Seven days after AI, a single embryo was transferred into the uterine horn contralateral to the ovary with a corpus luteum (AI/ET, n = 82). Artificial insemination at oestrus without further treatment was assigned as the control group (AI, n = 367). In 2007, frozen–thawed embryos of Japanese Black cattle were transferred, and the same cattle were used for ET of fresh embryos in 2008. The temperature-humidity index [0.8 × temperature + 0.01 ×relative humidity (temperature –14.4) + 46.4], rectal temperature, and diurnal highest or lowest and average ambient temperatures were measured at the time of AI and ET. Cows were diagnosed for pregnancy at 42 days after AI by palpation per rectum and were reexamined by transrectal ultrasonography at 60 days after AI. The CR was calculated as the number of cows diagnosed as pregnant 60 days after AI divided by the number of cows inseminated. Fetal loss was calculated as the number of cows that did not deliver calves after term divided by the number of cows diagnosed as pregnant. The CR, number of AI, fetal loss, and type of newborn (Holsteins, AI origin; Japanese Black, ET origin) were confirmed retrospectively. For statistical analysis, Fisher’s exact test and Student’s t-test were used for comparison of the CR, fetal loss, and body temperature by using a statistical software program for PC (Excel Statistics 2006). The CR for AI/ET was 30.4% and for AI was 13.8% in 2007 (P < 0.01), and the CR for AI/ET was 30.8% and for AI was 21.5% in 2008 (P = 0.294). The average diurnal temperature was 31.1°C in 2007 and 30.1°C in 2008, and the temperature-humidity index was 81.8 and 80.8, respectively. On Day 8, the pregnant cows had a lower rectal temperature than the open cows in 2007, but not in 2008 (38.9 v. 39.4°C in 2007; P < 0.05; and 39.1 v. 38.9°C in 2008; P > 0.05). The fetal loss was 38.1% in AI/ET v. 7.4% in AI in 2007 (P < 0.05) and 12.5% v. 0% in 2008 (P < 0.05), respectively. The AI/ET procedure could improve CR in dairy cows during the summer period in southern Japan. However, other problems may accompany AI/ET, such as higher fetal losses.
153 EFFECT OF DIFFERENT HOLDING AND TRANSPORT MEDIA ON CONCEPTION RATES FOLLOWING TRANSFER OF IN VIVO AND IN VITRO FERTILIZATION-DERIVED BOVINE EMBRYOS
Authors: Zanenga C. A. , Martins C. M. , Rodovalho N. C. , Aidar F. , Hasler J. F. , Santos I. C. C. , Valentim R.
Published: August 2011

Two experiments were conducted to compare conception rates following embryo transfer (ET) of bovine embryos held and transported in Syngro® holding medium (Bioniche, Belleville, Ontario, Canada) with other 2 holding media: Emcare® (ICPbio, Auckland, New Zealand) for in vivo-derived embryos and HEPES-buffered synthetic oviduct fluid (H-SOF) for IVF-derived embryos. The first trial was performed in the period from October through December 2006 at the Curitiba farm in Poços de Caldas, Minas Gerais, Brazil. A total of 140 in vivo-derived embryos were produced from 20 Nelore donor cows and transferred fresh at the same farm. After each donor recovery, embryos were equally separated per stage (morula or blastocyst) and classification (grades 1, 2, and 3) into 2 Petri dishes, each containing either Syngro or Emcare. The embryos were held for an average of 3 h after recovery, loaded into 0.25-mL straws, and transferred fresh into recipients heifers, which were all previously synchronized with the same hormonal protocol treatment and presented a corpus luteum on the day of transference. Conception rate was checked at approximately 60 days of conception by rectal palpation. The chi-square test was used for statistical analysis. The conception rate of embryos maintained in Syngro was significantly higher than those in Emcare: 64.2% (43/67) v. 47.9% (35/73; P < 0.05). A second experiment was performed between September and December 2008 at Embriza Biotechnology Laboratory, Campo Grande, Mato Grosso do Sul, Brazil. A total of 1689 IVF-derived embryos (stage = 7, quality = 1), produced from Nelore donor cows, were randomly assigned to be held and transported in either Syngro (769) or H-SOF transport medium (920). Transportation time ranged from 1 to 9 h, and the recipient farms ranged from 100 to 1200 km in distance from the Embriza Laboratory. Crossbred recipient heifers (Bos taurus × Bos indicus) were synchronized with prostaglandin or vaginal progesterone device protocols. Pregnancy diagnosis was performed by ultrasonography approximately 60 days after ET. Statistical comparisons were performed using the chi-square test. Conception rates resulting from embryos transported in Syngro (45.1%, 347/769) and in H-SOF (42.0%, 386/920) were not different (P = 0.19).

Financial support from Embriza Biotecnology, Tecnopec LTDA, and Bioniche Animal Health