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.     

Relationship between plasminogen activator production and bovine embryo development in vitro

The relationship of plasminogen activator (PA) production to cell stage, cell number and changes in overall diameter and zona pellucida thickness for bovine embryos developing in vitro was determined. Late morulae to blastocysts (n = 80) were collected nonsurgically from naturally mated, estrous-synchronized, superovulated crossbred beef cows. Embryos were cultured, one embryo per 25-microliters microdrop, for 6 d. At 24-h intervals, embryos were evaluated for stage of development and transferred to fresh microdrops; media were recovered for PA analysis. In addition, embryo diameter and zona pellucida thickness were measured with an ocular micrometer. Plasminogen activator production was determined using a caseinolytic assay with urokinase as the standard. Changes in diameter, zona pellucida thickness and PA production per 24-h interval for each embryo were plotted, and the graphs were cut out and weighed. Sixty-one embryos (76%) completed the hatching process. Total PA production was correlated positively (P less than .005) to embryonic size (r = .40), developmental stage (r = .35) and cell number (r = .35) and negatively, but weakly, correlated to zona pellucida thickness (r = -.13; P = .267). Hatched embryos produced more total PA than embryos that did not hatch (.140 +/- .011 vs .070 +/- .019 g; P less than .01). These results suggest that as embryonic size and cell number increase and development progresses, bovine embryos liberate more PA.     

Effect of Transgene Introduction and Recloning on Efficiency of Porcine Transgenic Cloned Embryo Production In Vitro

Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNβ-Z or LNβ-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro . Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNβ-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 ± 6.4%; 12.0 ± 5.7%) or EGFP (57.5 ± 6.3%; 10.1 ± 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 ± 8.2%; 12.3 ± 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.     

Development of one-cell porcine embryos to the blastocyst stage in simple media

Porcine embryos were flushed from mated donors and examined for cleavage stage. One- and two-cell embryos were randomly allotted to one of the five following in vitro treatments: M199 with Earle's salts, a modified Tyrode's medium (TL), TL supplemented with 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) (TLH), TLH supplemented with 5.5 mM glucose (TLHG), or TLH supplemented with 5 mM glutamine (TLHGL). The bicarbonate concentration of TLH, TLHG, and TLHGL was 2 mM, compared with the 25 mM concentration in M199 and TL. Embryos in M199 and TL were incubated in 95% air:5% CO2 at 39 degrees C. Those in the remaining three treatments were incubated in air at 39 degrees C. Embryos incubated in TL and M199 did not develop past the four- to eight-cell stage, whereas the proportions of embryos developing to the compact morula or blastocyst stage by d 7 of culture in the other treatments were as follows: TLHG, 49.1%; TLHGL, 59.4%; TLH, 63.5% (P less than .005). These results indicate that porcine embryos can be cultured from the one-cell stage to blastocyst in a simple HEPES-buffered medium in air. The ability of porcine embryos to develop without supplemental CO2 may be an important finding for use in situations in which embryos must be transported for long periods before embryo transfer.     

Development of porcine embryos in vitro: effects of culture medium and donor age.

We compared development of porcine embryos in three media and evaluated the effect of age of the donor on embryo development in vitro. In Exp. 1, embryos were collected from 35 postpubertal females on d 2 or 3 after onset of estrus. Embryos were cultured 144 h in Whitten's Medium (WM), North Carolina State University Medium-23 (NCSU-23), or Beltsville Embryo Culture Medium-3 (BECM-3) in 95% air: 5% CO2 at 39 degrees C. More (P < 0.01) embryos that were initially one cell or two cells developed to blastocysts when cultured in NCSU-23 (56%) and BECM-3 (43%) rather than in WM (7.5%). More (P < 0.01) embryos that were four cells at recovery developed to blastocysts in NCSU-23 (97%) than in BECM-3 (69%) or WM (69%). Blastocysts that developed from four-cell embryos cultured in BECM-3 had more (P < 0.01) nuclei than blastocysts that developed from four-cell embryos in the other two media. In Exp. 2, ovarian responses, fertilization rates, and in vitro embryo development in NCSU-23 and BECM-3 were compared for postpubertal (approximately 170-d-old) gilts vs gilts given exogenous gonadotropins at 102 d of age. Ovulation rate (P < 0.01), number of eggs recovered, and number of eggs fertilized per gilt (P < 0.001) were greater in the older gilts. The percentage of eggs fertilized, the number of unfertilized eggs, and the number of unclassifiable eggs were similar (P > 0.10) for both age groups. More (P < 0.10) blastocysts developed from embryos recovered from 170-d-old than from 102-d-old gilts, and more (P < 0.05) blastocysts developed in NCSU-23 than in BECM-3. Zona thicknesses and number of nuclei per embryo were similar (P > 0.10) for both ages. We conclude that embryos from prepubertal gilts do not have the same in vitro developmental potential as those from cyclic gilts. However, superior development of embryos in NCSU-23 from both 102-d-old and 170-d-old gilts indicates that media composition did not differentially affect embryos produced by younger vs older gilts.     

Follicle Ablation Improves the Ovarian Response and the Number of Collected Embryos in Superovulated Cows During the Early Stages of Lactation

A field study was designed to compare ovarian response and embryo yield in cows during early lactation when gonadotropin administration followed one of four treatments. In group 1A (n = 19) and 1B (n = 9), the estrouses were synchronized by two prostaglandin F2alpha (PG) injections given 11 days apart, and starting from day 9 of the synchronized cycle superovulation was conducted with eight decreasing dose of FSH. In group 1B, ablation of all follicles >3 mm was carried out on day 8. In group 2A and 2B (each n = 9), a progesterone plus oestradiol intravaginal device (PRID) was inserted for 11 days and gonadotropin administration started on day 9, while cows from group 2B had a follicle ablation on day 8. In all groups, two PG injections were given along with the sixth and the seventh dose of FSH, and the cows were twice inseminated 12 and 24 h after estrus detection. Embryos were collected on day 7. In cumulative results from aspirated and non-aspirated cows, follicular ablation significantly improved: the ovarian response (10 +/- 1.23 vs 6.69 +/- 0.60 corpora lutea per donor), the mean collected embryos (6.57 +/- 0.94 vs 2.46 +/- 0.53) and the mean transferable embryos (4.43 +/- 0.89 vs 2.18 +/- 0.47). Group 1B and 2B cows had better ovarian response than 1A (6.44 +/- 0.81, 12.25 +/- 4.11 and 9.44 +/- 0.93, for groups 1A, 1B and 2B, respectively, p < 0.05). Similarly, from groups 1B and 2B more (p < 0.05) embryos were collected in comparison with their respective group, while the mean transferable embryos from group 2B (5.22 +/- 1.13) was greater (p < 0.05) than that of group 1A (1.67 +/- 0.35), and tented to be greater than those of groups 2A (3.44 +/- 1.19, p = 0.062) and 1B (3.00 +/- 1.78, p = 0.066). The highest (p < 0.05) transferable embryo collection rate was recorded in group 2B (55.29%), followed by that of group 1B (41.33%). In summary, early in lactation, an acceptable number of transferable embryos can be collected from high producing dairy cows, when follicle ablation prior to superovulation is combined with progesterone and oestradiol administration.     

Transfer of porcine embryos after 3 days of in vitro culture

Two experiments were conducted to determine the viability of porcine embryos transferred after long-term in vitro culture. In Exp. 1, four-cell embryos were kept in culture for 120 h. Embryos that were exposed to fresh culture medium every 12 h survived better than embryos kept in the same medium throughout the culture period. In Exp. 2, four- and eight-cell embryos were cultured in vitro for 72 h before transfer to estrus-induced recipient gilts. Each gilt received, on average, 19 embryos. If recipients were synchronous with donors 3/32 (9%) recipients remained pregnant with an average of 4.0 +/- .6 viable young. If the sexual cycle of the recipients was 24 h behind that of the donors the pregnancy rate was 18/34 (53%) with 4.4 +/- .5 viable young. Average embryo survival rate for the two groups was 1.8 and 12.5%, respectively. A 24-hourly medium replacement during the in vitro culture period had no significant effect on transfer results. When transferring freshly collected blastocysts, pregnancy rate, number of viable young and survival rate of embryos were 6/10 (60%), 7.8 +/- 1.4, and 23.9% for synchronous recipients and 7/10 (70%), 9.3 +/- 1.8, and 32.9% for asynchronous recipients, respectively. Recipients with very high plasma progesterone levels or numerous follicular cysts at the time of transfer were less likely to remain pregnant than others.     

Effect of heat-stress on bovine embryo development in vitro.

Chronic elevation of uterine temperature has long been known to increase embryo mortality in dairy cattle. Short-term elevation in temperature of mouse embryos to 43 degrees C (acute) has been shown to induce intracellular production of heat-shock proteins. In this study, in vitro development of bovine embryos was assessed during short-term (60 h) coculture with oviduct epithelial cells at 38.6 degrees C (T1), 40 degrees C (T2), 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 5% CO2 (T3), or 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 100% CO2 (T4). During incubation, embryos cocultured at 40 degrees C had a greater (P < .05) mean embryo development score at 36 h than embryos cocultured at 38.6 degrees C. At 60 h of incubation, embryo development scores were greater (P < .05) for embryos cultured at 38.6 degrees C than for those cocultured at 40 degrees C. The number of embryos hatched at 60 h was similar after coculture at 38.6 degrees C (T1) or a prior pulse treatment with 5% CO2 and 43 degrees C (T3), but the embryo development score at 60 h was greater (P < .05) for the pulse-treated embryos. Embryos in T4 had greater (P < .05) embryo development scores than did T1 embryos from 36 through 60 h. Pulse treatment (T4) resulted in a greater (P < .05) number of hatched embryos at 60 h than T1, T2, and T3. These results indicate a detrimental effect of a chronic elevation in temperature that was evident shortly after embryo hatching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility of In Vivo- and In Vitro-produced Porcine Embryos to Classical Swine Fever Virus

The objective of this study was to investigate the susceptibility of in vivo- and in vitro-produced (IVP) porcine embryos to classical swine fever virus (CSFV). IVP zona pellucida (ZP)-intact porcine embryos (n = 721) were co-cultured with CSFV for 120 h. After washing according to the International Embryo Transfer Society guidelines (without trypsin) and transferring embryos to CSFV-susceptible porcine kidney cells (PK15 cell line), no virus was isolated. However, when 88 IVP ZP-intact porcine embryos were co-cultured with CSFV for only 48 h before being transferred to PK15 cells, virus was isolated in three of six replicates. Similarly, 603 in vivo-produced porcine embryos were co-cultured with CSFV for 120 h. Subsequently, CSFV was isolated in eight of 50 groups (16%) and the ability of these to form a blastocyst was significantly reduced when compared with the control group (68.2 +/- 19.9% vs 81.9 +/- 9.7%; p < or = 0.001). In contrast, the development of CSFV-exposed IVP porcine embryos was not affected when compared with control embryos (19.1 +/- 10.8% vs 18.9 +/- 10.6%; p > or = 0.05). After removal of the ZP of IVP embryos and subsequent co-culture with CSFV, the virus was isolated from all groups of embryos. These data suggest that virus replication had occurred in the embryonic cells. In conclusion, data indicate that in vivo- and in vitro-produced ZP-intact porcine embryos differ in their susceptibility to CSFV. Hatched or micro-manipulated embryos may increase the risk of transmission of CSFV by embryo transfer, which has to be confirmed by in vivo tests under isolation conditions.     

生长因子EGF、bFGF对猪孤雌胚体外发育的影响

在胚胎发育的不同阶段，分别在培养基中添加EGF和bFGF，研究EGF和bFGF对猪孤雌胚体外发育的作用。结果表明：在1细胞阶段添加EGF或bFGF，添加EGF能够显著提高孤雌胚的卵裂率（P〈0．05）；2～4细胞阶段添加EGF和bFGF，添加EGF组和添加bFGF组的囊胚率都显著高于对照组（P〈0．05），而添加bFGF组的囊胚率和囊胚细胞数都略高于对照组和添加EGF组。说明EGF和bFGF有利于猪孤雌胚的体外发育，而且，bF-GF能够通过提高囊胚细胞数而提高猪孤雌胚的质量。     

供体细胞对猪体细胞克隆胚胎早期发育的影响

以中国农业大学实验用小型猪香猪胎儿成纤维细胞、成年耳成纤维细胞和颗粒细胞3种细胞系为供体细胞进行核移植。比较了血清饥饿法和接触抑制法处理胎儿成纤维细胞诱导进入G0／G1期的效率，发现二者差异不显著（P〉0．05），血清饥饿2d和4d差异不明显，同样接触抑制2d和4d差异也不显著（P〉0．05）。系统研究了影响克隆胚胎发育的供体因素：血清饥饿与否、细胞形态、细胞类型及个体差异等，结果表明：血清饥饿处理对克隆胚的早期发育没有明显的促进作用；圆形光滑细胞有利于细胞融合，对早期发育无显著影响（P〉0．05）；不同个体、不同类型的供体细胞对克隆胚囊胚发育率有一定的影响。     

Effects of delipidation and oxygen concentration on in vitro development of porcine embryos

The effects of delipidation and the oxygen (O(2)) concentration in the atmosphere during culture on in vitro development and H(2)O(2) content were investigated in porcine in vivo fertilized embryos and embryos after in vitro maturation and in vitro fertilization (IVM/IVF embryos). There was no significant difference in the developmental rates to the blastocyst stage between the intact and delipidated IVM/IVF embryos. However, the mean number of cells in blastocysts derived from delipidated IVM/IVF embryos (19.8 +/- 0.8 cells) was significantly smaller than that from intact embryos (24.2 +/- 1.2 cells). Although there were no significant differences in the developmental rates to the blastocyst stage of intact and delipidated IVM/IVF embryos between the cultures under 5% O(2) and 20% O(2), the developmental rate of intact IVM/IVF embryos cultured under 5% O(2) (27.1%) was significantly higher than that of the delipidated embryos cultured under 20% O(2) (19.3%). On the other hand, there was no difference in the developmental rate to the blastocyst stage between in vivo fertilized embryos cultured under 5% O(2) and 20% O(2). Hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), is thought to cause damage to embryos. The H(2)O(2) content per embryo derived from oocytes cultured under 5% O(2) (in vivo fertilized, 58.0 +/- 2.5 pixels; IVM/IVF, 79.6 +/- 3.2 pixels) was significantly lower than that (in vivo fertilized, 100.2 +/- 3.8 pixels; IVM/IVF, 103.9 +/- 3.2 pixels) under 20% O(2). Furthermore, the level of H(2)O(2) in delipidated IVM/IVF embryos (94.7 +/- 3.9 pixels) was significantly lower than that in intact embryos (103.9 +/- 3.2 pixels) cultured under 20% O(2). The present results indicate that the delipidation of porcine IVM/IVF embryos and reduction of the O(2) concentration decreased the H(2)O(2) level rather than the in vitro developmental rate to the blastocyst stage.     

猪植入前胚胎体外培养条件的优化

探讨了更换胚胎培养液及添加FBS、高渗透压和不同浓度VE对猪卵母细胞体外受精(IVF)和孤雌激活(PA)胚胎体外发育的影响,进一步优化了猪植入前胚胎体外培养体系。试验一：在第2天、第4天更换新的培养液(换液组),在换液基础上第4天更换为添加10%FBS的培养液(FBS组)。试验二：胚胎分别在0.05 mol/L蔗糖(蔗糖组)和138 mmol/L氯化钠(氯化钠组)的PZM-3(300～320 mOsmol)中培养2 d后移至PZM-3(288 mOsmol)中培养5 d。试验三：在培养液中分别添加50、100和200 μmol/L VE。对照组均在PZM-3(288 mOsmol)中培养7 d。结果表明：试验一,IVF和PA胚胎FBS组囊胚率显著高于对照组和换液组(P<0.05);试验二,IVF胚胎氯化钠组卵裂率、囊胚率均显著高于对照组与蔗糖组(P<0.05);试验三,IVF胚胎添加100 μmol/L VE组囊胚率显著高于对照组(P<0.05)。结果提示,在换液的基础上添加FBS有利于猪IVF和PA胚胎的体外发育;氯化钠调节的高渗透压可以促进猪IVF胚胎的早期发育;添加100 μmol/L VE可以改善猪IVF胚胎的体外发育体系。     

Feline embryo transfer during the non-breeding season

To obtain normal kits by embryo treansfer (ET) during the non-breeding season, maintenance of pregnancy was carried out by administration of sustained action progesterone (P4) in queens. Embryos were recovered six days after mating from five donor queens in which ovulation was induced by administration of eCG and hCG. The number of embryos recovered ranged from 24 to 53 (mean: 37.2 +/- 6.4) per animal and most embryos were compacted morulae. The yield of embryos was 49.0-93.3% (mean: 73.8 +/- 9.6%). As for recipients, porcine pituitary gland preparation and hCG were administered to 19 queens and estrus and ovulation were induced in 18 queens (94.7%). These queens underwent intrauterine ET of five compacted morulae and 17 cats (94.4%) were impregnated. The number of implantations was 2-5 (mean: 3.7 +/- 0.3). Among these impregnated queens, 15 cats received P4 adminstration starting on day 24 of gestation and 1-5 newborns (mean: 3.4 +/- 0.3) were obtained by normal delivery or caesarean section on day 64-69 of gestation. However, two animals that were not treated with P4 underwent spontaneous abortion about the mid gestational period. Therefore, it is possible to obtain normal kits from queens in the non-breeding season by ET with maintenance of pregnancy by P4 administration.     

体细胞核移植技术生产猪植酸酶转基因胚胎的研究

为获得具有植酸酶腮腺特异性表达的猪转基因克隆胚胎,本研究使用植酸酶腮腺特异性表达的DNA质粒(包含腮腺分泌蛋白(parotid secretary protein,PSP)启动子与终止子序列、Neo筛选基因、绿色荧光蛋白(EGFP)报告基因和高比活的植酸酶appA基因),采用脂质体转染和基因素418(G418)药物抗性筛选的方法获取稳转细胞系,并利用体细胞核移植技术获得植酸酶转基因胚胎。结果表明,本研究构建的DNA质粒可用于细胞筛选,且质粒越小,细胞的转染效率越高,14.89 kb的YM6552仅获得了7.1%的转染率,EGFP质粒则获得了43.4%的转染效率。在单克隆形成上,较小的pYN3600也获得了更高的单克隆形成数(25个),其中表达EGFP的单克隆有14个,植酸酶PCR阳性集落有11个,高于YM6552的单克隆数(19、8和6)。转基因细胞构建重构胚胎后,所有的胚胎均能表达绿色荧光蛋白,虽其体外发育能力有所下降,但差异不显著(P>0.05)。综上所述,本研究所采用的植酸酶质粒、细胞筛选方法和核移植技术可生产植酸酶重构胚。     

Differences in Reproductive Performance, Embryo Development, Interferon-tau Secretion by the Conceptus and Luteal Function in Ewe Lambs Synchronized in Oestrus before or after the Spontaneous Onset of Luteal Activity Preceding Puberty

In mid-September, 1 month before the insertion of intravaginal pessaries to induce sexual activity, blood samples were collected every 4 days from 16 ewe lambs aged 7 months, in order to determine the incidence of ovulations by measurement of plasma progesterone concentrations. It has been studied whether the response to a progestagen treatment of ewe lambs apparently close to puberty could be modified by the onset of the ovarian events preceding puberty. The effect of the presence or absence of ovulations prior to progestagen treatment on the potential reproductive performance (fertility, litter size and fecundity), embryo development [embryo quality and interferon-tau (IFNτ) secretion], luteal function (progesterone secretion in vitro ) and endometrial progesterone content was studied in seven ovulating (Ov+) and nine nonovulating ewe lambs (Ov−) on day 14 after mating. The best potential reproductive results were obtained with Ov+ animals, although these differences could not be initially attributed to either different progesterone secretion in vitro or concentration of endometrial progesterone. Irrespective of the experimental groups, secretion of progesterone by luteal tissue from ewe lambs with normal embryos was significantly greater (p<0.05) than that of animals with abnormal embryos or with no embryos. Normal embryos secreted a higher amount of IFN-τ than those embryos classified as abnormal (p<0.07). In conclusion, ewe lambs which exhibit luteal activity before puberty have the highest levels of reproductive performances after a progestagen treatment. Corpora lutea from ewe lambs with normal embryos had higher rates of progesterone secretion in vitro and their embryos had a higher IFN-τ production by the embryos, indicating greater capacity for subsequent development.     

Growth of Preimplatation Bovine Embryos

Development of mammalian embryos in vitro is functionally and temporally inferior to embryo development as it occurs inside the female reproductive tract. The deficiencies of cultured embryos range from slow cleavage rates to complete developmental arrests or blocks, occurring at particular stages in many species. A variety of approaches have been used to overcome the blocks, including most extensively the coculture of preimplantation embryos with various somatic cells. However, even with coculture, development of embryos in vitro is still not equivalent to that in vivo. In most laboratories, only 25–40% of inseminated oocytes develop into morulae and blastocysts in spite of numerous variations on the basic technique. A better understanding of the factors governing embryonic growth is required before we can hope to achieve results comparable with those occurring in vivo.     

Iloprost对猪早期胚胎体外发育的影响

本研究旨在探讨PGI2类似物iloprost对猪胚胎体外发育的影响。试验以IVF胚胎为研究对象,分别在不同时期(0、24、48、72h)将不同浓度(0、0.2、0.5、1.0、2.0、5.0、10.0μmol.L-1)iloprost加入到猪胚胎培养液中,于156h时记录囊胚发育率和囊胚细胞数,筛选获得最佳添加方案,检测胚胎脂肪代谢速度和代谢相关基因(cox2、creb、pparδ、pdk、cpt2)的表达水平,分析iloprost影响胚胎发育的机制。结果显示,最佳的添加方案为,在受精后48h加入2.0μmol.L-1 iloprost,胚胎囊胚发育率(28%)和囊胚细胞数(49.42)显著高于(P<0.05)对照组的囊胚发育率(16%)和囊胚细胞数(28.22);添加iloprost后,胚胎脂肪酸降解速度也显著加快(P<0.05),脂肪酸代谢相关基因cox2、creb、pparδ、cpt2的表达量上升,糖代谢相关基因pdk表达量无显著变化。结果表明,PGI2类似物iloprost可以促进胚胎降解脂肪酸,为胚胎发育提供能量,提高了胚胎体外发育能力。     

Viability of stored equine embryos

Equine embryos were recovered nonsurgically 6.5 d after ovulation (Exp. 1) and those greater than 200 microns were stored in one of three media: 1) Ham's F10 + 10% fetal calf serum (FCS) under 5% CO2, 5% O2 and 90% N2 at 24 C (Ham's F10); 2) Minimal Essential Medium with Hank's balanced salts + 10% FCS in air (MEM) at 24 C or 3) MEM at 5 C n = 10/treatment). Embryos less than or equal to 200 micron (n = 10) were bisected microsurgically; one-half of each embryo was stored in Ham's F10 and the other half in either Dulbecco's phosphate-buffered saline + 10% FCS in air at 24 C (DPBS), or MEM in air at 24 C. At 0, 12 and 24 h, embryos were: 1) measured; 2) assigned a developmental score of 1 to 4 (1 = tight morula, 4 = expanding blastocyst) and 3) assigned a quality score of 1 to 5 (1 = excellent, 5 = degenerate). Whole embryos stored in MEM at 5 C or 24 C did not (P greater than .05) advance in development by 24 h, whereas those stored in Ham's F10 at 24 C were more (P less than .05) advanced (i.e., higher developmental score) by 24 h. From 0 to 24 h, 1 of 10, 6 of 10 and 7 of 10 whole embryos developed when stored in MEM 5 C, MEM 24 C and Ham's F10 24 C, respectively. Embryo quality was better at 24 h (P less than .05) for embryos stored in Ham's F10 at 24 C compared with MEM at 5 C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Function of berberine on porcine in vitro fertilization embryo development and differential expression analysis of microRNAs

The effect of berberine (Ber) on in vitro fertilization (IVF) embryo development in pigs and the associated differential expression of microRNAs (miRNAs) in the embryo were investigated. NCSU‐23 embryonic culture medium was used for a control group, while NCSU‐23 embryonic culture medium added with Ber was used for a Ber group. The embryo development rates in these groups were determined, and the zygotes, 4‐ and 8‐cell embryos, and blastocysts were collected for cDNA microarray analysis. The development rates of 2‐, 4‐, 8‐cell embryos and blastocysts were significantly higher in the Ber group than those in the control group (p < 0.01). The differentially expressed miRNAs in the 8‐cell versus the 4‐cell stage in control group as well as in the 8‐cell Ber group versus the 8‐cell control group overlapped, and it was found that nine miRNAs were commonly upregulated and two of them were downregulated, while there was no overlap among the other groups. The target genes of Ber‐regulated miRNAs at the 8‐cell stage were mainly associated with the molecular pathway of nucleic acid and protein synthesis. These findings suggest that Ber may regulate the expression of miRNAs at the 8‐cell stage, which is beneficial to provide material reserves for the maternal to zygote transition of porcine embryos, thereby increasing the porcine IVF embryo development rate.