Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.     

CUDC-101, a histone deacetylase inhibitor,improves the in vitro and in vivo developmental competence of somatic cell nuclear transfer pig embryos

The aim of the present study was to examine the effects of CUDC-101, a novel histone deacetylase inhibitor, on the in vitro development and expression of the epigenetic marker histone H3 at lysine 9 (AcH3K9) in pig SCNT embryos. We found that treatment with 1 μmol/L CUDC-101 for 24 hours significantly improved the development of pig SCNT embryos. Compared with the control group, the blastocyst rate was higher (18.5% vs. 10.3%; P < 0.05). To assess in vivo developmental potency, CUDC-101–treated SCNT embryos were transferred into two surrogate mothers, resulting in one pregnancy with six fetuses. We then investigated the acetylation level of histone H3K9 in SCNT embryos treated with CUDC-101 and compared them only against untreated embryos. The acetylation level of control SCNT embryos was lower than that of CUDC-101–treated embryos at pseudo-pronuclear stages, and immunofluorescent signal for H3K9ac in CUDC-101–treated embryos in a pattern similar to that of control group. In conclusion, we demonstrated that CUDC-101 can significantly improve in vitro and in vivo developmental competence and enhance the nuclear reprogramming of pig SCNT embryos.     

In vitro production of small ruminant embryos: Late improvements and further research

Beyond the potential use of in vitro production of embryos (IVP) in breeding schemes, embryos are also required for the establishment of new biotechnologies such as cloning and transgenesis. Additionally, the knowledge of oocyte and embryo physiology acquired through IVP techniques may stimulate the further development of other techniques such as marker assisted and genomic selection of preimplantation embryos, and also benefit assisted procreation in human beings. Efficient in vitro embryo production is currently a major objective for livestock industries, including small ruminants. The heterogeneity of oocytes collected from growing follicles by laparoscopic ovum pick up or in ovaries of slaughtered females, remains an enormous challenge for IVM success, and still limits the rate of embryo development. In addition, the lower quality of the IVP embryos, compared with their in vivo–derived counterparts, translates into poor cryosurvival, which restricts the wider use of this promising technology. Therefore, many studies have been reported in an attempt to determine the most suitable conditions for IVM, IVF, and in vitro development to maximize embryo production rate and quality. This review aims to present the current panorama of IVP production in small ruminants, describing important steps for its success, reporting the recent advances and also the main obstacles identified for its improvement and dissemination.     

The evolution of porcine embryo in vitro production

The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology.     

Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos

The objective was to evaluate pregnancy outcomes and birth rate of in vivo derived vs. in vitro produced ovine embryos submitted to different cryopreservation methods. A total of 197 in vivo and 240 in vitro produced embryos were cryopreserved either by conventional freezing, or by vitrification with Cryotop or Spatula MVD methods on Day 6 after insemination/fertilization. After thawing/warming and transfer, embryo survival rate on Day 30 of gestation was affected by the source of the embryos (in vivo 53.3%, in vitro 20.8%; P < 0.05) and by the method of cryopreservation (conventional freezing 26.5%, Cryotop 52.0%, Spatula MVD 22.2%; P < 0.05). For in vivo derived embryos, survival rate after embryo transfer was 45.6% for conventional freezing, 67.1% for Cryotop, and 40.4% for Spatula MVD. For in vitro produced embryos, survival rate was 7.3% for conventional freezing, 38.7% for Cryotop, and 11.4% for Spatula MVD. Fetal loss from Day 30 to birth showed a tendency to be greater for in vitro (15.0%) rather than for in vivo produced embryos (5.7%), and was not affected by the cryopreservation method. Gestation length, weight at birth and lamb survival rate after birth were not affected by the source of the embryo, the cryopreservation method or stage of development (average: 150.5 ± 1.8 days; 4232.8 ± 102.8 g; 85.4%; respectively). This study demonstrates that embryo survival and birth rate of both in vivo and in vitro produced ovine embryos are improved by vitrification with the minimum volume Cryotop method.     

Effect of dexamethasone on development of in vitro–produced bovine embryos

Studies in somatic cells have shown that glucocorticoids such as dexamethasone (DEX) may trigger or prevent apoptosis depending on the cell type in culture. Because the dysregulation of apoptosis may lower in vitro embryo production efficiency, we sought to investigate the effects of supplementing IVC medium with DEX (0.1 μg/mL) on embryo morphology, development kinetics, and apoptosis rates of in vitro–produced bovine preimplantation embryos. Embryo morphology was graded on Day 7, and development rates were assessed on Days 4 and 7 of IVC. Apoptosis was evaluated via annexin/propidium iodide staining under fluorescence microscopy where a cell labeled with annexin, propidium iodide, or both would be considered apoptotic. An embryo was counted in the apoptosis rates, if it displayed at least one such labeled cell. Although DEX supplementation did not reduce apoptosis rates, it had a positive impact on developmental kinetics and cell number both on Days 4 and 7 of embryo culture. Presumably, such effect resulted from increased cell proliferation rather than a direct inhibition of apoptosis. Further studies may evaluate the mechanisms by which glucocorticoids may affect embryo development, as DEX supplementation could become a tool to improve in vitro embryo yield in mammalian species.     

The new system of shorter porcine oocyte in vitro maturation (18 hours) using ≥8 mm follicles derived from cumulus-oocyte complexes

Despite recent efforts to improve in vitro maturation (IVM) systems for porcine oocytes, developmental competence of in vitro-matured oocytes is still suboptimal compared with those matured in vivo. In this study, we compared oocytes obtained from large (≥8 mm; LF) and medium (3–7 mm; MF) sized follicles in terms of nuclear maturation, intracellular glutathione and reactive oxygen species levels, gene expression, and embryo developmental competence after IVM. In the control group, cumulus-oocyte complexes (COCs) were aspirated from MF and matured for 22 hours with hormones and subsequently matured for 18 to 20 hours without hormones at 39 °C, 5% CO₂in vitro. In the LF group, COCs were obtained from follicles larger than 8 mm and were subjected to IVM for only 18 hours. The ovaries have LF were averagely obtained with 1.7% per day during 2012 and it was significantly higher in the winter season. The results of the nuclear stage assessment of the COCs from the LFs are as follows: before IVM (0 hours); germinal vesicle stage (15.2%), metaphase I (MI) stage (55.4%), anaphase and telophase I stages (15.8%), and metaphase II (MII) stage (13.6%). After 6 hours IVM; germinal vesicle (4.2%), MI (43.6%), anaphase and telophase I (9.4%), and MII (42.8%). After 18-hour IVM; MI (9.7%) and MII (90.3%). Oocytes from LF showed a significant (P < 0.001) increase in intracellular glutathione (1.41 vs. 1.00) and decrease in reactive oxygen species (0.8 vs. 1.0) levels compared with the control. The cumulus cells derived from LFs showed lower (P < 0.1) mRNA expression of COX-2 and TNFAIP6, and higher (P < 0.1) mRNA expression of PCNA and Nrf2 compared with the control group-derived cumulus cells. After parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (SCNT) using matured oocytes from LFs, the embryo development was significantly improved (greater blastocyst formation rates and total cell numbers in blastocysts) compared with the control group. In conclusion, oocytes from LFs require only 18 hours to complete oocyte maturation in vitro and their developmental competence is significantly greater than those obtained from MFs. Although their numbers are limited, oocytes from LFs might offer an alternative source for the efficient production of transgenic pigs using SCNT.     

Increasing glucose in KSOMaa basal medium on culture Day 2 improves in vitro development of cloned caprine blastocysts produced via intraspecies and interspecies somatic cell nuclear transfer

This study was conducted to evaluate the efficiency of potassium simplex optimization medium with amino acids (KSOMaa) as a basal culture medium for caprine intraspecies somatic cell nuclear transfer (SCNT) and caprine-bovine interspecies somatic cell nuclear transfer (iSCNT) embryos. The effect of increased glucose as an energy substrate for late stage development of cloned caprine embryos in vitro was also evaluated. Enucleated caprine and bovine in vitro matured oocytes at metaphase II were reconstructed with caprine ear skin fibroblast cells for the SCNT and iSCNT studies. The cloned caprine and parthenogenetic embryos were cultured in either KSOMaa with 0.2 mM glucose for 8 days (Treatment 1) or KSOMaa for 2 days followed by KSOMaa with additional glucose at a final concentration of 2.78 mM for the last 6 days (Treatment 2). There were no significant differences in the cleavage rates of SCNT (80.7%) and iSCNT (78.0%) embryos cultured in KSOMaa medium. Both Treatment 1 and Treatment 2 could support in vitro development of SCNT and iSCNT embryos to the blastocyst stage. However, the blastocyst development rate of SCNT embryos was significantly higher (P < 0.05) in Treatment 2 compared to Treatment 1. Increasing glucose for later stage embryo development (8-cell stage onwards) during in vitro culture (IVC) in Treatment 2 also improved both caprine SCNT and iSCNT embryo development to the hatched blastocyst stage. In conclusion, this study shows that cloned caprine embryos derived from SCNT and iSCNT could develop to the blastocyst stage in KSOMaa medium supplemented with additional glucose (2.78 mM, final concentration) and this medium also supported hatching of caprine cloned blastocysts.     

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality.     

Risk of Coxiella burnetii transmission via embryo transfer using in vitro early bovine embryos

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Domestic ruminants are the main source of infection for humans. The objectives of this study were to determine (1) whether C. burnetii would adhere to the intact zona pellucida (ZP-intact) of early in vitro–produced bovine embryos; (2) whether the bacteria would adhere to or infect the embryos (ZP-free) after in vitro infection; and (3) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. One hundred and sixty, eight- to 16-cell bovine embryos produced in vitro, were randomly divided into 16 batches of 10 embryos. Twelve batches (eight ZP-intact and four ZP-free) were incubated in a medium containing C. burnetii CbB1 (Infectiologie Animale et Santé Publique, Institut National de Recherche Agronomique Tours, France). After 18 hours of incubation at 37 °C and 5% CO₂ in air, the embryos were washed in 10 successive baths of a PBS and 5% fetal calf serum solution in accordance with the IETS guidelines. In parallel, four batches (two ZP-intact and two ZP-free) were subjected to similar procedures but without exposure to C. burnetii to act as controls. Ten washing fluids from each batch were collected and centrifuged for 1 hour at 13,000× g. The embryos and wash pellets were tested using conventional polymerase chain reaction. C. burnetii DNA was found in all ZP-intact and ZP-Free embryos after 10 successive washes. It was also detected in the first four washing fluids for ZP-intact embryos and in the 10th wash fluid for two of the four batches of ZP-free embryos. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. burnetii adheres to and/or penetrates the early embryonic cells and the ZP of in vitro bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of bovine embryos infected by C. burnetii would eliminate the bacteria from the ZP and to verify if similarly results are obtained with in vivo–derived embryos.     

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up–derived oocytes have different kinetics and requirements regarding maturation media

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.     

Large scale in vivo risk assessment of bovine viral diarrhea virus (BVDV) transmission through transfer of bovine embryos produced via somatic cell nuclear transfer (SCNT)

The objective was to use the bovine viral diarrhea virus (BVDV) as a model to assess the risk of infectious disease transmission in the system of in vitro embryo production and transfer via somatic cell nuclear transfer (SCNT) technology. The risks of BVDV transmission in the SCNT embryo production were previously evaluated [1]. In that in vitro study, following standard operating procedures (SOP), including pre-nuclear transfer donor cell testing, oocyte decontamination and virus-free cell and embryo culture conditions, SCNT embryos produced were free of detectable viral RNA. The current study focused on the evaluation of the potential risk of disease transmission from SCNT embryos to recipients, and the risk of producing persistently infected animals via SCNT embryo transfer. Blood samples were collected from 553 recipients of SCNT embryos and 438 cloned calves and tested for the presence of BVDV viral RNA via a sensitive real time PCR method. All samples tested were negative. These results, in conjunction with the previous in vitro study, confirmed that the established SCNT embryo production and transfer system is safe and presents no detectable risk of disease transmission.     

Dose-dependent effect of melatonin on postwarming development of vitrified ovine embryos

After cryopreservation, embryos become sensitive to the oxidative stress, resulting in lipid peroxidation, membrane injury, and structural destruction. The present study aimed to assess the effect of increasing concentration of melatonin during postwarming culture on embryo's ability to restore its functions after cryopreservation. In vitro–produced blastocysts were vitrified, warmed, and cultured in vitro in TCM 199 with 5 different supplementations: control (CTR): 10% fetal calf serum; bovine serum albumin (BSA): 0.04% (wt/vol) BSA; and MEL⁻³, MEL⁻⁶, MEL⁻⁹: BSA plus melatonin 10⁻³, 10⁻⁶, and 10⁻⁹ M. The medium with the highest melatonin concentration had the highest trolox equivalent antioxidant capacity, whose values were comparable with those determined in plasma sampled from adult ewes (8.7 ± 2.4 mM). The other media had lower trolox equivalent antioxidant capacity values (P < 0.01), below the range of the plasma. At the same time, embryos cultured with the highest melatonin concentration reported a lower in vitro viability, as evaluated by lower re-expansion and hatching rates, and lower total cell number compared with the other groups (P < 0.05). Their metabolic status was also affected, as evidenced by higher oxidative and apoptotic index and lower ATP concentration. The beneficial effects of melatonin on embryo development during postwarming culture were observed only at low concentration (10⁻⁹ M). These results suggest that melatonin at high concentration may exert some degree of toxic activity on pre-implantation embryos. Thus, the dose at which the embryos are exposed is pivotal to obtain the desiderate effect.     

Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer

Successful cryopreservation of porcine embryos offers a promising perspective in the fields of agriculture, animal science, and human medical research. The objective of the present work was to establish a system facilitating the cryopreservation of porcine embryos produced by somatic cell nuclear transfer (SCNT). Several key techniques including micromanipulator-based enucleation, noninvasive delipation, zona-free fusion, and activation were combined with high efficiency. After a partial zona digestion and high-speed centrifugation, 89.8+/-2.1% (mean+/-SEM) of enucleated oocytes were successfully delipated. Delipated cytoplasts were incubated for an additional 0.5 or 2 h before fusion with somatic cells. After activation and 6 days of in vitro culture, no significant difference in the rate of blastocysts per reconstructed embryo was observed between the two groups (33.1+/-1.8% and 26.0+/-4.3% for 0.5 and 2 h recovery time, respectively). Cryopreservation of the blastocysts was performed with a Cryotop device and factory-prepared vitrification and warming solutions. One hundred fifty-five vitrified SCNT embryos were transferred surgically into two recipient sows to test their developmental capacity in vivo. One recipient became pregnant and delivered six piglets. In conclusion, our simplified delipation and SCNT procedure resulted in viable piglets after vitrification and embryo transfer at the blastocyst stage.     

The ART of studying early embryo development: Progress and challenges in ruminant embryo culture

The study of preimplantation mammalian embryo development is challenging due to difficulties in accessing in vivo-derived embryos in large numbers at the early stages and the inability to culture embryos in vitro much beyond the blastocyst stage. Nonetheless, embryos exhibit an amazing plasticity and tolerance when it comes to adapting to the environment in which they are cultured. They are capable of developing in media ranging in composition from simple balanced salt solutions to complex systems involving serum and somatic cells. At least a proportion of the blastocysts that develop in culture are developmentally competent as evidenced by the fact that live offspring have resulted following transfer. However, several studies using animal models have shown that such embryos are sensitive to environmental conditions that can affect future pre- and post-natal growth and developmental potential. This review summarises some key aspects of early embryo development and the approaches taken to study this important window in early life.     

Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: Can supplementary progesterone rescue small embryos?

Conceptus size on Day 14 after multiple embryo transfer of Day 7 in vitro–produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development after the transfer of Day 7 blastocysts containing a low total cell number. The estrous cycles of crossbred beef heifers were synchronized using an 8-day progesterone (P4)–releasing intravaginal device (PRID) with the administration of a prostaglandin F_2α analog on the day before device removal. Only those heifers recorded in standing estrus (Day 0) were used. Heifers were randomly assigned to one of four treatment groups: (1) control: large blastocysts (high total cell number), (2) control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU human chorionic gonadotropin (hCG) on Day 2 after estrus, or (4) small blastocysts plus insertion of a vaginal P4 insert (PRID, 1.55 g P4) between Days 3 and 5 after estrus. In vitro–produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer), and conceptuses were recovered at slaughter on Day 14. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analyzed using the PROC MIXED procedure of SAS. Total cell number on Day 7 was significantly lower in small versus large blastocysts (72.4 ± 3.93 vs. 144.8 ± 3.90, P < 0.05). Conceptus recovery rate was 53.8% overall (140 of 260) and was highest in the large blastocyst group (68.3%, 41 of 60) compared with the other groups (45.7%–55.0%). Concentrations of serum P4 were similar in the two unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in the hCG-treated heifers and on Days 4 and 5 in the PRID group (P < 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm; P < 0.05), whereas insertion of a PRID from Days 3 to 5 increased conceptus length approximately fivefold (13.09 ± 2.11 mm; P < 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of “rescuing” poor-quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition.     

Expression patterns of sirtuin genes in porcine preimplantation embryos and effects of sirtuin inhibitors on in vitro embryonic development after parthenogenetic activation and in vitro fertilization

We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1-3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1-3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1-3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques.     

Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: A model for utilization of primordial oocytes

Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1^nu). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion.     

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro–produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro–matured oocytes and in vitro–produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.     

Promotion of Human Early Embryonic Development and Blastocyst Outgrowth In Vitro Using Autocrine/Paracrine Growth Factors

Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6–8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner’s criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.