scholarly journals The Influence of Developmental Stage and Morphological Quality of Frozen-Thawed Bovine Embryos on Pregnancy Rate in Bovine Embryo Transfer.

1999 ◽  
Vol 45 (4) ◽  
pp. 301-306 ◽  
Author(s):  
Masahiko Nishigai ◽  
Hideo Kamomae ◽  
Tomomi Tanaka ◽  
Yoshihiro Kaneda
Keyword(s):  
Developmental Stage ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Bovine Embryo ◽  
Bovine Embryos

Experience of using time-lapse microscopy in IVF and ICSI programs

2020 ◽  
pp. 47-50
Author(s):  
N. V. Saraeva ◽  
N. V. Spiridonova ◽  
M. T. Tugushev ◽  
O. V. Shurygina ◽  
A. I. Sinitsyna
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Selection Procedure ◽  
Time Lapse ◽  
Single Embryo Transfer ◽  
Main Group ◽  
Clinical Pregnancy ◽  
Control Group ◽  
Time Lapse Microscopy

In order to increase the pregnancy rate in the assisted reproductive technology, the selection of one embryo with the highest implantation potential it is very important. Time-lapse microscopy (TLM) is a tool for selecting quality embryos for transfer. This study aimed to assess the benefits of single-embryo transfer of autologous oocytes performed on day 5 of embryo incubation in a TLM-equipped system in IVF and ICSI programs. Single-embryo transfer following incubation in a TLM-equipped incubator was performed in 282 patients, who formed the main group; the control group consisted of 461 patients undergoing single-embryo transfer following a traditional culture and embryo selection procedure. We assessed the quality of transferred embryos, the rates of clinical pregnancy and delivery. The groups did not differ in the ratio of IVF and ICSI cycles, average age, and infertility factor. The proportion of excellent quality embryos for transfer was 77.0% in the main group and 65.1% in the control group (p = 0.001). In the subgroup with receiving eight and less oocytes we noted the tendency of receiving more quality embryos in the main group (р = 0.052). In the subgroup of nine and more oocytes the quality of the transferred embryos did not differ between two groups. The clinical pregnancy rate was 60.2% in the main group and 52.9% in the control group (p = 0.057). The delivery rate was 45.0% in the main group and 39.9% in the control group (p > 0.050).

scholarly journals Evaluation of Bovine Embryo Biopsy Techniques according to Their Ability to Preserve Embryo Viability

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
M. Cenariu ◽  
E. Pall ◽  
C. Cernea ◽  
I. Groza
Keyword(s):  
Pregnancy Rate ◽  
Genetic Diagnosis ◽  
Needle Aspiration ◽  
Embryo Biopsy ◽  
Embryo Cryopreservation ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Biopsy Methods ◽  
Biopsy Method

The purpose of this research was to evaluate three embryo biopsy techniques used for preimplantation genetic diagnosis (PGD) in cattle and to recommend the least invasive one for current use, especially when PGD is followed by embryo cryopreservation. Three hundred bovine embryos were biopsied by either one of the needle, aspiration or microblade method, and then checked for viability by freezing/thawing and transplantation to recipient cows. The number of pregnancies obtained after the transfer of biopsied frozen/thawed embryos was assessed 30 days later using ultrasounds. The results were significantly different between the three biopsy methods: the pregnancy rate was of 57% in cows that received embryos biopsied by needle, 43% in cows that received embryos biopsied by aspiration, and 31% in cows that received embryos biopsied by microblade. Choosing an adequate biopsy method is therefore of great importance in embryos that will undergo subsequent cryopreservation, as it significantly influences their viability after thawing.

scholarly journals Embryo transfer as an option to improve fertility in repeat breeder dairy cows

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Arkadiusz Nowicki
Keyword(s):  
Dairy Cows ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Oocyte Quality ◽  
Poor Quality ◽  
Early Embryo ◽  
Reproductive Disorder ◽  
Uterine Environment ◽  
Repeat Breeder

Abstract Repeat breeding is a serious reproductive disorder in dairy cattle. The causes of repeat breeding are multifactorial and there are two main mechanisms: failure of fertilisation or early embryo death, mainly due to poor quality of oocytes and an inadequate uterine environment. Many methods have been used to increase the pregnancy rate for repeat breeder cows, such as intrauterine infusion of antibacterial agents or antibiotics, hormonal treatments for oestrus synchronisation and induction of ovulation, and progesterone supplementation or induction of accessory corpus luteum; however, the results were inconsistent between studies. Embryo transfer (ET) has the capability to minimalise the effects of poor oocyte quality and unfavourable uterine environments on early embryo development during the first seven days after ovulation in repeat breeder cows, and several studies showed that ET significantly improved the pregnancy rate in this group of animals. Thus, ET can be considered an option to increase the conception rate in repeat breeder dairy cows.

scholarly journals Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

2021 ◽  
Vol 8 ◽  
Author(s):  
Yasumitsu Masuda ◽  
Ryo Hasebe ◽  
Yasushi Kuromi ◽  
Masayoshi Kobayashi ◽  
Kanako Urataki ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Preimplantation Embryos ◽  
Infrared Light ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

206 EFFECTS OF A COMBINATION OF A PRID, PGF2α AND eCG, ON ESTRUS SYNCHRONIZATION AND PREGNANCY RATE FOLLOWING EMBRYO TRANSFER IN HOLSTEIN HEIFERS

2007 ◽  
Vol 19 (1) ◽  
pp. 220 ◽  
Author(s):  
Y. Aoyagi ◽  
A. Ideta ◽  
M. Matsui ◽  
K. Hayama ◽  
M. Urakawa ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Plasma Concentrations ◽  
Pregnancy Rates ◽  
Bovine Embryo ◽  
Estrus Synchronization ◽  
Chi Square ◽  
End Of Treatment ◽  
Chi Square Analysis ◽  
Day Of Embryo Transfer

Successful bovine embryo transfer requires synchronization of luteolysis, estrus and ovulation. The objective of the present study was to evaluate the effect of a combination of a PRID, PGF2� and eCG, on estrus synchronization and pregnancy rate in recipient heifers. A PRID� (ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) was inserted into the vagina at random days of the estrous cycle for 7 (n = 35) or 9 (n = 43) days. Two days before removal of the PRID, the heifers were injected with PGF2� IM (2 mL Resipron�-C containing 0.25 mg mL-1 cloprostenol; ASKA). About half of the heifers in each group received 250 IU eCG IM (Serotropin�; ASKA) at the time of PRID removal. Blood was collected several times from the start of treatment for 7 (n = 9) or 9 (n = 9) days and on the day of embryo transfer by jugular venipuncture; plasma was immediately separated and stored at -20�C until assayed for plasma concentrations of estradiol-17α (E2) and progesterone (P4). The E2 and P4 determinations were performed by enzyme immunoassay after extraction by diethyl ether. Pregnancy was determined by ultrasonography on Day 30 (Day 0 = estrus). The rates of successful standing estrus (no. in estrus/PRID inserted), embryo transfer (no. transferred/estrus), and pregnancy (no. pregnancy/transferred) were compared between groups. Data were analyzed by chi-square analysis or Fisher's PLSD test following ANOVA. Injection of eCG at the time of PRID removal had no significant effect on the rates of successful standing estrus, embryo transfer, or pregnancy (P > 0.05). The proportion of heifers treated for 9 days that exhibited standing estrus (93%, 40/43) was significantly higher than the proportion of heifers treated for 7 days that exhibited standing estrus (66%, 23/35, P < 0.01). Of the heifers that were treated for 9 days, the proportion of heifers exhibiting standing estrus within 2 days after the end of treatment was significantly higher (93%, 37/40) than for heifers that were treated for 7 days (65%, 15/23; P < 0.01). Pregnancy rates of heifers treated for 9 days (84%, 32/38) and 7 days (81%, 17/21) were not significantly different. The E2 : P4 ratio normally increases during follicle growth and CL regression. The plasma E2 : P4 ratio between the time of injection of PGF2α and the time of PRID removal was significantly higher for heifers that were treated for 9 days than it was for heifers that were treated for 7 days (P < 0.01). These results suggest that a combination of PRID treatment for 9 days and injection of PGF2α 2 days before PRID removal successfully synchronized estrus in recipient heifers and led to high pregnancy rates following embryo transfer.

109 RELATIONSHIP BETWEEN ULTRASONIC MORPHOLOGY OF CORPUS LUTEUM IN HOLSTEIN HEIFERS AND PREGNANCY RATE AFTER EMBRYO TRANSFER

2012 ◽  
Vol 24 (1) ◽  
pp. 167
Author(s):  
A. Shirasawa ◽  
Y. Nakamura ◽  
A. Ideta ◽  
Y. Oono ◽  
M. Urakawa ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Blood Clot ◽  
Pregnancy Rates ◽  
Bovine Embryo ◽  
Tissue Area ◽  
The Mean ◽  
Rectal Palpation ◽  
Experimental Group

Recipient animals for bovine embryo transfer (ET) are routinely selected according to the morphology of the corpus luteum (CL) estimated by rectal palpation. However, rectal palpation is not a precise method of diagnosing the functional status of a CL. Ovarian ultrasonography (US) may be used to improve such diagnoses. The aim of this study was to evaluate the relationship between ultrasonographic images of CL and pregnancy rates after ET in Holstein heifers to determine whether US can be used to select recipients for ET. Recipient heifers (n = 285) were selected by detection of natural oestrus or following oestrus synchronization using a progesterone-releasing intravaginal device (PRID; ASKA Pharmaceutical, Tokyo, Japan). Transrectal US was performed immediately before ET, on Days 6 to 8 of the oestrous cycle (oestrus = Day 0), using a B-mode scanner (HS1500V; Honda Electronics Co. LTD, Aichi, Japan) equipped with a 7.5-MHz linear-array transducer designed for intrarectal placement. A cross-sectional image of the maximal area of the CL and luteal cavity was obtained. The areas of the CL and luteal cavity were each calculated using the formula for the area of an ellipse (height/2 × width/2 × π). (1) Ultrasonic morphology of CL was classified into 3 types: without cavity (n = 128), with cavity (n = 145) and with blood clot (n = 12). (2) The luteal cavity was categorized into 3 groups: small (<100 mm2, n = 93), medium (100 ≤ x < 200 mm2, n = 32) and large (≥200 mm2, n = 20). (3) Luteinized tissue area (total area of CL minus the area of the luteal cavity) was categorized into 3 groups: small (<250 mm2, n = 61), medium (250 ≤ x < 350 mm2, n = 128) and large (≥350 mm2, n = 84). In vivo–produced embryos were transferred nonsurgically into the uterine horn ipsilateral to the CL. Pregnancy was determined by transrectal US on Days 30 to 40 of gestation. The pregnancy rates of each experimental group were analysed by logistic regression. In this study, the pregnancy rate did not differ significantly in each experimental group: (1) without cavity: 77.3% (99/128), with cavity: 75.2% (109/145) and blood clot: 75.0% (9/12); (2) small cavity: 73.1% (68/93), medium: 75.0% (24/32) and large: 85.0% (17/20). The mean area of the cavity was 100.8 ± 110.3 mm2 (mean ± standard deviation) and recipients with 0 to 539.7 mm2 sized cavities had successful pregnancies (observational range was 0 to 539.7 mm2). (3) The pregnancy rates of recipients that had small, medium and large luteinized tissue were 77.0% (47/61), 75.0% (96/128) and 77.4% (65/84), respectively. The mean area of luteinized tissue was 318.9 ± 90.3 mm2 and 155.0 to 620.0 mm2 sized luteinized tissue had pregnancy success (observational range was 132.8 to 620.0 mm2). In conclusion, the results from this study indicate that the presence of a luteal cavity or blood clot has no detrimental effect on pregnancy success after ET in Holstein heifers. Furthermore, no relationship was found between luteinized tissue area at the time of ET and pregnancy rate.

scholarly journals Studies on factors affecting superovulation and embryo transfer in Hungarian Merino ewes

2001 ◽  
Vol 49 (4) ◽  
pp. 431-441 ◽  
Author(s):  
S. Cseh ◽  
L. Solti
Keyword(s):  
Developmental Stage ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Ovarian Stimulation ◽  
Corpora Lutea ◽  
Treatment Protocols ◽  
Factors Affecting ◽  
Factors Influencing ◽  
Significant Difference ◽  
One Year

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, unfertilised 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 nonsynchronised 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.

216 THE RELATIONSHIP BETWEEN OXYGEN CONSUMPTION RATE AND PREGNANCY RATE OF BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 225 ◽  
Author(s):  
N. Sakagami ◽  
K. Akiyama ◽  
Y. Nakazawa
Keyword(s):  
Oxygen Consumption ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Embryo Quality ◽  
Calf Serum ◽  
Pregnancy Rates ◽  
Bovine Embryos ◽  
Significant Difference ◽  
Consumption Rates ◽  
The Relationship

A precise evaluation of embryo quality is important to estimate the suitability of embryo transfer to recipient animal. Recently, an objective evaluation method was reported for bovine embryos, in which the oxygen consumption of embryos can be noninvasively determined by scanning electrochemical microscopy (SECM) (Shiku et al. 2001 Anal. Chem. 73, 3751–3758). Trimarchi et al. (2000 Biol. Reprod. 62, 1866–1874) suggested that the oxygen consumption reflects the cell number and mitochondrial activity of embryos. The objectives of this study were (1) to examine the oxygen consumption of in vivo-derived embryos by SECM, (2) to investigate the relationship between oxygen consumption and morphological estimation of embryos, and (3) to assess the correlation among the oxygen consumption, embryo viability, and pregnancy rates. Fifty-six embryos were collected from Japanese Black cattle, which were superovulated with a total dose of 20 mg porcine FSH (FSH-R; Kawasaki Pharmaceutical Co., Ltd., Tokyo, Japan) followed by AI. The qualities of collected embryos at the stage of compacted morulae (CM), early blastocysts (EB), and blastocysts (BL) on Day 7 after AI were categorized as grade 1 and grade 2, according to the IETS manual (2002). The oxygen consumption rates of embryos were evaluated by SECM, as previously described by Abe et al. (2004 J. Mamm. Ova Res. 21). Embryos were frozen by programmable freezer in Dulbecco&apos;s PBS containing 1.5 M ethylene glycol, 0.1 M trehalose, and 20&percnt; calf serum. They were thawed by holding the straws in air for 8 s and then immersing them in a 30&deg;C water bath for 15 s. After thawing, the embryos were examined for oxygen consumption. Twenty-eight embryos were then cultured in TCM-199 supplemented with 20&percnt; fetal bovine serum and 0.1 mM &beta;-mercaptoethanol for 24 h to assess the viability of embryos by re-expansion of blastocole. The remaining 28 embryos were transferred to recipients. The pregnancy rates were determined by rectal palpation on Day 70. Data were analyzed by ANOVA. The consumption rates of BL embryos on Day 7 were significantly higher (P &lt; 0.05) than those of CM collected on the same day (0.84 vs. 1.29 &times; 10&minus;14 mol s&minus;1, respectively). A significant difference was also observed in consumption rates between grade 1 and 2 embryos at the BL stage (P &lt; 0.05). After freezing&ndash;thawing, the average oxygen consumption rates of embryos were 0.52 &times; 10&minus;14 mol s&minus;1 for CM (n &equals; 9), 0.67 &times; 10&minus;14 mol s&minus;1 for EB (n &equals; 8), and 0.96 &times; 10&minus;14 mol s&minus;1 for BL (n &equals; 11). The CM embryos with rates of &lt; 0.5 &times; 10&minus;14 mol s&minus;1 and the EB and BL embryos with those &lt; 0.6 &times; 10&minus;14 mol s&minus;1 did not show good morphological appearance after 24 h in culture. Pregnant animals were not obtained from embryos with rates &lt;0.5 &times; 10&minus;14 mol s&minus;1 for CM (n &equals; 5) and &lt;0.7 &times; 10&minus;14 mol s&minus;1 for EB (n &equals; 9). A high pregnancy rate (67&percnt;) was obtained from embryos with rates &gt;1.0 &times; 10&minus;14 mol s&minus;1 for BL (n &equals; 14). These results suggest that the measurement of oxygen consumption of embryos after embryo freezing and prior to embryo transfer may be useful for estimating embryo quality and suitability of embryo transfer.

204 RELATIONSHIP BETWEEN RESPIRATORY ACTIVITY AND THE PREGNANCY RATE OF BISECTED BOVINE EMBRYOS IN VIVO

2007 ◽  
Vol 19 (1) ◽  
pp. 219 ◽  
Author(s):  
S. Moriyasu ◽  
H. Hirayama ◽  
K. Sawai ◽  
S. Kageyama ◽  
S. Aoyagi ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Respiratory Activity ◽  
Pregnancy Rates ◽  
Bovine Embryos ◽  
Chi Square ◽  
Important Indicator ◽  
Consumption Rates

Oxygen consumption is an important indicator of the metabolic activity of living cells, which may provide valuable information for evaluating embryo quality. We have found that the bovine embryos with high oxygen consumption possess stronger potential for further development. However, the relationship between respiratory activity and the pregnancy rate of embryos is still unclear. In this study, we investigated the respiration rates of bisected bovine embryos and the pregnancy rates of demi-embryos after embryo transfer. Compact morula-stage embryos were bisected evenly by micro glass needle. One hundred bisected embryos were incubated for 24 h in embryo culture medium (IVD101; Research Institute for the Functional Peptides, Yamagata, Japan) at 39�C under 5% CO2, 5% O2, 90% N2. After the incubation, demi-embryos were classified into 2 groups: blastocoel-formed (BC) and blastocoel-not-formed (CM) embryos. Oxygen consumption rates of demi-embryos were measured by scanning electrochemical microscopy (SECM; Hokuto Denko Corporation, Tokyo, Japan). Within 3 h after the measurement, 80 demi-embryos were transferred into recipient cows (one demi-embryo/one recipient) at 7–8 days after estrus. Recipient cows were diagnosed for pregnancy by ultrasonography approximately 40 days after estrus. Statistical difference was analyzed by Tukey's post-hoc test and chi-square test. A total of 27 recipient cows became pregnant; the pregnancy rates for cows with CM and BC demi-embryos were 40.6% (13/32) and 29.2% (14/48), respectively. Mean oxygen consumption rates (� 10-14 mol s-1) in pregnant and non-pregnant cows were 0.47 and 0.39 for CM demi-embryos and 0.63 and 0.52 for BC demi-embryos, respectively. Retrospective analysis showed that the respiratory activity of demi-embryos in the pregnant group was higher than those in the non-pregnant group. In particular, the pregnancy rates for demi-embryos with respiratory activity higher than 0.35 in CM and 0.40 in BC groups were 52.0% (13/25) and 35.9% (14/39), respectively. On the other hand, cows with demi-embryos having an oxygen consumption rate under 0.35 in CM (n = 7) and 0.40 in BC (n = 9) groups did not become pregnant. These results demonstrated that bovine demi-embryos with higher respiratory activity showed a high pregnancy rate after embryo transfer. It is generally known that the pregnancy rate after the transfer of bisected embryos is lower than that of whole embryos. The measurement of oxygen consumption by SECM procedures is a useful tool to assess the quality of pre-implantation embryos and may contribute to the improvement of the success rate for bisected embryo transfer.

51 ARTIFICIAL DORMANCY OF BOVINE EMBRYOS FOR A MAXIMUM OF 7 DAYS USING A SIMPLE MEDIUM

2014 ◽  
Vol 26 (1) ◽  
pp. 139 ◽  
Author(s):  
K. Tsuchiya ◽  
A. Ideta ◽  
Y. Nishimiya ◽  
S. Tsuda ◽  
Y. Aoyagi
Keyword(s):  
Pregnancy Rate ◽  
Storage Period ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Hypothermic Preservation ◽  
Mammalian Embryos ◽  
Simple Medium ◽  
Hypothermic Storage

The worldwide pregnancy rate using cryopreserved mammalian embryos has not improved over the past 2 decades, probably because the freeze-thawing processes cause significant damage. Therefore, it is now relevant to examine the feasibility of short-term non-freezing preservation, and whether this could be applied to embryos that have high vitality and are to be transferred into recipients within several days. We introduce here an artificial dormancy fluid that can extend the hypothermic storage period of bovine embryos for a maximum of 7 days. First, to examine the effect of different basal media and the optimal concentration of fetal bovine serum (FBS) for hypothermic preservation, bovine blastocysts produced in vitro were stored at 4°C in a plastic ministraw in 1 of the following 3 media: PBS, medium 199, or Leibovitz L15 with various amount of FBS (0, 5, 20, 50, or 100%) for 3 days. Second, to examine the effect of Good's buffers, bovine embryos produced in vivo (morula to blastocyst stages) were stored at 4°C in a plastic ministraw in medium 199 plus 50% FBS supplemented with various Good's buffers [HEPES, TES, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), MOPS, and 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS)] for 7 days. Following hypothermic preservation, the chilled embryos were squeezed out of the straw into PBS and washed 3 times in the same medium. Subsequently, the embryos were cultured in CR1aa medium supplemented with 5% FBS for 48 h at 38.5°C under 5% CO2 in air with high humidity. The viability rate of the embryos was assessed at the end of the culture period. Finally, to observe the pregnancy rate of chilled embryos, 32 embryos produced in vivo were stored at 4°C for 7 days in medium 199 plus 50% FBS supplemented with HEPES. Following hypothermic preservation, the chilled embryos were transferred into recipient heifers (1 embryo per recipient). Pregnancy was determined by real-time B-mode ultrasonography (Convex scanner HS-1500, Honda electronics Co. Ltd, Toyohashi, Japan) on Day 60 of gestation. Data were analysed using the chi-squared test. The viability rate of the embryos after hypothermic storage for 3 days was significantly increased for medium 199 plus 50% FBS [27/30 (90%)] compared with PBS [18/30 (60%)] or Leibovitz L15 [15/30 (50%)] plus 50% FBS (P < 0.05). Chilled embryos stored for 7 days in medium 199 plus 50% FBS supplemented with HEPES had much higher survival than embryos stored in the same medium with other Good's buffers. The pregnancy rate of the chilled embryos stored for 7 days was extremely high [24/32 (75%)] and normal live calves were delivered at term. In conclusion, maintaining artificial dormancy of bovine embryos for 7 days using a simple medium appears to be feasible. This is the first documented success of storing chilled mammalian embryos in a viable state for 7 days. To be of practical value, bovine embryo preservation at hypothermic temperatures must be able to maintain viability for periods longer than 7 days. This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry.

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