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1.
This study evaluated the effects of bovine serum albumin (BSA; 4 mg/ml) and estrous cow serum (ECS; 10%) in North Carolina State University (NCSU) 23 medium on the development of in vitro-matured and in vitro-fertilized porcine oocytes. Early cleavage rate was significantly (P < 0.05) higher in NCSU/ECS (71.3 +/- 14.7%) vs. NCSU/BSA (60.6 +/- 4.7%). Cleavage beyond the four-cell stage was not different between the two culture media (43.5 +/- 9.5% and 41.4 +/- 17.7%, respectively). The proportion of development to blastocysts was--with borderline significance (P = 0.05)--higher in NCSU/BSA (28.0 +/- 4.4%) than in NCSU/ECS (20.4 +/- 7.3%). Blastocysts produced in NCSU/BSA had significantly (P < 0.001) higher cell numbers than those cultured in NCSU/ECS (29.5 +/- 20.1 vs. 16.9 +/- 10.8). The ultrastructure of in vitro-produced blastocysts from both culture systems was compared vs. in vivo-derived blastocysts. The latter showed a clear differentiation between trophectoderm (TE) and inner cell mass (ICM) cells. The TE cells were anchored to other TE cells or ICM cells by long, well-developed junctional complexes. The apical membrane of trophoblast cells was covered with numerous microvilli. Mitochondria were abundant, round to elongated in shape, and showed clear transverse cristae. The ultrastructure of blastocysts cultured in NCSU/BSA mimicked that of in vivo-derived embryos closely. In contrast, blastocysts from the NCSU/ECS culture system displayed an irregular ultrastructure with reduced numbers of organelles and numerous cytoplasmic inclusions, such as lipid-yolk-vacuoles and vacuoles with lipid content. In some sections of these embryos, cellular debris was detected in cytoplasm. The shape of mitochondria was more ovoid and cristae were not visible. In summary, our results demonstrate a beneficial influence of ECS in the culture medium on initial cleavage of in vitro-produced porcine embryos. Clearly negative effects of ECS in the subsequent culture period are associated with marked ultrastructural changes of embryonic cells.  相似文献   

2.
Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM) or in mR1ECM supplemented with BSA (4 mg/ml; mR1ECM-BSA) or fetal bovine serum (FBS; 10%, v:v; mR1ECM-FBS) instead of polyvinylalcohol. There was no difference in percentages of embryos that developed to the 2-cell to blastocyst stages between mR1ECM and mR1ECM-BSA, but in mR1ECM-FBS, no development beyond the 2-cell stage was observed. When embryos were transferred to mR1ECM-FBS from mR1ECM after 24 to 64 h of culture, development of embryos to and beyond the 4-cell stage was inhibited. However, when transferred after 80 h of culture, more embryos developed to blastocysts and hatching or hatched blastocysts than in embryos cultured in mR1ECM. When 8-cell embryos and early morulae obtained after 72 and 80 h of culture in mR1ECM, respectively, were cultured in mR1ECM-FBS, a higher proportion of early morulae developed to the blastocyst stage than did 8-cell embryos. When morulae obtained after culture in mR1ECM or mR1ECM-BSA were transferred to recipient females, there was no difference in proportions of fetuses obtained. However, a higher proportion of blastocysts cultured in mR1ECM-FBS developed to fetuses compared with those obtained in mR1ECM. These results indicate that BSA has neither deleterious nor beneficial effects on development of rat 1-cell embryos. In contrast, FBS has deleterious effects on early cleavage of embryos but it promotes more rapid development of morulae to blastocysts, resulting in better quality blastocysts.  相似文献   

3.
This study examined the effects of different vitrification medium compositions and exposure times (2, 4 and 6min) on the post-thaw development of buffalo embryos produced in vitro (IVP). The compositions were (1) 40% ethylene glycol (EG); (2) 25% glycerol (G)+25% EG, and (3) 25% EG+25% dimethylsulfoxide (DMSO). The base medium was 25mM Hepes-buffered TCM-199+10% steer serum +50microg/mL gentamycin. The IVP embryos were cryopreserved by a two-step vitrification method at 24 degrees C. After warming, the embryos were cultured in vitro for 72h. The vitrification of morulae and blastocysts in 25% EG+25% DMSO with an exposure time of 2 and 4min, respectively, resulted in a better hatching rate than other combinations. The hatching rate of morulae vitrified in 25% EG+25% G, 25% EG+25% DMSO, and blastocysts vitrified in 40% EG, 25% EG+25% DMSO were negatively correlated with exposure time. However, the hatching rate of blastocysts vitrified in 25% EG+25% G was positively correlated with exposure time. The study demonstrated that the post-thaw in vitro development of IVP buffalo embryos was affected by the vitrification medium composition and exposure time.  相似文献   

4.
在动物卵母细胞体外成熟及胚胎体外培养体系中添加一定浓度的发情牛血清可能提高卵母细胞的成熟率及胚胎的发育率。本研究以屠宰场卵巢来源的绵羊卵母细胞为试验材料,探讨了发情牛血清对绵羊卵母细胞体外成熟及孤雌胚发育的影响。结果表明,成熟液中添加10%第1天的发情牛血清能显著提高绵羊卵母细胞的体外成熟率及孤雌胚卵裂率(P＜0.05);孤雌胚体外培养72 h后,向培养液中添加10% 第7天的发情牛血清能显著提高绵羊孤雌胚的桑囊胚率(P＜0.05)。结果表明,发情牛血清能够促进绵羊卵母细胞的体外成熟率及孤雌胚发育率。  相似文献   

5.
The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus × Bos taurus (Bi × Bt). Oocytes were aspirated post‐mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM‐199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi‐fine toluidine blue‐stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro‐cultured embryos.  相似文献   

6.
The efficacy of matching embryos with recipients on the basis of embryo stage and grade and donor-recipient oestrous synchrony was investigated using the records of 13,663 embryos that were collected and transferred at a commercial embryo transfer centre. The selection of early blastocysts for exact oestrous synchrony cows was effective and resulted in the highest pregnancy rates. Selection of early morulae was effective for recipients in oestrus after the donor but not when transferred into exact and negative recipients. The matching of late morulae with recipients in oestrus after the donor was not effective and had no influence on pregnancy rates. The selection of late, hatched and collapsed blastocysts for transfer into recipients in oestrus before the donor was ineffective and pregnancy rates were higher in exact and +12 hour recipients. Pregnancy rates declined 23.6 per cent in quality grades 1 to 4 whereas the range between stages was 13.3 per cent. Higher quality embryos of all stages gave the highest pregnancy rates. Examination of pregnancy rates of grades within stages suggested that the more developed the embryo the more difficult it is to grade. The difference in pregnancy rates between exact and -24 (6.9 per cent) and +24 (4.8 per cent) hour recipients was small and declined a further 4.7 per cent and 9.8 per cent in -36 and +36 hour recipients. Grade 3 and 4 embryos tolerated asynchrony better than grade 1 and 2, and early morulae tolerated asynchrony better than the other stages. It was concluded that the matching of certain embryo stages with the donor-recipient oestrous synchrony is advantageous but not always possible.  相似文献   

7.
用不同冷冻载体(玻璃管、塑料管和0.25 mL细管)及不同冷冻方法(程序化冷冻和玻璃化冷冻)对小鼠3.5 d~4 d桑椹胚和囊胚进行冷冻保存,并与不做任何冷冻保存处理直接培养进行对比。结果表明,使用玻璃管、塑料管和0.25 mL细管作为胚胎的承载材料进行玻璃化冷冻,效果差异不显著;采用程序化冷冻与OPS玻璃化冷冻法,对小鼠胚胎进行冷冻保存可以取得较好的结果。从而得出,用不同材质的冷冻载体进行玻璃化冷冻,可以获得与程序化冷冻相同的良好效果。  相似文献   

8.
The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.  相似文献   

9.
Maturation of oocytes and the subsequent outcome of the in vitro production (IVP) are affected by the composition of in vitro maturation (IVM) medium. To determine the use of serum interfering with effects of single molecules, we aimed at developing simplified IVM medium. The experimental IVM media were: (1) M199-medium supplemented with hormones and serum (control), (2) as 1 but serum was substituted with fatty acid-free serum albumin (FAFBSA) and (3) M199-medium without hormonal and serum supplementation (M199). The quality of embryos was assessed on day 7 by morphology and cryotolerance, as well as by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and differential staining. Results showed that the nuclear maturation was suppressed in M199 group alone. Embryo cleavage and development rates, and the proportion of quality 1 blastocysts were lower in the FAFBSA and M199 groups compared to the control. Differences in the cell allocation of fresh embryos were observed at the blastocyst stage, but not at the expanded blastocyst stage. The control group blastocysts had larger number of cells allocated to the inner cell mass (ICM), and the FAFBSA group blastocysts larger apoptotic cell proportion compared to the blastocysts derived from other groups. After cryopreservation, the reduction of ICM proportion and increase of apoptotic cell proportion of embryos were equal between the experimental groups. In conclusion, exclusion of serum from the IVM media reduces embryo development and may cause perturbations in blastocyst development. Differences in the cell allocation of blastocysts between IVM media may appear only when the developmental stages are taken into account.  相似文献   

10.
The present experiment was designed to identify possible effects of sera and steroid hormones added to a co-culture with bovine oviduct epithelial cells on embryonic development in vitro. Bovine oocytes were matured in vitro for 24 h and then fertilized in vitro using swim-up and heparin-treated, frozen-thawed spermatozoa. At 18 and 20 h after insemination, oocytes were cultured for 3 or 7 d in a co-culture system with bovine oviduct epithelial cells containing either fetal calf serum (FCS) or estrous cow serum (ECS) and one of six hormonal additions (none, 1 or 10 micrograms/ml estradiol [E]; 1 microgram/ml progesterone [P]; 1 microgram/ml E + P; and 10 micrograms/ml E + P). A total of 2,666 oocytes were cultured for 3 d and examined for cleavage. Of those, 2,280 oocytes were cultured up to 7 d for development to the late morula or blastocyst stage. Greatest cleavage rates for 2- to 8-cell and 8-cell stages were observed in FCS (71 and 24%) and ECS (66 and 23%) without steroid addition. For development into blastocysts, no serum effect was observed. Greatest rates for development into blastocysts were observed in FCS (14%) and ECS (16%) without steroid addition. These results indicate that addition of E and P at the doses and combinations tested did not enhance developmental capacity of in vitro fertilized bovine oocytes. Compared with FCS, ECS tended to increase cleavage rates and development into blastocysts.  相似文献   

11.
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12.
Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vitro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.  相似文献   

13.
The aim of this study was to evaluate if blastocysts arising from in vitro culture of Grade 3 bovine morulae produced in vivo can promote acceptable pregnancy rates when transferred into recipients. Embryos of different stages and qualities were recovered from superovulated Bos taurus and B. indicus donors. Grade 3 morulae were cultured in either Holding Plus® or TCM‐199 (supplemented with 10% bovine fetal serum) media for 24 h at 38.5°C. After this culture period, the resulting blastocysts were morphologically classified (Grades 1, 2 and 3) and transferred into recipients previously synchronized with the donors. Non‐cultured Grades 1 and 3 morulae were used as control. Pregnancy diagnosis was carried out 60 days after embryo transfer and the data were analysed by logistic regression, considering variables, such as embryo quality (Grade), donor breed, culture medium, donor‐recipient synchrony and seasonality. Embryo quality was the only variable, showing significant effect on the pregnancy rate. Pregnancy rates for non‐cultured Grade 1 and 3 morulae, and blastocysts arising from cultured Grade 3 morulae were 58.1% (n = 31), 17.1% (n = 35) and 51.1% (n = 47), respectively (p < 0.05). There were no statistical differences between non‐cultured Grade 1 morulae and cultured blastocysts. Pregnancy rates for Grades 1 (65.0%) and 2 (60.0%) were higher than Grade 3 (29.4%) cultured blastocysts (p < 0.05). It was concluded that short‐term in vitro culture is a very convenient method of identifying morphologically low quality morulae with higher chances of continuing development after the transfer into recipients.  相似文献   

14.
In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n=11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n=16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p<0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid.  相似文献   

15.
转基因兔胚胎玻璃化冷冻保存的研究   总被引：4，自引：0，他引：4  
在２５℃条件下,将兔体外受精精子载体转基因兔桑椹胚置于含有４０％乙二醇、１８％Ｆｉｃｏｌ、０．３ｍｏｌ蔗糖的ｍＰＢＳ溶液（ＥＦＳ４０）中平衡２分钟,然后直接投入液氮,成功地进行了玻璃化冷冻保存。解冻后桑椹胚发育至囊胚和孵化囊胚的比例分别为６５．８１％和３９．２４％,与未经冷冻的鲜胚发育比例（７１．０５％和４３．４２％）相比,没有明显的差异。７８枚经玻璃化冷冻和解冻的桑椹胚移植给５只受体,其中２只妊娠,共产下８只活仔兔。  相似文献   

16.
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.  相似文献   

17.
The impact of TCM‐199 supplemented with different proteins and heterologous hormones on the in vitro maturation (IVM) rate of bitch oocytes was evaluated by nuclear staining under fluorescence microscopy. Oocytes were recovered by slicing of ovaries from bitches presented at various stages of oestrous cycle to ovariohysterectomy. The basic culture medium was TCM‐199 supplemented with 25 mM Hepes/l, with 10% heat‐inactivated oestrous cow serum (ECS), 50 μg/ml gentamicin, 2.2 mg/ml sodium bicarbonate and 22‐μg/ml pyruvic acid, 1.0‐μg/ml oestradiol (E 8875; Sigma), 0.5‐μg/ml follicle‐stimulating hormone (FSH) (Folltropin‐V; Vetrepharm Inc., Ontario, Canada) and 0.03 IU/ml human gonadotropin (hCG) (Profasi HP; Serono, Aubonne, Switzerland). Oocytes were distributed randomly between basic culture medium (control) and the corresponding experimental treatment. Hormone treatments were: oocytes cultured in; (1) medium without FSH, (2) control medium supplemented with 20 μg/ml oestradiol, or (3) medium supplemented with 1 μg/ml human somatotropin (hST; Humatrope, Lilly, Saint Cloud, France). The second experiment consisted of oocytes cultured in medium supplemented with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Gibco Grand Island, NY, USA) instead of ECS, or oocytes cultured in medium with 10% inactivated oestrous bitch serum (EBS) instead of ECS. Oocytes were cultured in 100 μl droplets (up to 25 oocytes per drop) under mineral oil at 37°C in a 100% humidified atmosphere containing 5% CO2 in air. After 72 h of IVM, the highest rates (p < 0.05) of meiotic resumption were achieved with the 0.4% BSA supplementation. A positive influence on the metaphase II (MII) acquisition rate was observed with hST supplement. Oocytes cultured with 10% EBS supplementation did not develop to the MII stage. The results in this study show that the protein and hormone supplements to TCM‐199 culture medium tested did not promote the final steps of IVM of bitch oocytes.  相似文献   

18.
The objective of this study was to compare the embryo production and quality carried out entirely in vitro or partly in vitro combined with short‐ vs long‐term in vivo culture using the homologous cattle oviduct. The IVM oocytes were in vitro fertilized and cultured for 7 and 8 days (IVP‐Group), or after IVF and 2–3 days of IVC, 4–8 cell stage embryos were endoscopically transferred into oviducts of synchronized heifers (In Vivo‐Group) or IVM oocytes were co‐incubated with spermatozoa for 3–4 h and transferred into the oviducts of synchronized heifers (GIFT‐Group). Embryos of the In Vivo‐Group and the GIFT‐Group were recovered on day 7 from the oviducts and uterine horns. Embryos of all groups were either cryopreserved at day 7 (day 7 blastocysts) or cultured in vitro in CR1aa‐medium supplemented with 5% ECS for further 24 h and cryopreserved (day 8 blastocysts). The total blastocyst yield found in the in vivo cultured groups was similar to the results of the IVP‐Group. But the appearance of blastocysts was dependent on the duration of in vivo culture. The more time the embryos spent in the in vivo environment, the more blastocysts appeared at day 8. The quality of produced blastocysts assessed by cryo‐survival was also correlated to the culture conditions; the in vivo cultured embryos showed higher cryo‐tolerance. However, the duration of in vivo culture crucially influenced the cryo‐tolerance of produced blastocysts. It is concluded that tubal access is a promising tool to provide a further basis for studying embryo sensitivity to environmental changes.  相似文献   

19.
The present study was conducted to evaluate the possibility of using in vitro‐produced parthenogenetic (PA) embryos for co‐transfer with morulae that had been collected in vivo and cryopreserved. The proportion of PA blastocysts (20.5%) was higher than that of their in vitro fertilization (IVF) counterparts (16.6%). Although there were no differences in morphology or diameter between the two groups, the number of cells in early PA blastocysts after in vitro culture for 6 days was lower than for IVF blastocysts (25.7 and 30.4 cells, respectively), and the number in recovered PA blastocysts was also smaller than that in recovered IVF blastocysts (37.4 and 50.2 cells, respectively). When 10 morulae warmed after vitrification were co‐transferred with 10 PA blastocysts (total 20 embryos) to the uterus of five recipients, the rates of pregnancy and farrowing did not differ, but the average period until spontaneous abortion tended to be longer relative to the control (when 20 morulae were transferred). These data suggest that in vitro‐produced PA embryos offer the possibility of assisted pregnancy for cryopreserved embryos; further experiments will be needed to confirm the beneficial effect of this approach on piglet production.  相似文献   

20.
My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.  相似文献   

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