Vitro-fertilized Bovine Embryos Research Articles

RA33, an analogue of resveratrol, improves the development of in vitro-fertilized bovine embryos.

Oxidative stress is an undesirable effect of in vitro culture, which requires antioxidant supplementation. This study investigated the analogue of resveratrol (RA33) as an alternative to resveratrol, an antioxidant molecule, for the in vitro culture of in vitro-fertilized bovine embryos. The effect of different concentrations of RA33 on embryo development was evaluated and a comparison between RA33 and resveratrol was performed. The cleavage rate was higher (P < 0.05) with 2.5 μM (69.0 ± 4.4%) than at 0, 0.1 or 0.5 μM RA33 (62.1 ± 2.0%, 60.7 ± 5.9% and 56.7 ± 5.8%, respectively). The blastocyst rates on days 7 and 8 post-fertilization with 2.5 μM RA33 (19.4 ± 3.3% and 24.6 ± 3.3%, respectively) were higher (P < 0.05) than for 0 μM (12.4 ± 2.5% and 15.2±2.5%, respectively). When 2.5 μM RA33 was compared with 0.5 μM resveratrol, similar (P > 0.05) cleavage and blastocyst rates were found between them, but the cleavage rate was higher (P < 0.05) in the control (80.8 ± 3.4%) than for the resveratrol treatment (76.4 ± 3.6%). The numbers of apoptotic cells and the apoptotic index were lower (P < 0.05) with RA33 (6.5 ± 0.6 cells and 6.4 ± 0.7%, respectively) and resveratrol (5 ± 0.8 cells and 5.5 ± 1.0%, respectively) than in the control group (9.8 ± 1.2 cells and 8.9 ± 1.1%, respectively). In conclusion, RA33 can enhance the preimplantation development of in vitro-fertilized bovine embryos and be an alternative to resveratrol in embryo culture medium.

Gene expression at different cell stages of in vitro-fertilized bovine embryos

The birth rate of embryos produced in vitro (IVF) is still lower than that of embryos produced in vivo. Three major steps for the success of the IVF technique are maturation of immature oocytes, fertilization of matured oocytes, and culture of the resulting embryos. Studying mRNA expression in early embryonic development stages is important and can help to assess embryo quality and optimize production protocols in vitro. The current study aimed to determine the expression levels of developmentally important genes in different stages of bovine embryos produced in vitro. Cumulus-oocyte complexes (COCs) were collected from bovine ovaries and cultured in synthetic oviduct fluid (SOF) medium for 7 - 9 days. Embryos were collected at the time-points listed above, and mRNA expression of genes involved in pluripotency (OCT4), DNA methylation (DNMT1), apoptosis (BAX), and metabolism (GLUT1) and a heat shock protein (HSP70) was estimated from the 2-cell stage to the blastocyst stage of embryos. The results showed statistically significant differences in the relative abundance (RA) of OCT4, DNMT1, BAX, and GLUT1 gene transcripts among the different stages, whereas there were non-significant differences in the RA of HSP70 between these stages. In conclusion, gene expression levels differ among the developmental stages of embryos produced in vitro, possibly because of the timing of embryonic genome activation (EGA).

Efficient delivery of DNA into bovine preimplantation embryos by multiwall carbon nanotubes.

The pellucid zone (PZ) is a protective embryonic cells barrier against chemical, physical or biological substances. This put, usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells. Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection, however its ability to cross the PZ and mediated gene transfer is unknown. Our data confirm that multiwall carbon nanotubes (MWNTs) can cross the PZ and delivery of pDNA into in vitro-fertilized bovine embryos. The degeneration rate and the expression of genes associated to cell viability were not affected in embryos exposed to MWNTs. Those embryos, however, had lower cell number and higher apoptotic cell index, but this did not impair the embryonic development. This study shows the potential utility of the MWNT for the development of new method for delivery of DNA into bovine embryos.

Effect of Heat Stress in the Maturation, Fertilization, and Development Rates of In Vitro-Fertilized Bovine Embryos.

Effect of Heat Stress in the Maturation, Fertilization, and Development Rates of In Vitro-Fertilized Bovine Embryos.

166 EFFECT OF INDIVIDUAL CULTURE SYSTEM ON IN VITRO DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED BOVINE EMBRYOS

An individual in vitro culture system for bovine embryo needs to be developed for the study of embryo developmental competence. The objective of this study was to examine the effect of individual culture systems on the development of in vitro-matured–in vitro-fertilized bovine embryos. Two individual culture systems were compared. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter, 2 to 5 mm) obtained from a local abattoir. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL droplets of IVM media at 38.5°C under an atmosphere of 5% CO2 for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days (fertilization = Day 0). The presumptive zygotes were randomly assigned to 1 of the following 3 treatments: single culture (1 zygote was cultured in a 5-μL droplet), well-of-the-well (WOW; Sugimura et al. 2010 Biol. Reprod. 83, 970–978) culture (25 zygotes were cultured individually in each 125-μL droplet) and control culture (25 zygotes were cultured in a 125-μL droplet). Embryo development was evaluated for cleavage and blastocyst rates, on Day 2 and Day 7 to 9 after IVF, respectively. The rates of development up to the blastocyst stage and total cell number in the blastocysts, determined by an air-drying method, were investigated. The cleavage and blastocyst rates were analysed by the chi-square test and the total cell numbers were analysed by ANOVA. The cleavage rates were significantly higher in the control and WOW groups than in the single-culture group (P &lt; 0.01) and the blastocyst rates were significantly lower in the single-culture group than in the control culture group (P &lt; 0.05; Table 1). The total cell numbers (mean ± s.d.) of blastocysts did not significantly differ between the single culture (154.6 ± 21.8), control culture (155.2 ± 22.5) and WOW culture (159.8 ± 27.0) groups. These results indicate that although the blastocyst rate was lower in the single-culture system than in the WOW or group culture system, in vitro-matured–in vitro-fertilized bovine embryos can be cultured using the single-culture system. Moreover, the quality of blastocysts developed by the single-culture system is the same as that of blastocysts developed using the other 2 culture systems. Table 1.Effect of different culture methods for bovine embryo development

165 THE EFFECTS OF L-CARNITINE AND LINOLEIC ACID ALBUMIN SUPPLEMENTATION ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED/IN VITRO-FERTILIZED EMBRYOS IN IN VITRO CULTURE MEDIUM

The objective of this study was to investigate the effects of the supplementation of a lipid metabolism inducer, L-carnitine (LC) and a membrane stabilizer, linoleic acid albumin (LAA), on the developmental competence and cryosurvival of bovine in vitro-matured/in vitro-fertilized embryos in in vitro culture medium. Cumulus–oocyte complexes collected from the ovaries of slaughtered cattle were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH at 38.5°C in an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in CRlaa containing 5% CS at 38.5°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days. The culture medium was supplemented with 0.6 mg mL–1 of LC (LC group; n = 180) or with 0.25 mg mL–1 of LAA (LAA group; n = 180) or with both LC and LAA (LC + LAA group; n = 180) or without LC and LAA (control; n = 178). The cleavage rates were recorded on Day 2 and the blastocyst formation rates were recorded on Day 7 to 9. Expanded blastocysts harvested on Day 7 and 8 (LAA group: n = 31; LC group: n = 29; LC + LAA group: n = 25; control group: n = 33) were used for freezing in modified PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose and 20% CS. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol at 38.5°C under 5% CO2 in air for 72 h. The rates of re-expansion, hatching and formation of hatched blastocysts were determined at 24, 48 and 72 h after thawing, respectively. The rates of cleavage and blastocyst formation were expressed as mean ± s.e.m. and analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by chi-square test. The cleavage rate in the control group (69.1 ± 2.5%) was significantly lower than that in the LAA (81.8 ± 3.8%) and LC + LAA groups (77.9 ± 1.4%) but did not differ from that in the LC group (73.8 ± 2.4%). The blastocyst formation rate in the control group (21.7 ± 2.8%) was significantly lower (P &lt; 0.05) than those in the LAA and LC + LAA groups (33.5 ± 2.8% and 31.4 ± 2.4%, respectively), but it did not differ significantly from that of the LC group (32.1 ± 3.3%) despite a strong tendency (P = 0.06). There were no significant differences among the control, LC, LAA and the LC + LAA groups in post-thaw re-expansion rates (66.7, 75.9, 67.7 and 76.0%, respectively), hatching rates (48.5, 69.0, 58.1 and 64.0%, respectively) and rates of formation of hatched blastocysts (51.5, 62.1, 61.3 and 64.0%, respectively). These results indicate that the addition of LC and LAA to the medium for in vitro culture of in vitro-matured/in vitro-fertilized bovine embryos improved their ability to develop to the blastocyst stage; however, the effects on the freezing tolerance were not verified.

Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro

The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900mOsm) for 5min following 120min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (P<0.05) dehydration. Embryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P<0.05). In the experiment 2, the amount of Aqp3 and ATPase1 transcripts were quantified in blastocysts with high or low rehydration after exposure to hypertonic medium. No difference (P>0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (P<0.05) embryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (P<0.01) amount of Aqp3 transcripts was found in the vitrified-warmed embryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system.

Efeito do citrato e taurina em meio CR2aa no desenvolvimento de embriões bovinos fecundados in vitro

Avaliou-se o efeito do citrato em meio CR2aa suplementado com soro fetal bovino (SFB) ou livre de proteínas séricas e sua associação com taurina no desenvolvimento de embriões bovinos fecundados in vitro. Embriões foram cultivados em CR2aa contendo 0, 0,5, 1,0 e 3,0mM citrato, suplementado com 10% SFB (experimento 1) ou com álcool polivinil (PVA; experimento 2). No terceiro experimento, embriões foram cultivados em meio com 0,5mM citrato, ou 7mM taurina, ou com a associação de ambos, suplementado com SFB. Os cultivos foram realizados com células do cumulus em ambiente a 38,8ºC com 5% de CO2 em ar atmosférico. Melhora no desenvolvimento embrionário foi observado no cultivo de embriões em CR2aa com 0,5 e 1,0mM citrato na ausência de SFB (P&lt;0,05), 8,6% e 11,3% de blastocistos, respectivamente, porém com valores mais baixos (P&lt;0,05) que embriões cultivados em CR2aa com SFB (31,9%). Associação de citrato com taurina em meio com SFB não influenciou (P&gt;0,05) a produção de embriões ou o número de células. Citrato em meio CR2aa pode ser uma alternativa para cultivo embrionário em condições atmosféricas com 5% de CO2 em ar na ausência de proteína sérica.

315 SELECTION OF BOVINE OOCYTES BY BRILLIANT CRESYL BLUE BEFORE IN VITRO MATURATION IMPROVES BLASTOCYST DEVELOPMENT

The morphological identification of immature oocytes has commonly been used to select the bovine oocytes for IVF. However, &amp;lt;30% of the recovered oocytes reach the blastocyst stage after fertilization, and this is probably due to the quality of the oocytes at the beginning of maturation. The brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. The aim of this study was to evaluate the effect of the BCB stain on the selection of bovine oocytes and on the subsequent embryo development for in vitro production (IVP). Cumulus–oocyte complexes (COCs) were collected by the aspiration of 2- to 6-mm follicles. A total of 559 oocytes were divided into 2 groups: (1) a control group, immediately cultured, and (2) a BCB-incubated group. After 90 min of BCB staining (Pujol et al. 2004 Theriogenology 61, 735–744), the oocytes were divided into oocytes with blue cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB−). The COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg mL−1 FSH at 38.5°C under an atmosphere of 5% CO2 in air. The matured COCs were inseminated with 5 × 106 sperm mL−1. After 18 h of gamete co-culture, the presumed zygotes were cultured in CR1aa supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (proportion of ≥5-cell stage, the total cleavage rates) and on Days 7 to 9 (blastocyst rate). The experiment was replicated 5 times, and the data were analyzed by a chi-square test and ANOVA. The results are presented in Table 1. The proportion of embryos with ≥5-cell stage was significantly higher (P &amp;lt; 0.01) in the BCB+ group than in the BCB− group, but not in the control group. The total cleavage rate for the BCB+ embryos was significantly higher than that of either the BCB− or the control group (P &amp;lt; 0.01). There were also significant differences (P &amp;lt; 0.01) in the blastocyst development between the BCB+ and BCB− embryos and between the BCB− and the control embryos (P &amp;lt; 0.05). This result showed that the selection of bovine oocytes by BCB staining before in vitro maturation may be useful for selecting oocytes that are developmentally competent up to Day 9 for IVP. Table 1.Effect of selection of oocytes by brilliant cresyl blue (BCB) staining on the subsequent embryo development of in vitro-matured/in vitro-fertilized bovine embryos

280 EXPRESSION OF HSP70.1 GENE IN IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN CR2 MEDIUM SUPPLEMENTED WITH KNOCKOUT™SR

The exposure of embryos to serum during in vitro culture can affect morphology, metabolism, tolerance to cryopreservation, and expression of specific transcripts. On the other hand, serum-free medium seems to avoid some of those serum effects. KnockoutTMSR (GIBCO Laboratories, Grand Island, NY, USA) is a serum replacer optimized to support embryonic stem cells in culture and can also be used to replace serum during culture of in vitro-fertilized bovine embryos. The expression of genes associated with stress response, such as heat shock proteins (HSP), can be affected by in vitro culture conditions, being easily induced by a variety of stress agents, including culture medium components. This study aimed to determine whether KnockoutSR or serum in culture medium alters the relative abundance of HSP70.1 transcripts in in vitro-fertilized bovine embryos. Cumulus–oocyte complexes obtained from slaughterhouse ovaries were matured and feritlized in vitro. Presumptive zygotes were randomly cultured with their own cumulus cells in CR2aa medium supplemented with 10% fetal calf serum (GIBCO-BRL, Paisley, UK; FCS group), 10% KnockoutSR (GIBCO-BRL; KSR group), or 3 mg mL-1 of polyvinyl alcohol (PVA group). All steps were performed at 38.5�C, under 5% CO2 in air and 95% humidity. Blastocysts on Day 8 post-fertilization were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction (3 replicates for each group). Total RNA extraction was performed using an Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and the first strand was synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000 Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) with cDNA equivalent to 0.8 embryos and gene-specific primers. Expression of the glyceraldehyde 3-phosphate dehydrogenase gene was used as endogenous reference. Calculations of relative quantification were performed by comparative Ct method, using the value found in the PVA group as calibrator. Expression levels for the FCS and KSR groups were 1.2 � 0.06- and 1.4 � 0.08-fold differences relative to the PVA group without differences (P &amp;gt; 0.05). These data show that bovine embryos cultured in medium supplemented with KSR have the same HSP70-1 expression pattern as those in medium with added FCS, suggesting that embryos in both groups are under the same stress conditions. This work was supported by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogenetica, Vi�osa, Brazil, for the real-time PCR machine.

170 POST-THAW VIABILITY OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED EITHER IN HOST CAPRINE REPRODUCTIVE TRACTS OR IN VITRO

In vitro culture of bovine embryos is usually associated with poor pregnancy rate following cryopreservation. The objective of this study was to compare the post-thaw viability of in vitro-produced bovine zygotes, cultured in vitro or in the reproductive tract of a host goat. Cumulus-oocyte complexes were matured in vitro, and in vitro fertilization was carried out with frozen-thawed semen as per standard laboratory procedures. At 18-20 h post-fertilization, zygotes were stripped of remaining cumulus cells and randomly separated into culture treatments. In three replicates, a total of 606 embryos were surgically transferred 12 to 24 h post-ovulation to the oviducts of an estrous-synchronized goat (VIVO) and 550 embryos were cultured in G1.3 for 72 h and then moved to G2.3 medium for 96 h and in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 (IVC). On Day 7, embryos were flushed from the excised tract with a 69.5% recovery rate or removed from culture. Embryos were classified according to IETS criteria with grades and stages recorded. All data were analyzed using the one-way analysis of variance and means were compared using Student's t-test. No differences were seen in the percentage of freezable quality embryos per total recovered between the two groups (34.3% vs. 32.3% for IVC and VIVO, respectively). However, there was a significant difference in the pre-freezing stage between the two culture groups (Stage 5.5 � 0.22 vs. Stage 4.8 � 0.26 for IVC and VIVO, respectively; P &lt; 0.05), but no difference in the quality grade. All embryos greater than Stage 4, Grade 2 were frozen in groups of 5-10 in ethylene glycol with sucrose (Vigro Ethylene Glycol Freeze Plus; Bioniche Animal Health, Belleville, Ontario, Canada) in 0.25-mL straws. After thawing, embryo groups were washed, rehydrated, and incubated in G2.3 as above. Morphology was assessed by assigning grade and stage objectively at 24 h and 48 h post-thaw. Post-thaw viability in vitro was not different between groups (73.4% vs. 72.7% for IVC and VIVO, respectively). The average changes in morphology post-thaw from pre-freezing to 24 h and from 24 h to 48 h within each freezing group were determined. There was no significant difference in the mean change in stage (0.67 � 0.15 vs. 0.82 � 0.17 at 24 h and 0.31 � 0.09 vs. 0.37 � 0.10 at 48 h for IVC and VIVO, respectively) or grade (0.60 � 0.15 vs. 0.41 � 0.17 at 24 h and 0.03 � 0.06 vs. 0.14 � 0.07 at 48 h for IVC and VIVO, respectively) at either observation point. These results suggest that culture of in vitro-fertilized bovine embryos in the caprine reproductive tract did not alter post-thaw development or improve post thaw viability compared to in vitro cultured controls. However, morphological evaluation is too subjective to successfully predict pregnancy rate after transfer; therefore, further study is needed to determine if there are differences in pregnancy rates between these culture methods.

Effects of epidermal growth factor on maturation, fertilization and development of bovine follicular oocytes

When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 0 to 50 ng/ml EGF, the rate of metaphase II oocytes of 30 ng/ml EGF (97%) was significantly (P < 0.05) higher than that of the control (77%), 10 (85%), and 50 ng/ml EGF (82%). After in vitro fertilization, the rate of monospermic oocytes of 30 ng/ml (75%) and 50 ng/ml EGF (77%) was significantly (P < 0.05) higher than that of the control (56 %). When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 30 ng/ml EGF and/or 10% FCS and fertilized with frozen-thawed spermatozoa, the rate of monospermic oocytes was significantly (P < 0.05) higher in EGF + FCS (82%) than in EGF (61%) and FCS (67%). The rate of oocytes with 2 pronuclei was significantly (P < 0.05) higher in EGF + FCS (54%) than in EGF (27%). When in vitro-fertilized bovine embryos were cultured for 8 d with granulosa cells in TCM 199 containing 0, 10, 30 and 50 ng/ml EGF, the rate of embryos developing to the blastocyst stage was not significantly different among the control (22%), 10 ng/ml (20%), 30 ng/ml (18%), and 50 ng/ml (20%) EGF groups. These results indicate that EGF has a beneficial effect on in vitro maturation and fertilization of oocytes and that EGF plus FCS also have a beneficial effect on normal fertilization of oocytes. However, EGF had no beneficial effect on in vitro development of embryos when they were co-cultured with granulosa cells in medium with FCS.

Postthaw Viability of the Inner Cell Mass of in Vitro-Matured/in Vitro-Fertilized Bovine Embryos Frozen in Various Cryoprotectants

A study was conducted to examine the viability of inner cell mass (ICM) cells of frozen-thawed in vitro-matured (IVM)/in vitro-fertilized (IVF)-derived embryos using various cryoprotectants. Expanded blastocysts were frozen and thawed in 1.4 M glycerol with 0.25 M sucrose (GL), 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), or 1.3 M ethylene glycol monomethyl ether (EME) as cryoprotectants using a one-step method. After thawing, the embryos were cocultured for 24 h with cumulus cells in TCM199. Embryos wbich were viable after thawing and developed beyond the blastocyst slage were treated by immunosurgery and differential fluorochrome staining for ICM cell counts. Overall, there were no significant differences in the development to blastocyst stage after 24 h culture in each cryoprotectant (P < 0.05. X2 analysis). The viability of ICM cells of frozen-thawed embryos with each cryoprotectant was lower (GL, 72.7%; PG, 67.8%; EG, 77.5%; EME, 74.7%) than that of unfrozen embryos (84.4%). In the case of PG as a cryoprotectant, viability of ICM cells was significantly lower than that or unfrozen embryos (P < 0.05, ANOVA analysis). Our results suggest that the viability of ICM cells of frozen-thawed bovine embryos tend to be lower than that of unfrozen embryos irrespective of the cryoprotectants used. PG was significantly more toxic to the ICM cells compared with the other cryoprotectants.

Porcine oviductal cells support in vitro bovine embryo development

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.

Expression of 6WTK-LacZ gene construct in in vitro produced bovine embryos following microinjection into pronuclei or cytoplasm

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.

Production of chimeric calves by aggregation of in vitro-fertilized bovine embryos without zonae pellucidae

Bovine embryos produced by in vitro maturation (IVM), fertilization (IVF) and culture (IVC) were used to produce aggregation chimeras. An aggregated chimera was produced by combining bovine IVF embryos (Holstein × Japanese Black and Japanese Brown × Limousin breeds) which were cultured in vitro without the zonae pellucidae. Forty-eight hours after IVF, embryos at the 8 cell-stage were used to produce aggregation chimeras. In Experiment I, the zonae pellucidae was removed by a microsurgical method using a microblade or by treatment with 0.25% pronase. Holstein × Japanese Black embryos were aggregated with Japanese Brown × Limousin embryos after zonae removal by hand manipulation in culture medium. In Experiment II, the viability of the aggregated embryos developing into blastocysts was examined by measuring the extent of development. The number of aggregated embryos and embryos developed into blastocysts was 34 (91.9%) and 24 (70.6%), respectively, when the zonae pellucidae was removed by the microsurgical method; and 12 (92.3%) and 6 (50.0%), respectively, when the zonae pellucidae was removed using the 0.25% pronase treatment. The size of the aggregated embryos was significantly different from that of the normal embryos when cultured in vitro until Day 10, but not different thereafter. Five aggregated embryos were transferred nonsurgically to the recipients, resulting in 1 pregnancy and the birth of 2 chimeric calves. Skin color was used as evidence of chimerism.

Stimulation of development of in vitro-matured and in vitro-fertilized bovine embryos by platelets.

In vitro-fertilized bovine embryos were incubated in Menezo's B2 medium (MB2) supplemented with 2 mg/mL of BSA. In Exp. 1, eight-cell stage embryos were allotted to one of the following groups: control medium (MB2), MB2 with 20 ng/mL of platelet-activating factor (PAF), 1 x 10(7) bovine blood platelets (Platelets), oviductal cells (BOEC), BOEC and 20 ng/mL of PAF (BOEC+PAF), or BOEC and 1 x 10(7) platelets (BOEC+Platelets). In Exp. 2, eight-cell embryos were allotted to one of the following groups: control medium (MB2), MB2 with 1 x 10(7) platelets (Platelets), 1 x 10(7) platelets and 10 micrograms/mL of platelet-derived growth factor antibody (Platelets+anti-PDGF), 1 x 10(7) platelets and 1 microgram/mL of indomethacin (Platelets+Indomethacin), or 1 x 10(7) platelets and 3 micrograms/mL of mianserin (Platelets+Mianserin). Embryos were incubated at 39 degrees C in 5% CO2 in groups of five until 8 d after in vitro fertilization (IVF). In Exp. 1, Platelets stimulated embryo development to the morula, blastocyst, and expanded blastocyst stages. Embryo development was greatest in the BOEC+Platelets group on d 7 and 8 after IVF. Only embryos incubated in the BOEC+Platelets treatment group reached the hatched blastocyst stage on d 8. In Exp. 2, embryos incubated in the Platelets treatment group had the greatest (P < .05) proportion develop beyond the eight-cell stage. Embryos incubated in the Platelets + anti-PDGF group had less (P < .05) development beyond the eight-cell stage and to the morula stage. These results indicate that the stimulatory effects of PDGF on bovine embryo development may be derived from both the oviductal epithelium and platelets.