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This study was conducted to examine the potential for implantation and sustainable fetal development of mouse embryos cultured from the pronuclear to blastocyst stage. Pronuclear embryos from ICR mice (Harlan Sprague‐Dawley) were cultured in Sydney IVF sequential media (Cook) to the blastocyst stage in medium only or co‐cultured with autologous cumulus cells. We also experimented with co‐culture in 100 µL drops. Drop co‐culture produced blastocyst formation rates with a mean of 47.0%, which was significantly higher (P < 0.05) compared to embryos cultured in identical culture conditions except without cumulus cells at 27.3%. Blastocysts obtained in vitro in Cook medium only and co‐cultured in Cook medium with cumulus cells were transferred to pseudopregnant females of ICR strain. The day of blastocyst transfer into surrogate females was designated as post‐transfer of blastocyst day 1 (PT 1). The implantation and fetal development was compared to embryo transfer of in vivo derived blastocysts, which served as controls. There were no statistical differences for implantation and fetal development rates for blastocysts cultured in vitro in either Cook medium only or co‐culture in Cook medium with cumulus cells compared to in vivo‐derived blastocysts. The advantage of the co‐culture system is in generating more blastocysts available for transfer. 相似文献
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Utilization of porcine in vitro‐produced parthenogenetic embryos for co‐transfer with vitrified and warmed embryos 
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下载免费PDF全文 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
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In vitro development of OPU‐derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen‐thawed embryos after transfer 下载免费PDF全文
下载免费PDF全文 Tomohiro Isobe Yoshihisa Ikebata Lanh Thi Kim Do Fuminori Tanihara Masayasu Taniguchi Takeshige Otoi 《Animal Science Journal》2015,86(7):661-665
The optimization of single‐embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick‐up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen‐thawed embryos after a direct transfer. When two‐cell‐stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen‐thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen‐thawed embryos after transfer to recipients. 相似文献
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Kato M Ishikawa A Hochi S Hirabayashi M 《The Journal of reproduction and development》2004,50(2):191-195
The present study was conducted to examine the developmental potential to offspring of rat embryos cultured from 1-cell to morula/blastocyst stage. Pronuclear zygotes from Wistar x Wistar or (SD x DA) x Wistar strains were cultured in modified rat 1-cell embryo culture medium (mR1ECM) for 96 h in 5% CO(2) in air at 37 C. The proportion of the 3-way cross hybrid zygotes developing into morula/blastocyst stage (74%) was higher than that of the Wistar zygotes (66%). Day-5 morulae/blastocysts developed in vitro were transferred into Day-3 or -4 pseudopregnant recipients of Wistar or SD x DA strain. The transfer of cultured embryos resulted in the birth of offspring at 13-59%, while that of non-cultured control blastocysts showed birth rates of 35-65%. The best offspring rate of cultured embryos (59%) was obtained when the hybrid 1-cell zygotes were cultured in mR1ECM medium and transferred into the 2-days earlier uteri of SD x DA recipients. These results suggest that genetic background of recipients as well as donors is a possible factor affecting full-term development of rat morulae/blastocysts derived from 1-cell stage zygotes cultured in vitro. 相似文献
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Matching of embryo stages and grades with recipient oestrous synchrony in bovine embryo transfer 总被引：1，自引：0，他引：1
L E Donaldson 《The Veterinary record》1985,117(19):489-491
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
6.
Successful Vitrification of In vivo Embryos Collected from Superovulated Japanese Black Cattle (Wagyu) 下载免费PDF全文
下载免费PDF全文 L An PP Ling X Zhu Y Liu F Zhang X Ma B Xu Y Wang Z Du L Yang F Xue A Bella GA Presicce F Du 《Reproduction in domestic animals》2016,51(2):255-261
The aim of this study was to determine whether vitrification is an effective method when used for Japanese Black Cattle (Wagyu) in vivo‐derived embryos, collected following a superovulation treatment and embryo transfer (MOET) programme. In vivo‐derived morula and blastocysts collected on day 7 after artificial insemination, were vitrified using a modified droplet vitrification (MDV) procedure and subsequently warmed for transfer (ET) into synchronized recipients. Fresh embryos, and embryos cryopreserved using a standardized slow freezing procedure (direct thaw/direct transfer, DT) served as ET controls. Two different follicle‐stimulating hormone (FSH) sources, Folltropin® Canada (FSH BAH, 24 donors) and a brand prepared by the Chinese Academy of Science (FSH CAS, 16 donors), were compared in a series of superovulation outcomes following well‐established FSH administration protocols. Following data analysis, the total number of ovulations recorded at the time of embryo flushing (10.5 vs 8.5; p = 0.28) and the total number of transferable embryos (6.2 vs 5.1; p = 0.52) were similar between the two FSH sources. ET for MDV (39.7%, n = 78), DT (35.2%, n = 71) and fresh controls (47.1%, n = 34) resulted in similar pregnancy rates (p > 0.05). When MDV was used, a higher pregnancy rate (42.6%) resulted from the transfer of vitrified morulae, when compared to the DT counterparts (24.3%), (p = 0.05). Transfer of vitrified morulae resulted also in higher pregnancy rate, when compared to the transfer of vitrified blastocysts (42.6% vs. 29.4%; p < 0.05). Transfer of DT blastocysts resulted in higher pregnancy rate than morulae, similarly cryopreserved (47.1% vs. 24.3%, p < 0.05). In conclusion, MDV is an effective alternative methodology for cryopreservation of in vivo‐derived embryos. This study gives also indication that, compared to vitrified blastocysts, MDV of morula stage embryos results in higher pregnancy rates following warming and transfer into synchronized recipients. 相似文献
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V Havlicek A Kuzmany S Cseh G Brem U Besenfelder 《Reproduction in domestic animals》2010,45(5):832-837
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
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山羊胚胎分割及同卵双生试验 总被引：10，自引：1，他引：10
选择山羊晚期桑椹胚、囊胚、孵出囊胚和孵出增大胚泡,用简化分割法二分。将19对裸半胚移植于18只受体羊,结果有12只妊娠,其中两只胚胎消失,两只流产,其余8只足月分娩,共产半胚羔11只。晚期桑椹胚、囊胚、孵出囊胚和孵出增大胚泡各组的半胚发育为羔羊的发育率分别为12.5％(1/8)、20％(2/10)、25％(3/12)和62.5％(5/8)。前三组均未获得同卵双生羔羊。在第四组,将4对裸半胚移植于4只受体,有3只妊娠,足月分娩半胚羔5只,其中两对为同卵双生。本研究证明,对称分割山羊孵出增大胚泡,不仅其半胚在体内仍可继续发育形成正常胎儿,而且不装透明带移植其裸半胚,仍能获得较高的同卵双生率。山羊孵出增大胚泡更适宜用简化分割法分割。 相似文献
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Effects of Addition of Tissue‐Type Plasminogen Activator in In Vitro Fertilization Medium on Bovine Embryo Development and Quality 下载免费PDF全文
下载免费PDF全文 F Krania E Dovolou CA Rekkas EK Theodosiadou I Pappas GS Amiridis 《Reproduction in domestic animals》2015,50(1):112-120
Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue‐type plasminogen activator (t‐PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus‐oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t‐PA and/or its inhibitor epsilon‐aminocaproic acid (control, t‐PA, t‐PA+ε‐ACA, ε‐ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t‐PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε‐ACA. PAA in the treated group was significantly reduced by ε‐ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t‐PA‐modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t‐PA. In conclusion, it appears that excessive t‐PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena. 相似文献
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Manabu OZAWA Yukiko YAMASAKI Miho HIRABAYASHI Yukio KANAI 《Animal Science Journal》2003,74(3):181-185
Mammalian preimplantation embryos are susceptible to heat stress. This present study examined how maternal heat stress affects the development of mouse zygotes in vivo and in vitro. In Experiment 1, zygotes collected from female mice that were heat‐stressed for 12 h on day 1 of pregnancy were cultured in vitro. Maternally heat‐stressed zygotes developed normally to the two‐cell stage, but the majority of embryos failed to develop into morulae or blastocysts. In Experiment 2, pregnant mice were heat‐stressed on day 1 or from day 1 to day 3 of pregnancy. The number of living fetuses on day 14 of pregnancy was lower in heat‐stressed mice than in non‐stressed mice, but the difference was significant only in successively heat‐stressed mice. These results demonstrate that maternally heat‐stressed zygotes have reduced in vitro viability, but this phenomenon does not necessarily lead to embryo loss in the maternal environment. 相似文献
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Yijin Pei 《Veterinary research communications》2010,34(1):11-18
This study aimed to investigate the effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos
in vitro. Zygotes were collected from superovulated New Zealand rabbits 19 h after injection of hCG and immediately mating and cultured
in TCM-199 plus fatty-acid free BSA with different concentrations of NGF. Zygotes not treated with NGF served as control.
At 24 h, 48 h, 72 h and 96 h of the culture, the numbers of the early cleavage stage, morulae, blastocysts and hatching blastocysts
were determined. The intrazonal diameter of the blastocyst and the total cell numbers per blastocyst were measured after 96 h
of culture. The results showed: (1) NGF at 100 ng/mL and 1000 ng/mL could improve the numbers of the hatching blastocysts
which developed compared to the control treatment (p < 0.05); (2) All concentrations of NGF increased the total cell numbers in the blastocysts compared to the control treatment
(p < 0.05); (3) NGF had no significant effect on the blastocyst intrazonal diameter of the blastocysts at 96 h of culture (p = 0.493); (4) The proportion in the early cleavage stage at 24 h of culture (p = 0.635), of morulae at 48 h of culture (p = 0.812) and of blastocysts at 72 h of culture (p = 0.812) in all treatments were not significantly different. 相似文献
16.
Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super‐cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non‐vitrified control group, (ii) vitrified in normal (?196°C) liquid nitrogen (LN2) and (iii) vitrified in super‐cooled LN2 (≤?200°C). Open‐pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro‐matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN2 state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super‐cooled LN2, resulted in viable blastocysts and live calves following in vitro embryo production. 相似文献
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B Trigal M Muoz E Gmez JN Caamao D Martin S Carrocera R Casais C Diez 《Reproduction in domestic animals》2013,48(2):200-206
This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp‐70) were also examined. Day 7 and 8 bovine in vitro‐produced blastocysts were submitted to an HHP treatment (60 MPa, at 32°C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post‐warming) and hatching (48 h post‐warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP‐treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32°C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP‐treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp‐70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post‐warming. 相似文献
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Trasorras V Giuliano S Chaves G Neild D Agüero A Carretero M Pinto M Baca Castex C Alonso A Rodríguez D Morrell JM Miragaya M 《Reproduction in domestic animals》2012,47(4):562-567
The aim of this study was to carry out in vitro fertilization using spermatozoa selected with Androcoll‐E? and to evaluate the efficiency of the culture medium DMEM‐F12 for in vitro embryo development in the llama. Twelve adult females from 18 superstimulated (67%) were used as oocyte donors. They were superstimulated with 1500 IU of eCG and after 5 days, received a single dose of buserelin. Twenty hours post‐injection, follicular aspiration was conducted by flank laparotomy. Semen collections were performed under general anesthesia by electroejaculation of the male. The ejaculates were processed with a solution of collagenase (0.1%) and an Androcoll‐E? column was used to improve the sample. Sixty nine COCs were recovered from 79 aspirated follicles (87% recovery). Only expanded COCs were used (n = 67); they were randomly placed in groups of 1–5 in Fertil‐TALP and the sperm suspension (20 × 106 live spermatozoa/ml) was added to each fertilization microdroplet. After 24 h, they were randomly placed in one of two culture media: SOF (n = 34) or DMEM‐F12 (n = 33) and incubated for 6 days in humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38°C. The blastocyst rate was 20% (7/34) in SOF medium (3 hatched, 2 expanded and 2 early blastocysts) and 15% (5/33) in DMEM medium (all expanded blastocysts). In conclusion, using Androcoll‐E? it is possible to select good quality spermatozoa from llama ejaculates for in vitro fertilization and to produce blastocysts in DMEM‐F12 medium. This is also the first time that hatched llama blastocysts have been produced after culture in a defined medium such as SOFaa. 相似文献
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EA Ordoñez‐Leon H Merchant A Medrano M Kjelland S Romo 《Reproduction in domestic animals》2014,49(2):306-314
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