In Vitro Culture Medium Research Articles

Effect of L‐carnitine on maturation, cryo‐tolerance and embryo developmental competence of bovine oocytes

In this study, the effects of the addition of L-carnitine in in vitro maturation (IVM) medium for bovine oocytes on their nuclear maturation and cryopreservation were investigated; they were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of L-carnitine (control, 0.3, 0.6 and 1.2 groups, respectively) and some of them were vitrified by Cryotop. Moreover, the effects of L-carnitine during in vitro fertilization (IVF) and in vitro culture (IVC) on the developmental potential and quality of IVF embryos were also examined. A significantly higher maturation rate of oocytes was obtained for 0.3 and 0.6 mg/mL groups compared with the control (P < 0.05). The blastocyst formation rate in the 0.6 group was significantly improved, whereas the rate in the 1.2 group was significantly decreased when compared with the control group (P < 0.05). No significant difference was found in embryo development between the control and the L-carnitine group after oocyte vitrification. Supplementation of IVF and IVC media with L-carnitine had no effect on development to the blastocyst stage of IVM oocytes treated with 0.6 mg/mL L-carnitine. In conclusion, the supplementation of L-carnitine during IVM of bovine oocytes improved their nuclear maturation and subsequent embryo development after IVF, but when they were vitrified the improving effects were neutralized.

Supplementation of insulin–transferrin–selenium to embryo culture medium improves the in vitro development of pig embryos

Insulin, transferrin and selenium (ITS) supplementation to oocyte maturation medium improves the post-fertilization embryonic development in pigs. ITS is also commonly used as a supplement for the in vitro culture (IVC) of embryos and stem cells in several mammalian species. However, its use during IVC of pig embryos has not been explored. This study investigated the effect of ITS supplementation to IVC medium on the in vitro development ability of pig embryos produced by parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). We observed that ITS had no significant effect on the rate of first cleavage (P > 0.05). However, the rate of blastocyst formation in ITS-treated PA (45.3 ± 1.9 versus 27.1 ± 2.3%), IVF (31.6 ± 0.6 versus 23.5 ± 0.6%) and SCNT (17.6 ± 2.3 versus 10.7 ± 1.4%) embryos was significantly higher (P < 0.05) than those of non-treated controls. Culture of PA embryos in the presence of ITS also enhanced the expansion and hatching ability (29.1 ± 3.0 versus 18.2 ± 3.8%; P < 0.05) of blastocysts and increased the total number of cells per blastocyst (53 ± 2.5 versus 40.9 ± 2.6; P < 0.05). Furthermore, the beneficial effect of ITS on PA embryos was associated with significantly reduced level of intracellular reactive oxygen species (ROS) (20.0 ± 2.6 versus 46.9 ± 3.0). However, in contrast to PA embryos, ITS had no significant effect on the blastocyst quality of IVF and SCNT embryos (P > 0.05). Taken together, these data suggest that supplementation of ITS to the IVC medium exerts a beneficial but differential effect on pig embryos that varies with the method of embryo production in vitro.

130 EFFECT OF HYALURONIC ACID SUPPLEMENTATION ON OVINE EMBRYO DEVELOPMENT IN VITRO AND ON SURVIVAL AFTER VITRIFICATION

Studies have shown that supplementation with hyaluronic acid (HA), a glycosaminoglycan found in mammalian follicular, oviduct, and uterine fluids, improves in vitro development and post-thaw survival of bovine embryos. In this study, we examined the effect of HA supplementation on ovine embryo development and on survival after vitrification using the minimum volume cooling (MVC) cryotop method. Abattoir sourced ovine oocytes were in vitro matured and fertilized as per routine procedures (Walker et al. 1996 Biol. Reprod. 55, 703–708). In Experiment 1 (5 replicates), presumptive zygotes were randomly allocated to IVC medium supplemented with 0.8 mg mL–1 BSA, amino acids, and 0, 0.5, 1.0, 1.5 or 2.0 mg mL–1 HA. Cleavage rates were recorded and blastocyst development evaluated on Day 7 (Day 0 = day of IVF). In Experiment 2 (3 replicates), presumptive zygotes were placed in in vitro culture (IVC) medium with or without 1.0 mg mL–1 HA. Embryos were vitrified using the MVC cryotop method (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172) on either Day 5 (morula–blastocyst stages), Day 6 (compact morula–hatching blastocyst stages), or Day 7 (blastocyst–hatching blastocyst stages). Vitrified embryos were thawed 7 days later and placed into IVC medium. Embryo survival (assessed by blastocoele re-expansion) and hatching rates were recorded on Day 8. Variables were assessed using procedure CATMOD in SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, the addition of HA did not affect cleavage or blastocyst formation rates but hatching rates were significantly (P &lt; 0.05) improved at concentrations of 0.5 to 1.5 mg mL–1 (Table 1). In Experiment 2, HA supplementation (1.0 mg mL–1) compared with control medium did not affect cleavage (96.8 and 97.1%, respectively) or blastocyst formation rates (68.6 and 70.2%, respectively). HA significantly (P &lt; 0.05) improved survival after thawing of embryos vitrified on Day 5 (100 v. 85.6%, n = 85 and 90). However no effect was observed when embryos were vitrified on either Day 6 (97.8 v. 97.8%, n = 91 and 92) or Day 7 (96.7 v. 97.9%, n = 92 and 94). Within day, HA supplementation did not affect hatching rate compared with control medium (Day 5, 54.1 v. 53.2%; Day 6, 62.9 v. 65.6%; Day 7, 71.9 v. 62.0%, respectively). These results demonstrate that HA supplementation of IVC medium significantly improves hatching rate of ovine embryos and we speculate that this improvement may correlate with comparable improvements in pregnancy rates after transfer. Hyaluronic acid binds to CD44, a glycoprotein expressed on the surface of preimplantation ovine embryos and shown to play a role on embryo development (Luz et al. 2012 Genet. Mol. Res. 11, 799–809). Hyaluronic acid plays a role in cell migration and we suggest that, in the early blastocyst, it affords an advantage to the trophectoderm cells. Table 1.Effect of hyaluronic acid (HA) supplementation in IVC medium on cleavage, blastocyst, and hatching rates

94 USE OF β-MERCAPTOETHANOL AND CYSTEINE FOR IN VITRO MATURATION AND CULTURE OF SHEEP EMBRYOS

During in vitro production (IVP), embryos are sensitive to suffering negative effects from catabolites, such as reactive oxygen species (ROS). Under physiological conditions, the action of the ROS is blocked by antioxidants such as glutathione, but glutathione's concentration is reduced during the main steps of the IVP process. The objective of the present study is to evaluate the effect of the supplementation of the media for in vitro maturation (IVM) and in vitro culture (IVC) with β-mercaptoethanol and cysteine on the rates of embryo development and viability after vitrification in open pulled straws (OPS). Ten IVP routines were conducted for IVP, using ovaries form pubertal sheep collected in a slaughterhouse. The ovaries were kept in a saline/antibiotic solution at 30°C during transport to the laboratory. The cumulus oophurus–oocytes complexes (COC) selected for IVM were allocated to 2 treatments: T1 (control), including no antioxidants in the IVM and IVC media (n = 676); and T2, including 50 μM β-mercaptoethanol and 600 μM cysteine, in the IVM and IVC media (n = 729). The IVM was conducted using the TCM 199 medium including oestradiol, FSH, LH, pyruvate, heat inactivated sheep serum and antibiotics, for 22 to 24 h. Sperm selection was conducted by swim-up in medium with tris-glucose-citric acid with fresh semen. For IVF, conducted for 18 to 22 h, 1 × 106 spermatozoa per mL were used in SOF medium including 2% heat-inactivated sheep serum. Both IVM and IVF were conducted with incubation with 5% CO2 at 39°C with saturated humidity. After IVF, the probable zygotes were denuded and cultured for 8 days in SOF medium with 0.4% BSA, at 39°C, in bags with 3 gases (5% CO2, 90% N2 and 5% O2). The criteria considered for embryo viability were: cleavage rate at Day 2 (cleaved/inseminated), embryo development at Day 7 (blastocysts/cleaved) and the reexpansion rate 24 h post-vitrification. Such frequencies were compared between treatments by the chi-squared test. The cleavage rate did not differ (P &gt; 0.05) for T1 (60.3%) and T2 (64.3%). The rate of embryro development at Day 7 was also similar (P &gt; 0.05) for T1 (33.6%) and T2 (36.6%). The reexpansion rate for T1 (76.9%) and T2 (54.1%) were also similar (P &gt; 0.05). Thus, supplementation of IVM and IVC media with β-mercaptoethanol and cysteine presented no effect in the development and viability of vitrified sheep embryos. CAPES, MARFRIG Group.

90 INTRACELLULAR REACTIVE OXYGEN SPECIES IN BOVINE EMBRYOS CULTURED IN VITRO WITH CATALASE UNDER VARIOUS OXYGEN TENSIONS

The production of reactive oxygen species (ROS), such as superoxide anion (O2–), hydroxyl radical (OH–) hydrogen peroxide (H2O2) and organic peroxides, is a normal process that occurs in the cellular mitochondrial respiratory chain. The high oxygen tension in in vitro culture (IVC) conditions is believed to induce oxidative stress, as a result of increase in ROS intracellular production, that can be correlated with embryonic developmental failure. Supplementation with antioxidants during IVC appears to increase the resistance of bovine embryos to the oxidative stress and consequently improve embryo development. The aim of this study was to evaluate the effects of antioxidant (catalase) and oxygen tensions during IVC on the embryonic development and quantification of intracellular ROS. Cumulus–oocyte complexes (COC; n = 337) were in vitro matured (IVM) in TCM-199 supplemented with 0.2 mM pyruvate, 25 mM sodium bicarbonate, 75 μg mL–1 gentamicin, 10% FCS and hormones for 24 h at 38.5°C and 5% CO2 in air. Then they were fertilized and the presumptive zygotes were cultured in SOFaa medium without (control) or with 100 UI catalase (CAT) for 7 days at 38.5°C in one of 2 types of humified atmosphere: 5% CO2 in air (≈20% O2) or in gaseous mixture (7% O2, 5% CO2 and 88% N2). The cleavage rate was evaluated at 72 hours post-insemination (hpi) and the embryonic development at 168 hpi. At this time, the level of intracellular ROS was measured using the fluorescent probe 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Invitrogen, Oakville, Canada), at 5 μM (Bain et al. 2011 Reprod. Fertil. Dev. 23, 561–575). Stained embryos were imaged immediately using an inverted microscope and analysed by Q-Capture Pro image software (QImaging, Surrey, BC, Canada). The signal intensity values of embryos were subtracted by the average of backgrounds in the images. Embryo development was analysed by chi-squared test and means of the intensity of fluorescence were compared by ANOVA followed by Tukey's test (P &lt; 0.05). The cleavage rates were 84.04%a (control 20% O2), 77.55%a (CAT 20% O2), 77.03%a (control 7% O2) and 71.83%a (CAT 7% O2). The embryonic development rates were 40.43%a (control 20% O2), 33.67%a (CAT 20% O2), 20.27%b (control 7% O2) and 16.90%b (CAT 7% O2). The fluorescent intensity were 3.9 ± 0.4a (control 20% O2), 1.8 ± 0.2b (CAT 20% O2), 2.7 ± 0.2ab (control 7% O2) and 2.8 ± 0.2ab (CAT 7% O2). Although catalase did not significantly affect blastocyst frequencies (P &gt; 0.05), embryo development was adversely affected by reduced O2 tension (P &lt; 0.05). H2DCFDA staining indicated a significant (P &lt; 0.05) reduction in the levels of intracellular ROS within embryos cultured with catalase under 20% O2 compared with the control group in the same O2 tension. Additionally, a consistent but insignificant reduction in intracellular ROS within embryos cultured under 7% O2 was found. We can conclude that supplementation with catalase to IVC medium at 20% O2 is suitable for lowering intracellular ROS levels in IVP bovine embryos, without lowering the rates of blastocysts production. This finding corroborates with theory that antioxidants are beneficial to embryo quality. Alta Genetics Brazil, Deoxi Biotecnologia.

Establishment of An Advanced Chemically Defined Medium for Early Embryos Derived from In Vitro Matured and Fertilized Bovine Oocytes

A chemically defined medium would be useful for analyzing promoters or inhibitors in in vitro culture (IVC) of bovine embryos. However, an IVC system for bovine embryos in a chemically defined medium has not been fully established. The present study was carried out to establish an advanced chemically defined medium for bovine embryos that supports a high rate of embryo development to the blastocyst stage. In the first experiment, we examined the effects of addition of Medium RD (RPMI1640 and Dulbecco's MEM, 1:1 v/v) to mKSOM/aa on developmental competence. The addition of 10% RD to mKSOM/aa with BSA improved the rate of development to the blastocyst stage; however, 10% RD-mKSOM/aa with PVP, which is a chemically defined medium, caused a reduction in the percentage of hatching blastocysts. In the second experiment, embryos were cultured in the chemically defined medium of 10% RD-mKSOM/aa containing 11.7, 23.4, 46.8, 70.2 or 96.8 µM inositol. Inositol at the concentration of 70.2 µM improved the rate of development to the hatching blastocyst stage. In the third experiment, the optimal RD concentration in the IVC medium was evaluated. Embryos were cultured in the chemically defined medium supplemented with 10, 20 or 30% (v/v) RD. The rate of development to the blastocyst stage was highest with 20% RD. In the fourth experiment, the effects of N-acetylglucosamine (GlcNAc) as an IVC medium supplement on developmental competence were examined. The rate of development to the blastocyst stage with 1.0 mM GlcNAc was significantly higher than that without GlcNAc, but the rate of development with 1.2 mM GlcNAc was not different from that without GlcNAc. We also evaluated the ability of blastocysts produced in RD-mKSOM/aa to develop to normal calves after being transferred into recipients. Ten of the 16 recipients became pregnant, with 9 delivering normal calves. These results indicate that 20% RD-mKSOM/aa containing 70.2 µM myo-inositol and 1 mM GlcNAc is useful as a chemically defined medium for IVC of bovine embryos.

213 THE EFFECTS OF SERICIN SUPPLEMENTATION IN IN VITRO CULTURE MEDIUM ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED - IN VITRO-FERTILIZED EMBRYOS

The objective of this study was to investigate the feasibility of the silk protein sericin as an alternative protein supplement for bovine embryo cultures. The effects of sericin supplementation in in vitro culture (IVC) medium on the development and cryosurvival of bovine IVM-IVF embryos were investigated. Cumulus–oocyte complexes were collected from 2- to 8-mm follicles of ovaries obtained from a local abattoir. They were matured for 20 to 22 h in TCM-199 supplemented with 5% fetal bovine serum (FBS), 0.002 AU mL–1 of FSH, and 1 μg mL–1 of oestradiol-17β at 38.5°C under an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in SOF medium (IVC medium) containing amino acids and supplemented with either 5% FBS (FBS group: n = 400) or sericin at 3 different concentrations (wt/vol; 0.05%: n = 493; 0.1%: n = 419; or 0.15%: n = 520; sericin groups) at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2 for 5 days. They were then transferred into each IVC medium supplemented with 0.1 mM β-mercaptoethanol and cultured for an additional 4 days (9 days in total). Cleavage rates were recorded on Day 2 of IVC. The excellent expanded blastocysts harvested on Days 7 and 8 were used for freezing (FBS group: n = 51, 0.05% sericin group: n = 56, 0.1% sericin group: n = 44, 0.15% sericin group: n = 44). They were frozen in m-PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose, 20% calf serum, and 4 mg mL–1 of BSA. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol under the same atmosphere used for IVC for 72 h. Rates of the embryos that reexpanded and developed to the hatching and hatched blastocyst stages were determined at, respectively, 24, 48, and 72 h after thawing. Rates of cleavage and blastocyst formation were expressed as mean ± SD and were analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by Fisher’s exact test and chi-square test. Four replications were performed. Cleavage and blastocyst formation rates did not differ among the groups (FBS: 61.6 ± 15.1 and 22.1 ± 3.5%; 0.05% sericin: 71.6 ± 12.0 and 19.4 ± 6.7%; 0.1% sericin: 70.3 ± 4.7 and 18.4 ± 5.6%; 0.15% sericin: 66.9 ± 10.3 and 17.9 ± 6.9%, respectively). There were no significant differences in post-thaw survival rates after 24 h among the FBS (88.2%), 0.05% sericin (92.9%), 0.1% sericin (84.1%), and 0.15% sericin (93.2%) groups. However, post-thaw survival rates after 72 h in the 0.05% sericin (83.9%) and FBS (82.4%) groups were significantly higher than those in the 0.1% sericin (56.8%) and 0.15% sericin (61.4%) groups (P &lt; 0.05). These results indicate the feasibility of sericin as an alternative protein supplement for bovine embryo culture. Additionally, in this study, 0.05% sericin was shown to be the best concentration for survival of the resultant embryos after freezing and thawing.

218 ANTIBIOTIC-RESISTANT MICROBIAL CONTAMINATION (ENTEROBACTER CLOACAE) DERIVED FROM EGG YOLK, FROZEN SEMEN EXTENDER, IN PORCINE IN VITRO-FERTILIZED EMBRYOS

The semen storage medium and the embryo culture environment are important for the further development of pre-implantation embryos, and these environments provide an ideal habitat for the propagation of a variety of microorganisms. Even though all precautions are taken to prevent contamination, this happens frequently during IVF and in vitro culture (IVC). Therefore, the aim of the present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen (liquid semen was prepared according the method of (Weitze 1990 Reprod. Dom. Anim., 231–253 suppl. 1), and purchased from the Veterinary Service Laboratory, Department of Livestock Research (Yongin city, Gyeonggi-do, Korea) in our laboratory for IVF experiments because of a lack of fresh semen. Antibiotics were added to the frozen semen extender (kanamycin and gentamicin) and IVC medium (gentamicin) to further inhibit the growth of microorganisms. Nevertheless, microorganisms were observed to proliferate in the IVC drop when culturing IVF embryos using frozen semen. Three samples were randomly taken from the liquid semen, frozen semen, and egg yolk. Contaminated IVC medium, frozen–thawed semen, liquid semen, and egg yolk were cultured in de Man, Rogosa, and Sharpe agar medium. Identical whitish colonies were detected in the contaminated IVC drop, frozen–thawed semen samples, and egg yolk, but no colonies were formed in liquid semen samples. Identical gram-negative and rod-shaped bacteria were found in both the frozen–thawed semen sample and the contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to the manufacturer’s instructions, with an identification value of 94.3% and a T index of 0.88. Antibiotic susceptibility tests were done according to CLSI (Wayne, PA) by using an ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole, trimethoprim, norfloxacin, and ciprofloxacin test. Among them, E. cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, and kanamycin but were susceptible to tetracycline, oxytetracycline, sulfamethoxazole and trimethoprim, norfloxacin, and ciprofloxacin. We suggest, based on these findings, that the sources of contamination might be from egg yolk. Therefore, synthetic semen extenders, which are free of egg yolk, might be considered during semen preparation. This work was supported by a grant (20070301034040) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

Adding Ascorbic Acid to Vitrification and IVC Medium Influences Preantral Follicle Morphology, but Not Viability

In this study, we analysed the effect on morphology and viability of ovine primordial follicles, when ascorbic acid (AA) was added to vitrification and in vitro culture (IVC) media. For morphological analysis, ovarian tissue was vitrified using DMSO or ethylene glycol (EG), to which AA was added or omitted. After warming, the tissue was fixed for histology or 1-day cultured in the presence or absence of AA. Isolated primordial follicles from ovine ovarian tissue vitrified with DMSO or EG, both supplemented with AA were stained with trypan blue for viability analysis, or 5-day cultured with or without AA followed by a viability analysis. In this study, we report on the successful vitrification protocol developed for ovine ovarian tissue using EG. Vitrification using DMSO reduced the percentage of morphological normal primordial follicles, whereas addition of AA to the vitrification and culture media did enhance these results (p < 0.05). However, vitrification in a DMSO + AA medium followed by 5-day IVC resulted in a significant decrease in the follicular viability, independently of the presence of AA in the IVC medium.

Effects of histone hyperacetylation on the preimplantation development of male and female bovine embryos

Trichostatin A (TSA) induces histone hyperacetylation by inhibiting histone deacetylases and consequently increasing gene expression. The hypothesis was that TSA supplementation during the in vitro culture (IVC) of bovine embryos would increase the blastocyst rate, particularly in low-quality and female embryos. Oocytes were fertilised separately with X and Y spermatozoa and, 70 h after IVF, the IVC medium was supplemented with 5 nM and 15 nM TSA for 48 or 144 h. Incubation of female embryos with 5 nM and 15 nM TSA resulted in similar increases in acetylated histone H3K9 levels. However, to see comparable effects on acetylated histone H3K9 levels in male embryos, the culture medium needed to be supplemented with 15 nM TSA (as opposed to 5 nM TSA for female embryos). Treatment of male and female embryos with 5 nM TSA for 48 h or female embryos with 5 nM for 144 h had no effect on blastocyst rates, although 15 nM TSA compromised embryonic development. The terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay revealed increased apoptosis in female embryos treated with 5 nM TSA for 144 h, as well as in male and female embryos treated with 15 nM TSA for 48 h, but this increase in apoptosis was not observed in low-quality embryos. The results of the present study suggest that TSA treatment promotes histone hyperacetylation, but has no beneficial effects on the in vitro production of male and female bovine embryos during preimplantation development.

γ-LA-Supplementation to IVC for IVP Bovine Embryos

The present study aimed to examine the effects of γ-linolenic acid (GLA) supplementation to in vitro culture (IVC) medium on in vitro developmental competence, freezability and morphology of in vitro matured and fertilized bovine embryos. In vitro produced (IVP) bovine zygotes were cultured in IVC medium supplemented with 0 (negative control), 15, 31, 62, 125, 250, 500 or 1,000 ppm GLA, 250 ppm linoleic acid albumin (LAA) and without any supplement as a control. Day 6 blastocysts derived from culture control were cultured in IVC medium containing either 62, 250 GLA or 250 LAA for 24 h, and at Day 7 were subjected to freezing or morphological examination by electron microscope. GLA 15 showed a tendency to have a higher cleavage rate at Day 2 (70.3%) than other groups. The hatching rate at Day 9 in LAA (38.2%) was significantly higher than the control and all treatment groups (p<0.05), while the blastocyst rate in LAA (32.4%) did not differ from those of 15 (30.5%), 31 (27.1%), and 62 GLA (33.1%) or the control (35.1%). GLA in concentrations of 125, 250, 500, and 1,000 ppm had significantly detrimental effect on the blastocyst rate compared to 15, 31 and 62 ppm GLA, LAA, and control groups (p<0.05). In contrast, the highest post-thaw survival rate (100%) was observed in the control group (p<0.01). Large lipid droplets were observed in the cytoplasm of trophoblastic cells, even in the control, but were abundant in GLA groups. Taking the results of the study into consideration, the addition of GLA to the culture medium for IVP bovine embryos at the dose of 15 ppm increased the developmental competence of zygotes and enhanced the cleavage rate up to Day 2. However, blastulation rate and post-thaw survival were not increased when GLA was added to the culture media.

Amphiregulin promotes the proliferation of trophoblast cells during preimplantation development of porcine embryos

Our objective was to investigate the effects of in vitro culture (IVC) medium supplemented with amphiregulin (AREG) on the preimplantation embryonic development of porcine (Genus: Sus domestica, Species: Landrace) embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA). In vitro fertilization and PA embryos at the 1-cell stage were cultured in IVC medium supplemented with 0, 0.5, 5, or 50 ng/mL AREG for 7 d. There were significantly greater total numbers of cells in blastocysts of IVF and PA embryos cultured with 50 ng/mL AREG compared with that of controls. In vitro fertilization and PA embryos were then cultured in NCSU-23 medium supplemented with 50 ng/mL AREG on Days 1 through 7, Days 1 through 3 (early stage), or Days 4 through 7 (late stage), or without AREG. There were significantly greater numbers of trophoblast cells in the late-stage and full-term groups of IVF and PA embryos than in the early-stage and control groups. The presence of AREG protein in IVF-derived blastocysts was detected using a polyclonal AREG antibody and indirect immunofluorescence. Amphiregulin protein was localized in both the cytoplasm and nucleus. Using real-time polymerase chain reaction, we detected the expression of AREG mRNA in all developmental stages of IVF and PA embryos; however, the expression level varied according to stage. Thus, the incubation of porcine IVF and PA embryos in AREG-supplemented culture medium mainly at the late preimplantation stage increases the numbers of trophoblast cells.

119 LEPTIN ENHANCED THE DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) EMBRYO CULTURED IN VITRO

Buffalo is an important livestock resource in many Asian and Mediterranean countries. In vitro embryo production (IVEP) and transfer of the embryos to produce calves with high genetic merit would be of great interest in buffalo species. The efficiency of the IVEP in buffalo is low compared to that in bovine. It may be due to the reproductive physiology of buffalo or the technical factors in IVEP procedures. Recent research revealed that supplementation of leptin in the in vitro culture (IVC) medium could significantly increase embryo development (2005 Mol. Cell Endocrinol. 229, 141–147; 2006 Reproduction 132, 247–256). In this study, the effect of leptin on buffalo embryo development in vitro was assessed by supplementation of the leptin into the IVC medium. Methods: Buffalo oocytes were aspirated form 2 to 6 mm follicles from slaughterhouse ovaries and washed in TCM199 and once more with in vitro maturation (IVM) medium (TCM199, 5% ECS, 15 μg mL–1 FSH). Oocytes with compact cumulus cells were matured in IVM medium at 38.5°C, 5% CO2 for 22–22 h. The frozen–thawed buffalo sperm underwent a centrifugation in Percoll gradient to remove the dead sperm. Ten to 15 matured oocytes were added to a drop of 40 μL modified Tyrode’s medium supplemented with 0.6% BSA, 2.0 mm caffeine and 20 μg mL–1 Heparin. Concentration of sperm added into the fertilization medium was 1 to 2 million per mL. Eight to 10 h after insemination, the presumptive zygotes were transferred to IVC medium (TCM199, 10% newborn cow serum) supplemented with 0 ng mL–1 (control), 10 ng mL–1, 100 ng mL–1 or 500 ng mL–1 of leptin. Cleavage and blastocyst development rate was recorded on Day 2 and Day 6 to 8 after insemination. The experiment was repeated 10 times, and a total of 831 oocytes were used with the IVF procedures. The results revealed that the cleavage rates in the group of 0 ng mL–1, 10 ng mL–1, 100 ng mL–1 and 500 ng mL–1 of leptin were 50.1 ± 3.5%, 55.0 ± 1.3%, 50.0 ± 1.8% and 52.9 ± 2.2%, respectively. No statistical difference was observed regarding cleavage rates between treatments (P &gt; 0.05). The percentage of oocytes developing to blastocysts in the group of 10 ng mL–1 and 100 ng mL–1 leptin were 26.1 ± 1.5% and 23.5 ± 1.2%, respectively, significantly higher than that of 17.5 ± 2.1% in the control (P &lt; 0.05). The blastocyst development rate in the group of 500 ng mL–1 leptin was 20.9 ± 1.4%, less than that of 10 ng mL–1 (P &lt; 0.05). In conclusion, the results of this study indicated that supplementation of leptin in the IVC medium could enhance the blastocyst development in buffalo species and the optimal concentration of leptin in the present procedures was 10 ng mL–1. This work was jointly supported by National Science and Technology Supporting Program (No. 2006BAD04A18), Guangxi Science Foundation (0832012) and Guangxi University Key Research Program (No. 2005ZD05).

Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 microm cysteamine. Supplementation of IVM medium with 50 microm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 microm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 microm (P < 0.05) cysteamine, whereas 200 microm cysteamine was ineffective. Supplementation of both IVM medium with 50 microm cysteamine and of IVC medium with 100 microm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.

α-Tocopherol and l-ascorbic acid increase the in vitro development of IVM/IVF swamp buffalo (Bubalus bubalis) embryos

This study was conducted to investigate the effects of capacitating agents added at in vitro fertilization (IVF) and antioxidants supplemented during in vitro culture (IVC) on the development of buffalo embryos. In experiment I, in vitro embryo development of buffalo embryos was compared when the IVF medium was supplemented with heparin, caffeine and calcium ionophore A23187 either alone or in combination. There was no significant difference (P > 0.05) in the cleavage rates of oocytes among the treatment groups but the development rate to the blastocyst stage and the cell numbers of blastocyst in the heparin-treated group were significantly higher (P < 0.05) than that of other treatments. In experiment II, in vitro embryo development of buffalo embryos was compared when IVC medium was supplemented with either α-tocopherol (250 and 500 μM) or l-ascorbic acid (250 and 500 μM). The rate of development to the blastocyst stage of embryos cultured in medium supplemented with 250 μM α-tocopherol (33%, 41/123) and 250 μM l-ascorbic acid (31%, 38/123) was significantly higher (P < 0.05) than that of those cultured in medium alone (19%, 20/108) but not significantly different (P < 0.05) from medium supplemented with either 500 μM α-tocopherol (24%, 30/123) or 500 μM l-ascorbic acid (25%, 33/133). These results suggest that buffalo spermatozoa treated with heparin were suitable for IVF and that α-tocopherol and l-ascorbic acid added during IVC increased the rate of buffalo embryo development.

Effect of plasmin, plasminogen activators and a plasmin inhibitor on bovine in vitro embryo production

In the present study, four experiments were conducted to investigate the possible effects of plasminogen activators (urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA)), plasmin, and a plasmin inhibitor (epsilon-aminocaproic acid (epsilon-ACA)) on different stages of bovine in vitro embryo production (IVP). The concentrations of these modifiers in IVP media were conditioned according to the plasminogen activator activity of bovine preovulatory follicular fluid. Media were modified in a single phase of IVP with an 18 h or 24 h incubation for in vitro maturation (IVM) and a 24 h or 48 h incubation for the IVF or in vitro culture (IVC), respectively. After IVM the oocytes were either fixed and stained or underwent IVF and IVC. The main findings were: (1) plasmin added to the 18 h IVM medium increased maturation rate without affecting fertilisation or embryo development rates; (2) t-PA added to the IVF medium significantly increased cleavage; (3) u-PA added to the IVC medium significantly increased embryo development rates; (4) the efficiency of all phases of IVP was reduced after the addition of epsilon-ACA; and (5) plasminogen addition had no effect in any IVP phase tested. We conclude that the members of the plasminogen activator-plasmin system contribute in different ways to bovine IVM, IVF and IVC.

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 microM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4-64.3%) and blastocyst formation (6.5-22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 microM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively). The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 microM Cys is not necessary and TCM199 plus Cys (100 microM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

Treatment of Porcine Oocytes with MEM Vitamins During In Vitro Maturation Improves Subsequent Blastocyst Development Following Nuclear Transfer

This study was carried out to investigate the effects of minimum essential medium (MEM) vitamins during in vitro maturation (IVM)/in vitro culture (IVC) of porcine nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. Porcine cumulus-oocyte complexes (COCs) were divided into five groups, matured for 44 h in maturation medium with various concentrations of MEM vitamins (0, 0.05, 0.1, 0.2 and 0.4%), and observed for maturation rate. Also, COCs were matured in NUSU-23 media without MEM vitamins for 44 h and cultured in PZM-3 media with various concentrations of MEM vitamins (0, 0.05, 0.4 and 1.0%) for 6 days following nuclear transfer. Factorial (IVM/IVC) experiments were also performed in NCSU-23 medium with or without 0.05% MEM vitamins and PZM-3 medium with or without 0.4% MEM vitamins. They were then tested by examining in vitro development of the porcine reconstructed embryos. The maturation rates of the COCs treated with the MEM vitamins did not differ significantly among the MEM vitamin-treated groups. Addition of vitamins to culture medium did not affect development of porcine reconstructed embryos in vitro. However, addition of low concentrations of MEM vitamins only to maturation medium increased (P<0.05) the proportion of NT embryos developing into blastocysts compared with the control group. Addition of MEM vitamins to IVC medium did not enhance the developmental rate compared with the control group. Thus, addition of MEM vitamins to IVM medium could improve subsequent blastocyst development of porcine NT embryos.

293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples&amp;apos; ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P &amp;lt; 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

300 BLASTOCYST PRODUCTION BY IN VITRO MATURATION AND DEVELOPMENT OF PORCINE OOCYTES IN DEFINED MEDIA FOLLOWING INTRACYTOPLASMIC SPERM INJECTION

Most culture media for in vitro production (IVP) of porcine embryos contain serum or bovine serum albumin (BSA); especially in the case of in vitro maturation (IVM) porcine follicular fluid is added. The present study was carried out to establish porcine-defined IVP. In Experiment 1, we investigated the efficacy of additional 0.6 mM cystine, 100 �M cysteamine (Cys), or cystine + Cys to a defined TCM-199 maturation medium with regard to the developmental competence of in vitro-matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM-199 containing 0.05% (w/v) polyvinyl alcohol (PVA). In Experiment 2, the effect of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development following ICSI. As positive and negative controls, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the basic medium. Five or 10 ng mL-1 of EGF was supplemented in the negative control medium (mPZM-4). All percentage data on cleavage and blastocyst development were subjected to arcsine transformation before statistical analysis using the STATVIEW program (Abacus Concepts, Berkeley, CA). The mean cell numbers per blastocyst (assessed by Giemsa staining method) were analyzed by one-way ANOVA, and the differences among the groups were also analyzed using Tukey-Kramer multiple comparison tests. In Experiment 1, there were no significant differences in the rates of cleavage (31.4 to 64.3%) and blastocyst formation (6.5 to 22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups, without significant differences. In Experiment 2, the rates of cleavage (43.0 to 58.0%) and blastocyst formation (14.0 to 23.6%), and the mean cell numbers per blastocyst (30 to 37) were not significantly different among the groups. However, the addition of 5 ng mL-1 of EGF to the mPZM-4 resulted in a significantly (P d 0.05) higher blastocyst rate (48.6%) to cleaved embryos than the other two defined groups (mPZM-4 and mPZM-4 with 10 ng mL-1 EGF: 23.4% and 23.1%, respectively), but not significantly different with mPZN-3 (40.1%). The present results indicate that TCM-199 with added cysteamine (100 �M) can be used as a defined IVM medium for porcine oocytes, and further addition of cystine into the IVM medium is not necessary. The addition of 5 ng mL-1 of EGF to a defined IVC medium enhanced subsequent development after ICSI. These results show that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI, and IVC). This study was partly supported by a grant-in-aid for Scientific Research (17580242) from the Japan Society for the Promotion of Science.