Improved in vitro development of porcine embryos with different energy substrates and serum

The effect of replacing 5.5 mM glucose in North Carolina State University (NCSU)-23 medium with 0.5 mM pyruvate/5.0 mM lactate on porcine IVF embryo development was investigated in Experiment 1. Culturing embryos with pyruvate/lactate for 7 days or with pyruvate/lactate from Days 0 to 2, and then glucose from Days 2 to 7 improved cleavage rates. In Experiment 2, embryos were cultured for 7 days in pyruvate/lactate containing NCSU-23 medium supplemented with 0.05% PVA, 0.4% BSA or 10% fetal bovine serum (FBS). The BSA supplement increased the rates of cleavage, blastocyst formation, and the number of total cells in blastocysts. In Experiment 3, embryos were cultured in pyruvate/lactate containing NCSU-23 medium supplemented with 0.4% BSA for 7 days (BSA-PL), 0.4% BSA from Days 0 to 4 and then 10% FBS from Days 4 to 7 (BSA-PL-->F ) or 0.4% BSA from Days 0 to 7 with addition of 10% FBS (BSA-PL + F ) at Day 4. More blastocysts in BSA-PL--> F and hatching or hatched blastocysts in BSA-PL-->F and BSA-PL+F were obtained. Total cell number in blastocysts derived from BSA-PL-->F and BSA-PL+F were increased. Our results demonstrated that supplementing pyruvate/lactate containing NCSU-23 medium with 0.4% BSA for 4 days and replacing it with 10% FBS for another 3 days improved porcine IVF embryo development.     

Changes of lipid composition in non-cultured and cultured porcine embryos

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

New technique to quantify the lipid composition of lipid droplets in porcine oocytes and pre-implantation embryos using Nile Red fluorescent probe

The principal objective of this study was to develop a novel method based on confocal microscopy and a solvatochromic fluorescent dye, Nile red (NR) to quantify the main types of lipids in a single mammalian oocyte and embryo. We hypothesize that NR staining followed by the decomposition of NR-spectra identifies and quantifies the triglycerides, phospholipids, and cholesterol in a single oocyte and embryo. We analyzed the lipid droplets in porcine oocytes and pre-implantation embryos up to the hatched blastocyst stage developed in vivo and in cultured blastocysts. The emission spectrum of NR-stained mixture of different lipid types is a convolution of several component spectra. The principal component analysis (PCA) and a multivariate curve resolution-alternating least squares method (MCR-ALS) allowed to decompose the emission spectrum and quantify the relative amount of each lipid type present in mixture. We reported here that the level of the triglycerides, phospholipids and cholesterol in lipid droplets significantly decreases by 17.7%, 26.4% and 23.9%, respectively, from immature to mature porcine oocytes. The content of triglycerides and phospholipids remains unchanged in droplets of embryos from the zygote up to the morula stage. Then the triglyceride level decreases in the blastocyst by 15.1% and in the hatched blastocyst by 37.3%, whereas the amount of phospholipids decreases by 10.5% and 12.5% at the blastocyst and hatched blastocyst stages, respectively. In contrast, the content of cholesterol in droplets does not change during embryo cleavage. The lipid droplets in the blastocyst produced in vivo contain lower amounts of triglycerides (by 26.1%), phospholipids (by 14.2%) and cholesterol (by 34.8%) than those in the blastocyst cultured in NCSU-23 medium. In conclusion, our new technique is suitable to quantify the content of triglycerides, phospholipids and cholesterol in individual mammalian oocytes and embryos. Our findings indicate an important role for lipids during porcine oocyte maturation and early embryonic development, and suggest an altered lipid metabolism in cultured embryos.     

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.     

Set up of a serum-free culture system for bovine embryos: embryo development and quality before and after transient transfer

It is well known that serum in culture medium negatively affects blastocyst quality. The objective of this work was to develop and test a serum-free culture medium which could improve embryo quality, measured by the resistance to freezing, lipid and glutathione content of the resulting blastocysts, as well as the ability of the blastocysts to elongate after transient transfer to recipient cows. In a first experiment we showed that adding a mixture of insulin, transferrin and selenium to serum-free Synthetic Oviduct Fluid medium (SOF-ITS) improved embryo development and quality. In the second experiment, the addition of BSA to SOF-ITS further improved blastocyst development. Moreover, a reduction in lipid content of morulae was observed in SOF-ITS-BSA by comparison with morulae cultured with serum (SOF-FCS). The resistance to freezing measured by hatching rates 24h post-thawing was also improved for blastocysts with a diameter between 160 and 180 microm cultured in SOF-ITS-BSA by comparison to those produced with serum. In order to evaluate the redox potential of the embryos, reduced glutathione content (GSH) was evaluated both before and after cryopreservation. A significant decrease in glutathione was observed after freezing, whatever the culture medium, but no difference was observed between culture conditions. Transient transfers were performed and elongated D-13 embryos were recovered. Elongation was more pronounced and the embryonic disk more often visible in embryos cultured in SOF-ITS-BSA than in embryos cultured with FCS. In conclusion, the serum-free system we developed to produce in vitro bovine embryos meets the developmental and qualitative requirements for a large-scale use.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Developmental responses of 2-cell embryos to oxygen tension and bovine serum albumin in Wistar rats.

To improve rat embryo culture conditions, responses of Wistar 2-cell embryos from 2 breeders to oxygen tension (5 vs 20%) and bovine serum albumin (BSA) (0 vs 3 mg/ml) were examined using rat 1-cell embryo culture medium (mR1ECM). Supplementation of 3 mg/ml BSA significantly stimulated and accelerated development to the blastocyst and expanded blastocyst stages during 72 and 96 h culture, while reduced oxygen tension stimulated cell division. Fetus development after transfer of blastocysts obtained from 72 h culture under 5% O2 with BSA was significantly higher than those cultured under atmospheric oxygen without BSA. However, the nuclear numbers of in vitro cultured blastocysts and fetus development after embryo transfer were still significantly lower than in vivo developed blastocysts, indicating the current culture condition is still suboptimal.     

Birth of cloned pigs from zona-free nuclear transfer blastocysts developed in vitro before transfer

The objective of this work was to obtain cloned pig offspring by uterine transfer of blastocysts produced by zona-free manipulation. We started by defining the most suitable culture media for growing pig nuclear transfer embryos produced by zona-free micromanipulation comparing NCSU-23aa with Synthetic Oviduct Fluid (SOFaa) and with in vivo culture in the sheep oviduct. We found that parthenogenetic development to day 7 blastocyst in NCSU-23aa and sheep oviduct was significantly superior as compared to SOFaa (61.8%, 64% and 42.4 respectively) although blastocyst cell number was higher in the latter. Interestingly, when we compared the two media for the culture of nuclear transfer (NT) embryos derived from 3 different donor cell lines, we observed lower rates of development with NCSU-23aa (from 24.5% to 32.4%) while with SOFaa the development was significantly higher for two donor cell lines as compared to the third (44.4%, 48.9% and 20.6% respectively). A total of 244 blastocysts grown in SOFaa were transferred in four synchronized sows on day 5 or 6 of development. Two recipients farrowed 6 and 8 piglets corresponding to an efficiency of development to term of 8% and 16% of the transferred embryos respectively. Eleven pigs are now 10 month of age and those that have reached puberty have been proven to be fertile. Finally, this is the first report on the production of cloned pigs derived from the transfer of NT embryos at the blastocyst stage.     

Development and quality of porcine embryos in different culture system and embryo-producing methods

In this study, the developmental ability and cellular composition of porcine IVF, parthenote and somatic cell nuclear transfer (SCNT) embryos were evaluated following different in vitro culture systems. Group 1, embryos were cultured in NCSU-23 with 5.55 mM D-glucose (NCSU+) until day 6 on 20% O(2) or 5% O(2) (Group 2). Group 3, embryos were cultured in D-glucose-free NCSU-23 (NCSU-) with 0.17 mM Na pyruvate/2.73 mM Na lactate for 58 h and subsequently cultured in NCSU+ until day 6 (NCSU -/+) on 20% O2 or 5% O(2) (Group 4). IVF blastocysts did not differ significantly with O(2) concentrations, but differed significantly with major energy source (glucose and pyruvate/lactate). In Group 3 and 4 IVF blastocysts, the total cell number and apoptosis rates were not significantly different with different O(2) concentrations. Blastocyst rate, total cell number and apoptosis rate in Groups 3 and 4 parthenote embryos also were not significantly different. Parthenote and SCNT, under the same culture treatment, exhibited significant differences in blastocyst and apoptosis rates (47.5 +/- 16.1 vs. 24.0 +/- 4.0 and 4.9 +/- 9.0 vs. 22.8 +/- 23.3). Apoptosis-generating rate increased in the order parthenote, IVF and then SCNT. In conclusion, in vitro development of porcine embryos was not affected by O(2) concentrations but was affected by major energy source. Even so, the concentration of each major energy source and the timing of its inclusion in culture could accomplish relatively high embryonic development, the apoptosis rate stressed that more work still needs to be done in developing a better defined culture system that could support SCNT embryos equivalent to in vivo preimplantation porcine embryos.     

Development of porcine embryos produced by IVM/IVF in a medium with or without protein supplementation: effects of extracellular glutathione

This study was designed to examine the effects of extracellular reduced glutathione on development of pig embryos, produced by in vitro maturation (IVM) and in vitro fertilisation (IVF), in a chemically defined North Carolina State University (NCSU) 23 medium or in NCSU 23 medium with bovine serum albumin (BSA). Microfilament distribution, as a marker of embryo quality, was also examined by immunocytochemical staining and confocal microscopy. When the inseminated oocytes were cultured in the defined medium containing 0-0.5 mM glutathione, blastocyst formation as observed only in the media with glutathione (8.5-16.0%). Increased numbers of blastomeres were observed in the blastocysts as the concentration of glutathione was increased (18.8 +/- 7.2 to 31.0 +/- 8.6). In NCSU 23 medium with 4 mg BSA/ml, addition of glutathione at concentrations of 0.125-0.5 mM significantly increased the proportions of oocytes that developed to blastocysts (39.2-52.5%) compared with the control (29.5%). However, no difference was observed in the average cell number in the blastocysts (41.9 +/- 15.6 to 49.1 +/- 15.5). There were no significant differences in the microfilament distribution in the embryos produced in the defined medium and in the BSA-containing medium. These results indicate that pig embryos produced by IVM/IVF can develop to the blastocyst stage in a defined medium. BSA and glutathione have a synergistic effect on pig embryo development.     

Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation

This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P < 0.01). No differences were found for additives (P > 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P < 0.05) between additives and hexoses for blastocyst production; although trends were similar, the benefit of fructose compared to glucose was greater for controls than for FCS or PES. Culture of embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively.     

In vitro production of pig embryos: comparisons of culture media and boars

The utilization of in vitro produced pig embryos for commercial production or research is dependent upon the development of improved methodology. Our objective was to establish a consistent in vitro embryo production (IVP) system and subsequently utilize the procedures to evaluate culture system components and boar effects. To summarize the IVP system, 403 inseminated oocytes from a total of 2243 were analyzed across 17 replicates for maturation and fertilization efficiency, while 1838 zygotes were cultured in 26 replicates for developmental data. Penetration, cleavage and blastocyst development rates were determined at 18, 44 and either 144 or 168 h post insemination, respectively. Monospermic penetration averaged 31.8+/-7.3% while polyspermy was 30.8+/-17.2%. Cleavage rate was 44.9+/-16.1%, with 21.8+/-7.5% of fertilized oocytes and 51.9+/-15.9% of cleaved embryos developing to blastocysts. For culture medium comparison, fertilized oocytes were cultured in either BECM-6, BECM-7, NCSU-23 or NCSU-23aa and supplemented on Day 5 post insemination (pi) with 10% FCS. These treatments resulted in 4.0, 4.9, 19.8 and 13.6% (+/-3.2%) blastocysts by Day 7 pi, with an average cell number of 44.4+/-9.0, 65.1+/-8.2, 61.3+/-4.5 and 64.4+/-4.8, respectively. These IVP procedures consistently produced zygotes from semen of several different boars, capable of forming blastocysts in vitro. Comparison of developmental rates among the boars indicated that this system is variable among boars but not strictly boar-dependent. Culture media comparisons suggest that NCSU-23 yielded a higher percentage of blastocysts than the other media in this IVP system.     

In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres

This study investigated the effect of culture media and gas atmospheres on the development of porcine nuclear transfer embryos. Oocytes derived from a local abattoir were matured for 42-44 h and enucleated. Fetal fibroblasts were prepared from a Day 35 porcine fetus. Confluent stage fetal fibroblasts were introduced into the perivitelline space of enucleated oocytes. Fusion and activation were induced simultaneously with two direct current (1.2 kV/cm for 30 micros) in 0.3 M mannitol medium. For parthenogenetic activation, the same pulses were used. In Experiment 1, parthenogenetically activated oocytes were cultured in North Carolina State University-23 (NCSU-23), Porcine Zygote Medium-3 (PZM-3), or Beltsville Embryo Culture Medium-3 (BECM-3). Parthenogenetically activated oocytes cultured in PZM-3 had a higher (P < 0.05) developmental rate to the blastocyst stage (15.2% versus 3.7-9.6%) as compared to BECM-3 or NCSU-23. The number of nuclei in Day 6 blastocysts was higher (P < 0.05) in PZM-3 (23.6) and NCSU-23 (21.4) than BECM-3 (14.2). In Experiment 2, parthenogenetically activated oocytes were cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air for 6 days (T1), 5% CO(2), 5% O(2), 90% N(2) for 6 days (T2), 5% CO(2) in air for 3 days, then 5% CO(2), 5% O(2), 90% N(2) for 3 days (T3), or 5% CO(2), 5% O(2), 90% N(2) for 3 days, then 5% CO(2) in air for 3 days (T4). Blastocyst formation rates were not different among treatments (12.9 =/-3.6 %, 13.5 +/- 4.2%, 10.8+/-2.4%, and 12.6+/-2.7%, respectively). However, T2 (36.7+/-2.9) and T3 (33.8+/-3.0) resulted in more nuclei per blastocyst than T1 (23.2+/-2.1) or T4 (26.0+/-2.1 ). In Experiment 3, reconstructed porcine nuclear transfer (NT) embryos were cultured in NCSU-23 or PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2). Developmental rates to blastocyst stage for porcine NT embryos cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) were 7.2+/-1.4% and 12.3+/-1.4%, and the number of nuclei was 12.2=/-0.8% and 19.4+/-1.0, respectively. NT embryos cultured in PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) had developmental rates to blastocyst stage of 18.8+/-1.9 %, and 17.8+/-3.8% the nuclei number was 20.9 +/- 1.9 and 21.9+/-3.3, respectively. NT embryos cultured in NCSU-23 had a higher developmental rate to the blastocyst stage in 5% CO(2), 5% O(2), 90% N(2) than in 5% CO(2) in air (P < 0.05). Regardless of gas atmospheres, NT embryos cultured in PZM-3 had a higher developmental rate (18.3 =/- 1.7% versus 16.9 +/- 1.2%) and nuclei number (21.4 +/-1.8 versus 16.9 +/- 1.2) than in NCSU-23 (P < 0.05). In conclusion, a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2) supported a higher development rate of porcine NT embryos than 5% CO(2) in air when the porcine NT embryos were cultured in NCSU-23. Furthermore, regardless of atmosphere, PZM-3 supported a higher development rate of porcine nuclear transfer embryos than NCSU-23.     

Prediction of porcine blastocyst formation using morphological, kinetic, and amino acid depletion and appearance criteria determined during the early cleavage of in vitro-produced embryos

The determination for early cleavage-stage embryos of noninvasive morphologic and metabolic criteria that are predictive of blastocyst development and/or full-term viability remains an important research target. We describe the derivation of a logistic regression model that predicts the probability of porcine blastocyst formation in vitro. Pig zygotes, derived by in vitro maturation and fertilization of slaughterhouse oocytes, were cultured in NCSU-23 medium that was supplemented with a mixture of 20 amino acids (NCSU-23(aa)). On Day 1, at 21, 23, 25, 27, 29 and 31 h postinsemination, cleaving embryos were evaluated morphologically in terms of the: i) number of blastomeres, ii) evenness of division, and iii) degree of fragmentation. These embryos were then placed in 1.5-microl drops of NCSU-23(aa) for 24 h, after which time the three morphologic criteria were re-evaluated and 1.2 microl of spent medium were removed for analysis by HPLC, in order to determine the net rates of amino acid depletion and appearance. Embryos were then cultured singly in NCSU-23(aa) by placing them between the filaments of a woven polyester mesh until Day 6, in order to permit the identification of individual embryos. Of 256 cleaved embryos, 28.7 +/- 6.2% (n = 5 replicates) developed into blastocysts. Discriminant analysis was used to select a subset of amino acids (threonine, valine, lysine, and phenylalanine) that discriminated optimally between embryos that became blastocysts or degenerated. These discriminant scores were entered into the logistic regression. Significant univariate relationships were established between the probability of blastocyst development and amino acid score (odds ratio [OR] 0.53, 95% confidence interval [CI] 0.40-0.69, P < 0.001), cleavage time (OR 0.79, 95% CI 0.71-0.87, P < 0.001), degree of fragmentation on Day 1 (OR 0.55, 95% CI 0.35-0.84, P = 0.009) and Day 2 (OR 0.53, 95% CI 0.35-0.78, P = 0.002), evenness of division on Day 2 (OR 0.66, 95% CI 0.46-0.96, P = 0.028), and categorical values of blastomere number on Day 2 (all P < 0.02), although no single variate could accurately predict blastocyst formation. However, multivariate analysis of the cell numbers on Day 1 and Day 2 correctly classified 51.9% of the predicted blastocysts. The inclusion of cleavage time in the regression analysis raised this rate to 63.5%, which was increased to 66.2% by the addition of evenness of division and degree of fragmentation. Finally, the full logistic regression model, which incorporated amino acid score together with all the other morphologic and kinetic variables, correctly classified 80.8% of the predicted blastocysts. This represented 51.2% of the observed blastocysts. Our data are novel in that they not only define in a quantitative manner the influence of previously undescribed predictors of porcine blastocyst formation, but they also provide a simple model of preimplantation development with reasonable predictive accuracy. The present study also provides a basic model for the examination and incorporation of additional early morphologic and metabolic correlates of developmental competence and could potentially be applied to the selection of human embryos for transfer in clinical IVF.     

Protein-free culture medium containing polyvinylalcohol, vitamins, and amino acids supports development of eight-cell hamster embryos to hatching blastocysts

In vitro development of eight-cell hamster embryos to hatching blastocysts requires the presence of amino acids and a group of water-soluble vitamins in the culture medium. The present studies investigated the effect of type of macromolecule on blastocyst hatching and on the requirement for vitamins. Embryos were cultured for 3 days in the presence of the synthetic macromolecule polyvinylalcohol (PVA) and of different types of bovine serum albumin (BSA), both with and without vitamins. The results showed th at eight-cell embryos develop to hatching blastocysts in the presence of vitamins and amino acids with PVA as the only macromolecule in the medium. The presence of certain types of BSA reduced but did not eliminate the need for vitamins. Glutamine alone was as efficient as a complete amino acid supplement in supporting blastocyst hatching. These results demonstrate for the first time that eight-cell hamster embryos can be cultured to hatching blastocysts in a chemically defined medium.     

培养基中能量底物对猪胚胎发育的影响

旨在探讨丙酮酸和乳酸对猪(Susscrofa)胚胎早期发育的影响,将NCSU-23培养基中的5.56mmol/L葡萄糖替换为0.2mmol/L丙酮酸、5.7mmol/L乳酸,并将此培养基命名为mNCSU-23。根据实验设计,孤雌胚及核移植胚转移到mNCSU-23或NCSU-23中培养。激活第2天统计孤雌胚及核移植胚中的5～8细胞胚胎数。激活第6天统计孤雌胚及核移植胚囊胚形成率及囊胚细胞数。实验结果表明,mNCSU/NCSU处理组的5～8细胞胚胎数及囊胚数显著高于对照组(P0.05);单纯使用mNCSU培养猪胚胎时,囊胚率最低,发育结果最差(P0.05)。本研究证实,在体外培养前两天,用乳酸和丙酮酸代替培养基中的葡萄糖对胚胎发育有利。     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

The objective of this study was to determine the effect of fetal calf serum (FCS) on the quality of in vitro produced bovine embryos. Cumulus oocyte-complexes (COCs, n = 2 449) recovered by ovum pick-up from Bos taurus indicus donors were randomly assigned to experimental groups. Sperm selected by Percoll gradient was used for in vitro fertilization (insemination = Day 0). In Experiment 1 (n = 1 745 COCs), zygotes were cultured in vitro in Synthetic Oviduct Fluid + 4 mg/mL of bovine serum albumin (BSA), or BSA + 2% FCS (BSA+FCS). In Experiment 2 (n = 704 COCs), the COCs were cultured in SOF + BSA, BSA + 2% FCS, or BSA + 2% FCS on D4 (BSA + FCSD4). In Experiment 1, blastocyst yield (51%) and Quality I blastocysts (41%) at Day 7 were higher (P < 0.05) in the BSA + FCS treatment than in BSA (42 and 30%, respectively). In Experiment 2, blastocyst yield was higher (P < 0.05) in the BSA+FCS (47%) treatment. Quality I blastocyst yield was higher (P < 0.05) for BSA + FCS (34%) and BSA+FCSD4 (32%) compared to the BSA treatment (20%). A total of 820 embryos were transferred, with no significant differences among groups in pregnancy rates. In conclusion, in vitro culture in SOFaaci + BSA + FCS enhanced blastocyst yield and Quality I blastocysts; adding FCS to the culture medium increased the efficiency of IVP of bovine embryos.