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1.
This study was performed to produce transgenic Korean native goat (Capra hircus) by laparoscopic embryo transfer (ET) to overcome the limitations of ET performed by laparotomy. Transgenic embryos were produced by DNA pronuclear microinjection of in vivo zygotes. The recipient goats were synchronized for estrus by using an introvaginal progesterone devices as a controlled internal drug-releasing insert (CIDR) for 13 days and injection of 400 IU PMSG 48 h before removal of the insert. Embryos were transferred on day 3 and 4 after removal of the insert. Recipient goats were deprived of feed for 48 h, then suspended in a laparotomy cradle at an angle of 45°. After obtaining a sufficient pneumoperitoneum, the laparoscope and forceps were inserted abdominally through 5 mm trocar sleeves. Examination of the ovaries and uterus was performed and then 213 embryos were transferred into the oviducts via the infundibula of 76 recipient goats. To compare pregnancy rates, ET was also performed by laparotomy in 82 recipient goats. The pregnancies in the recipient goats were diagnosed by ultrasound on day 30 after embryo transfer. The pregnancy rate with laparoscopic ET was significantly higher than with ET performed by laparotomy (46.1% vs. 28.6%, p < 0.05). In addition, the pregnancy rates were compared between ovulated and non-ovulated ovaries of the recipient goats in the laparoscopic ET group. No significant difference was observed between the pregnancy rates of ovulated and non-ovulated ovaries (41.3% vs. 33.3%, p < 0.05) suggesting that ET may also be possible in non-ovulated recipients through artificial rupture of Graafian follicles. These results suggest that laparoscopic ET is a highly efficient method for the transfer of goat embryos.  相似文献   

2.
试验通过使用人工授精后绵羊作为受体进行胚胎移植,以寻找一种更加有效的方法提高绵羊胚胎移植的经济效益。试验中使用FSH对10只无角道赛特绵羊进行超数排卵处理,同时对60只受体小尾寒羊进行同期发情。供体羊在发情配种后4.5～5.0d从子宫角收集胚胎。同时,将胚胎移植到同期发情并进行人工授精的受体羊子宫内。总共有57枚可用胚移植给44只受体小尾寒羊,32只怀孕到分娩,共产下羔羊51只(无角道赛特羔28只,道赛特与小尾寒羊杂种羔23只)。此外,经人工授精但未进行手术移植的7只小尾寒羊产下15只杂种羔羊。移胚植受体妊娠率72.7%(32/44),移胚受体繁殖率118%(51/44),受体利用率88.3%(53/60)。移胚受体总妊娠率和受体利用率均显著高于常规ET组(P<0.01)。与常规胚胎移植相比,受体羊人工授精后移植胚胎不仅提高了无角道赛特母羊的繁殖率,而且提高了受体羊的利用率。  相似文献   

3.
In the present study, we investigated the spent culture media of in vitro produced (IVP) bovine embryos which did (group Pregnant) or did not (group Non-pregnant) establish pregnancy after transfer. For that purpose, IVP embryos on D5 were transferred to individual droplets for the last 48 h of culture. Embryos at the blastocyst stage were then transferred to synchronized recipients, while respective culture media drops were collected and evaluated individually. The list of metabolites present in spent culture media was obtained by electrospray ionization mass spectrometry (ESI-MS) and analysed with Metaboanalyst® to characterize the metabolic profile of each group. The spectrometric analysis showed that pathways related to lipid metabolism, particularly fatty acids degradation via beta-oxidation, were more present in the Pregnant group whereas no significant pathway was identified in the group Non-pregnant. By using this method, we were able to identify a metabolic signature in culture media that allows for a better comprehension of preferential metabolic routes taken by the most viable embryos. These findings offer great insights into the biochemistry of embryo development and reveal a potential target for the development of better-quality IVP systems, as well as tools to identify bovine embryos with greater chances to establish and maintain pregnancy.  相似文献   

4.
Reasons for performing the study: During embryo transfer (ET) the equine embryo can tolerate a wide degree of negative asynchrony but positive asynchrony of >2 days usually results in embryonic death. There is still confusion over whether this is due to the inability of the embryo to induce luteostasis or to an inappropriate uterine environment. Objectives: To assess embryo survival and development in an advanced uterine environment. Hypothesis: Embryo–uterine asynchrony, not the embryo's inability to induce luteostasis, is responsible for embryonic death in recipient mares with a >2 days chronologically advanced uterus. Methods: Experiment 1: Thirteen Day 7 embryos were transferred to the uteri of recipient mares with luteal prolongation, occasioned by manual crushing of their own conceptus, such that donor–recipient asynchrony was between +13 and +49 days. Experiment 2: Day 7 embryos were transferred to recipient mares carrying their own conceptus at Days 18 (n = 2), 15 (n = 2), 14 (n = 4), 12 (n = 4) or 11 (n = 4) of gestation. In addition, Day 8 embryos were transferred to 4 pregnant recipient mares on Day 11 of gestation. Results: No pregnancies resulted following transfer of Day 7 embryos to recipients in prolonged dioestrus with asynchronies between +13 and +49 days. However, the use of early pregnant mares as recipients resulted in 5/20 (25%) twin pregnancies, 4 of which came from the transfer of a Day 8 embryo to a Day 11 recipient. All transferred embryos showed retarded growth, with death occurring in 4/5 (80%). Conclusions and potential relevance: The results emphasise the importance of an appropriate uterine environment for embryo growth and the inability of equine embryos to survive transfer to a uterus >2 days advanced even when luteostasis is achieved. It is possible that in normal, non‐ET equine pregnancy, embryo–uterine asynchrony may account for some cases of embryonic death.  相似文献   

5.
Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non‐surgical ET and short‐ and long‐term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non‐surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short‐term use in pig production.  相似文献   

6.
This study was performed to evaluate reproductive performance after non‐surgical embryo transfer (Ns‐ET) of 10–15 porcine expanded blastocysts (ExBs) that had been vitrified and warmed (V/W) using the micro volume air cooling (MVAC) method. The effect of asynchrony between the donor and recipient estrous cycle was investigated. Ns‐ET was conducted in recipients whose estrous cycle was asynchronous to that of donors by a delay of 2, 1, or 0 days. In the 2‐day and 1‐day groups, the similar farrowing rates (27.3% and 25.0%) and survival rates to term (13.9% and 15.7%) were obtained after Ns‐ET of V/W ExBs. None of the recipients in 0‐day group farrowed. Artificial insemination (AI) prior to Ns‐ET was then evaluated. Ten–15 V/W ExBs were transferred non‐surgically to 12 recipients whose estrous cycles were asynchronous to that of donors by a 2‐day delay. All of the recipients produced piglets, and all (100.0%) delivered piglets were derived from the transferred V/W ExBs. The survival rate of V/W ExBs to term was 25.2%. These results demonstrate that Ns‐ET of V/W ExBs using MVAC can facilitate piglet production, even if 10–15 embryos are transferred. Moreover, piglets were obtained stably when AI was performed prior to Ns‐ET.  相似文献   

7.
试验旨在研究添加羧乙基锗倍半氧化物（carboxyethylgermanium sesquioxide，Ge-132）对牛孤雌激活后胚胎发育率、胚胎细胞数、早期胚胎内活性氧（reactive oxygen species，ROS）水平及胚胎内相关凋亡基因的影响。在牛早期胚胎体外培养基中添加不同浓度的Ge-132（0、10、100和200 μg/mL），观察其对牛体外孤雌激活胚胎发育的影响；应用Hoechst对孤雌激活后第8天胚胎进行染色后制作装片，在显微镜下对细胞进行计数；用DCFH-DA染色检测早期胚胎内ROS水平，并用Image J测量荧光强度后对数据进行统计分析，用RT-PCR对胚胎内细胞凋亡相关基因（Caspase-3、Bax、Bcl-xl和Survivin）进行分析。结果显示，10 μg/mL Ge-132处理组胚胎率与对照组间无显著差异（P>0.05），但可降低1细胞期的ROS水平；10 μg/mL Ge-132处理组较对照组早期胚胎细胞数显著增加（P<0.05）；通过检测细胞凋亡相关基因mRNA转录水平发现，与对照组相比，10 μg/mL Ge-132处理组胚胎促凋亡基因Caspase-3表达水平显著降低（P<0.05），抑制细胞凋亡基因Survivin表达水平显著升高（P<0.05）。结果表明，在早期胚胎培养基中添加10 μg/mL Ge-132可降低细胞内ROS水平，减少胚胎中氧化应激诱导的细胞凋亡，从而提高孤雌激活后牛胚胎的发育潜能。  相似文献   

8.
This study aimed to compare the efficiency of non‐surgical embryo transfer (ET) using a newly developed catheter, which enables transferring embryos into a proximal site of the uterus (mostly uterine body), and surgical ET of vitrified porcine embryos. In Experiment 1, the catheter was inserted into 12 gilts, with each half of the group allocated to skilled or novice operators. The time required for insertion into the uterus did not differ between skilled and novice operators (4 min 9 s and 4 min 6 s, respectively). In Experiment 2, 12 gilts were used as recipients for non‐surgical and surgical ET with vitrified embryos (n = 6, each). There was no significant difference in the rate of piglet production based on the number of transferred embryos between surgical and non‐surgical ET (25.8% vs. 15.4%, p = .098). The results suggest that non‐surgical ET catheter allowed for easy insertion and transfer of embryos without special training. Although the catheter is effective for deposition of embryos into the proximal site of uterus, the efficiency of piglet production is not enhanced compared with surgical ET. The ET method using this catheter, being labor‐saving and less‐invasive, may contribute to the improvement of ET in pigs.  相似文献   

9.
To obtain data concerning the survival of embryos and calves derived from somatic cell nuclear transfer (SCNT) in Japan, a nationwide survey was carried out in April, 2009. As a result, data concerning 3264 embryo transfers (ETs) with SCNT embryos which produced 301 calves were accumulated and their survival was analyzed. The present survey revealed that survival rates of transferred bovine embryos and produced calves derived from SCNT had not improved over a decade (1998–2007). A remarkable feature of the pregnancies with SCNT embryos was a high incidence of spontaneous abortions. When the decade was divided by the occurrence of bovine spongiform encephalopathy (BSE) in 2001, significant decreases in the ‘after BSE’ period (2002–2007) were observed in the percentages of calves born (P < 0.01), calves living at birth (P < 0.05), calves living for 24 h (P < 0.05) and 6 months (P < 0.01). Abortions that occurred during 61–99 days after ETs were significantly increased (P < 0.01) in the ‘after BSE’ period. Certain kinds of regeneration that occurred in oocytes during the 15–20 h of storage of bovine ovaries at 10–15°C as a part of BSE inspection might have had some negative effects on SCNT embryos when these oocytes were used as recipients of SCNT.  相似文献   

10.
Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24–48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP–embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.  相似文献   

11.
This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN2). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN2 for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN2.  相似文献   

12.
选择9头母牛先行人工授精、7天后再移植冷冻胚胎以生产双犊。试验结果表明,母牛产双犊达44.44%(P<0.01)。提示:采用A(I人工授精)+ET(胚胎移植)是提高母牛双犊率的有效技术措施之一。  相似文献   

13.
In dogs, embryo transfer (ET) techniques such as induciton of excessive ovulation and synchronization of estrus have not progressed well. Therefore, using embryos at various developmental stages, ET was investigated in dogs from a beagle colony in which the ovulation days were close, as estimated by the progesterone level. Embryos were, recovered 8-11 days after ovulation (4-9 days after mating) by excising the oviducts and uteri (excision method) in 16 animals and by surgical flushing of the uteri at laparotomy (surgical method) in 3 animals. In 24 dogs with -4 to +2 days of difference in the timing of ovulation between donor and recipient dogs, 1-10 embryos at the 8-cell to blastocyst stages were transferred per animal. The mean embryo recovery rate by the excision method (97.1%) was significantly higher than that by the surgical method (42.5%) (p<0.01). Twelve (57.1%) of 21 animals with -1 to +2 days difference in ovulation day became pregnant after the transfer of 8-cell to blastocyst stage embryos. Although 3 dogs with -4 to -2 days of difference of ovulation day underwent ET of morula or compacted morula, none of these dogs became pregnant. The mean ratio of the number of newborns to the number of transferred embryos was only 51.9%. The mean duration of the period between ovulation and delivery in the pregnant recipients was 65.8 days, which tended to be longer than that in natural mating. These results demonstrate that pregnancy can be induced by ET at the 8-cell to blastocyst stage in dogs with -1 to +2 days difference in ovulation day.  相似文献   

14.
The objectives of this study were (a) to assess the ovulatory response and embryo production of Hungarian Merino ewes after superovulation, (b) to investigate the factors influencing the efficiency of embryo transfer (ET) in Hungarian Merino ewes, (c) to compare the results of two ovarian stimulation protocols (PMSG and PMSG + FSH treatment) in Hungarian Merino ewes, and (d) to study how superovulation, laparoscopic insemination and surgical embryo retrieval (ER) affect the subsequent reproduction of Hungarian Merino donor females after an ET programme. There was no significant difference between the ovarian stimulation protocols in the ratio of donor ewes responding to superovulation nor in the average number of corpora lutea. However, the number of transferable embryos recovered per donor ewe was higher in the PMSG + FSH group. The proportion of transferable embryos, unfertilized oocytes and degenerated embryos did not differ between the treatment protocols. The total pregnancy rate was 53.4% (179/335). Neither the developmental stage of the embryo nor the number of transferred embryos affect the implantation of embryos. However, the increased number of transferred embryos positively influenced the pregnancy rate. No difference was found in the pregnancy rate between synchronised and non-synchronised groups of recipients. Thirty-six out of 45 donor ewes (80%) became pregnant within one year after the ET programme, indicating that ovarian stimulation and surgical ER did not affect adversely their reproduction.  相似文献   

15.
旨在基于高通量SNP芯片建立一种可评估牛早期胚胎染色体质量和生产性能的方法,为胚胎牛育种提供技术支撑。本研究通过胚胎切割技术分别对荷斯坦奶牛体内囊胚(n=27)、体外囊胚(n=21)、体外2细胞胚胎(n=6)和8细胞胚胎(n=5)进行切割取样并统计发育率,其中体内囊胚组分为1/2体内胚组(切取半个胚胎)和体内滋养层(trophoblast cells, TE)组(切取体内胚胎少量TE),体外囊胚组、体外2和8细胞胚胎分别为体外TE组(切取体外胚胎少量TE)、体外2细胞(切取体外2细胞胚胎的1个卵裂球)和8细胞组(切取体外8细胞胚胎的1个卵裂球)。切割取样后进行全基因组扩增(whole-genome amplification, WGA),对扩增成功且DNA量大于1 000 ng的样品进行SNP芯片检测,芯片检出率大于90%的进行生产性能评估,低于90%的进行染色体片段缺失分析和数据填充。结果显示：1)切割部分TE后,体内TE组剩余部分和体外TE组剩余部分继续培养24 h后的发育率分别为(94.4±5.6)%和(90.5±6.6)%,而1/2体内胚组剩余部分的发育率显著降低,仅为(22....  相似文献   

16.
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17.
Pregnancy rates after embryo transfer (ET) are disappointing in donkey species. This study aims to report two successful ET of mini-donkey embryos using Brazilian Northeastern jennies as recipients. Eighteen embryo flushes were performed 9 days post-ovulation in two non-pregnant mini-donkeys jennies (11 and 7 cycles per jenny). Eleven embryos (61%, 11/18) were collected and transferred to Brazilian Northeastern jennies 4–6 days post-ovulation by conventional (n = 6) or an alternative (n = 5) technique. The alternative method consisted of inserting a Polansky equine vaginal speculum smeared with lubricant in the vagina of the recipient jenny. The arms of the speculum were extended to allow the visualization of the cervix. Then, using an adapted crafted, elongated, toothed tissue grasping forceps, the external cervical os was held, and the cervix was gently pulled backward, aiming to straight the cervical canal. The ET gun was inserted through the vagina and cervix by visual inspection, and the embryo was released into the uterine lumen. All embryos collected were Grade 1 and classified as Expanded Blastocysts. No jennies become pregnant after conventional ET (0/6), whereas two recipient jennies (40%, 2/5) become pregnant and delivered offspring in the following year after ET using the alternative technique. In conclusion, Brazilian Northeastern jennies can be used as embryo recipients using the alternative method proposed in the present study. However, further investigations are needed to improve the knowledge and results of ET in donkey species.  相似文献   

18.
The objectives of the present study were to establish a nonsurgical transfer method for elongating bovine conceptuses and to combine this method with biopsy and sexing. Bovine conceptuses were recovered from donor cows on days 13–14 of the estrus cycle. In experiment 1, day 13 conceptuses were transferred to recipient cows using a standard day 7 embryo transfer (ET) method. The pregnancy rate of day 13 conceptus transfer (CT) is comparable to that of day 7 ET. In experiment 2, day 14 conceptuses were transferred using modified methods (balloon catheters or ET guns with modified sheaths). Using the standard ET method, no pregnancies were obtained; however, when balloon catheters or ET guns with modified sheaths were used, the pregnancy rates after CT were 48.0% and 44.8%, respectively. In experiment 3, day 14 conceptuses were biopsied without a micromanipulator, sexed using the loop-mediated isothermal amplification method and transferred to recipient cows. The pregnancy rate of biopsied conceptuses was 46.2% and did not differ significantly from that of unbiopsied conceptuses. Moreover, all pregnant cows transferred conceptuses following biopsy and sexing delivered calves with the expected sexes. These results suggested that the nonsurgical bovine CT method was comparable to day 7 ET and that this technique enables biopsy and sexing without expensive equipment such as a micromanipulator or specialized skills.  相似文献   

19.
The objective of this study was to develop an in‐straw dilution method suitable for direct transfer of vitrified bovine sexed embryos. Embryo sexing was performed by molecular diagnosis. Several sexed and vitrified‐warmed embryos were transferred after evaluation of morphologically embryonic survival at warming and in‐straw dilution (Evaluation group). The other embryos were immediately directly transferred to recipients without first being expelled from the straws after in‐straw dilution (Non‐evaluation group). The pregnancy rates of vitrified sexed embryos were 38.7% and 34.8% in the Evaluation group and Non‐evaluation group, respectively, which were not significantly different. The viability of lower quality embryos before vitrification tended to be lower (P = 0.087) than that of the higher quality embryos regardless of evaluating embryos after warming and in‐straw dilution. The abortion rates were similar, and there was no difference between the two groups (13.9% and 12.5%, respectively). These results demonstrate that vitrified bovine sexed embryos can be vitrified and diluted by the in‐straw method and that the vitrified and warmed sexed embryos can develop to term.  相似文献   

20.
This study was designed to evaluate the effect of estrus synchronization treatments on recipient heifers for embryo transfer (ET). Holstein heifers were separated into the following three groups: (i) an administration of 50 µg GnRH (gonadotropin‐releasing hormone) analog was given to heifers at a random stage of the estrus cycle, followed 7 days later by two administrations of 7.5 mg prostaglandin F2 alfa analog (PG) as control; (ii) another administration of 100 µg GnRH was given to the control group at 48 h after the administration of PG as the second GnRH group; and (iii) an administration of 0.75 mg estradiol benzoate (E2) was given to the control group at 24 h after the administration of PG as the E2 group. Each method caused estrus synchronization. Fresh embryos were nonsurgically transferred into the suitable recipients that had a functional corpora lutea (CL) 7 days after estrus. The E2 group showed a significantly higher (P < 0.01) rate of estrus synchronization (98.9%) at 1–3 days after PG administration and the final pregnancy rate of the E2 group (50.6%) was also significantly higher than the other groups (37.1%, P < 0.05 and 30.9%, P < 0.01, respectively). These findings demonstrate that E2 administration 24 h after PG protocol is effective for estrus synchronization of Holstein heifers, thus improving the productivity of ET.  相似文献   

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