140 UTILIZATION OF LASER-ASSISTED HATCHING TO IMPROVE THE PREGNANCY RATE OF IN VITRO-FERTILIZED BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 150 ◽  
Author(s):  
S. Menges ◽  
H. Wei ◽  
D. Faber ◽  
D. Kraemer ◽  
C. Long
Keyword(s):  
Pregnancy Rate ◽  
Pulse Length ◽  
Pregnancy Rates ◽  
Live Cells ◽  
Assisted Hatching ◽  
Laser Exposure ◽  
Bovine Embryos ◽  
Embryo Viability

In vitro-produced (IVP) bovine embryos are known to produce a lower pregnancy rate when compared to conventional in vivo-produced embryos. The inability of the IVP embryo to hatch from the zona pellucida (ZP) after embryo transfer is thought to be one contributing factor. This study was designed to evaluate the utilization of a microscope objective-mounted laser to cut the ZP to assist hatching prior to transfer into the recipient. Preliminary data were acquired to evaluate the effect of laser treatment on in vitro development and blastomere survival following treatment. In six replicates, bovine oocytes were in vitro-matured, fertilized, and cultured as per standard laboratory procedures (TransOva Genetics, Sioux Center, IA, USA). On Days 5, 6, and 7 of in vitro culture, embryos were randomly divided into 3 treatment groups: no treatment (Control; n = 63), sham ZP cut (Sham; n = 68), or ZP cut (Cut; n = 70). Control embryos were immediately returned to the incubator following selection. Sham embryos were exposed to all conditions as Cut except laser-assisted hatching. The XYClone� system is a 300-mW, class 1 laser that emits a 3.5-µm beam at a wavelength of 1480 nm (Hamilton Thorne Biosciences, Beverly, MA, USA). This laser was used to produce the Cut group, using a pulse strength of 90% and pulse length of 600 µs. Embryos were returned to culture until Day 8 when rates of embryonic development and the percentage of live cells were determined. Chi-square was used to analyze all data. No significant effect of treatment or day of exposure was noted in either the total number of developing embryos or the ratio of live cells in each embryo. Mean live cells ranged from 89 to 96% across all treatments regardless of day of treatment. To investigate IVP embryo viability after laser-assisted hatching, commercially produced embryos (TransOva Genetics, Sioux Center, IA, USA) were randomly divided into two groups on the day of transfer, Control or Cut. The ZP of treated embryos were cut with slightly reduced laser exposure of 80% pulse strength and pulse length of 500 µs on Day 7, immediately prior to transfer into estrus-synchronized recipients. Pregnancy rates were determined via ultrasonagraphy at Day 30 (n = 337) and, due to the commercial nature of this project, only a subset of the Day 30 pregnant cows was checked at Day 60 (n = 289). The 30-day pregnancy rates were 49.2% and 54.1% for Control (n = 189) and Cut (n = 148) embryos, respectively, and were not statistically different (P > 0.05). However, at Day 60, the pregnancy rates for the Control (45.7%; n = 166) and Cut groups (57.7%; n = 123) were statistically different (P < 0.05). These results demonstrate that laser-assisted hatching using the XYClone system can improve 60-day pregnancy rates for in vitro-produced embryos.

171 LASER-ASSISTED ZONA PELLUCIDA HATCHING IN FROZEN - THAWED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 244 ◽  
Author(s):  
M. K. Chiasson ◽  
J. A. Carter ◽  
K. R. Bondioli ◽  
R. A. Godke ◽  
G. T. Gentry
Keyword(s):  
Calf Serum ◽  
Pregnancy Rates ◽  
Water Bath ◽  
Assisted Hatching ◽  
Direct Transfer ◽  
Pulse Treatment ◽  
Bovine Embryos ◽  
Hatching Rates

Incomplete zona hatching or failure of the zona to rupture compromises post-transfer embryo viability and conceptus development. Assisted hatching prior to the transfer of frozen-thawed bovine embryos has been proposed as a means to increase recipient pregnancy rates. The objective of this study was to determine if laser-assisted hatching would improve in vivo derived frozen-thawed bovine embryo hatching rates. In Exp. 1, direct-transfer beef cattle embryos were air-thawed for 15 s, placed in a 30°C water bath for 15 s, then held in TALP-HEPES, evaluated for stage and grade (1 = good to 3 = poor) and randomly applied to treatments. Embryos (n = 156) received either 2 or 3 symmetrical rents 40% through the outer zona surface using the XYClone diode laser (Hamilton Thorne, Beverly, MA, USA) at 90% power with a 600 μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were then cultured in vitro in CR1aa supplemented with 10% calf serum at 39°C in 5% CO2 and 5% O2 for 4 d. Embryo hatching rates were 47% for Treatment A and 53% for Treatment B. In Exp. 2, in vivo produced, nonsurgically collected direct-transfer Hereford embryos (n = 64) were utilized. In Exp. 3, in vivo produced nonsurgically collected glycerol frozen Brangus embryos (n= 46) were utilized. Embryos utilized in Exp. 2 and 3 were air-thawed for 15 s, placed in a 30°C water bath for 15 s, and then held in 1 M sucrose for 7 min. Embryos were then held in phosphate-buffered saline with 10% calf serum (Exp. 2) or ViGRO Holding Plus (Bioniche, Pullman, WA, USA) (Exp. 3), evaluated for stage and grade before being randomly assigned to either Treatment A or B. Embryos received either 3 symmetrical rents 40% through the outer zona surface using the XYClone laser at 90% power with a 600-μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were transferred nonsurgically (1 embryo/female) by the same technician into synchronized mixed breed recipient beef cows on Day 7 of the estrous cycle. Pregnancy status was determined at 35 days and 60 days via ultrasonography. In Exp. 2, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Also, treatment did not affect pregnancy rates at 35 or 60 days (41% and 28% for Treatment A and 44% and 41% for Treatment B, respectively). Likewise, there was no difference in calving rate for recipients confirmed pregnant at 60 days for Treatment A (89%) and Treatment B (77%). In Exp. 3, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Pregnancy rates at 35 and 60 days were not affected by treatment (65% and 65% for Treatment A and 76% and 59% for Treatment B, respectively). Calving rates for those recipients in Exp. 3 were not available at the time of abstract preparation. Based on the data presented herein, it does not appear that laser-assisted hatching with the XYClone laser increases the number of in vivo derived frozen-thawed embryos that hatch following in vitro culture or increase pregnancy rates after transfer to recipient cattle.

scholarly journals 183PREGNANCY RATE FOLLOWING TRANSFER OF IN VITRO- AND IN VIVO-PRODUCED BOVINE EMBRYOS TO LH-TREATED RECIPIENTS

2004 ◽  
Vol 16 (2) ◽  
pp. 213 ◽  
Author(s):  
J. Small ◽  
M. Colazo ◽  
D. Ambrose ◽  
R. Mapletoft ◽  
J. Reeb ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Animal Health ◽  
Beef Cows ◽  
Water Bath ◽  
Bovine Embryos ◽  
Chi Square ◽  
Embryo Survival ◽  
Frozen Embryos

The objective was to evaluate the effect of pLH treatment on pregnancy rates in recipients receiving in vivo- or in vitro-produced bovine embryos. Heifers (n=37) and lactating (n=28) and non-lactating (n=150) beef cows were treated at random stages of the cycle with 100μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day −9, 500μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day —2 and GnRH on Day 0 (66h post-PGF; without estrus detection). Cattle were placed at random, by class, into three groups: no further treatment (Control; n=71), or 12.5mg pLH (Lutropin-V, Bioniche Animal Health, Belleville, Ontario, Canada) on Day 5 (n=72) or on Day 7 (n=72) after the second GnRH. On Day 7, cattle with a CL &gt;10mm in diameter (determined ultrasonically) received in vivo-produced, fresh (Simmental) or frozen (Holstein), or in vitro-produced frozen (Holstein) embryos (embryo type balanced among groups). Embryos were cryopreserved in 10% ethylene glycol; in vivo-produced frozen embryos were thawed 5 to 10s in air, 15s in a water-bath at 30°C and then “direct-transferred” nonsurgically. In vitro-produced frozen embryos (donated by IND Lifetech Inc., Delta, British Columbia, Canada) were thawed in a water-bath at 27°C for 10s and placed in ViGro Holding Plus medium (AB Technology, Pullman, WA, USA) at room temperature, evaluated and then transferred nonsurgically. Pregnancy was determined by ultrasonography on Day 35. Data were analyzed with CATMOD, chi-square and GLM procedures (SAS Institute, Cary, NC, USA.). Twenty cattle (9.3%) did not receive embryos; five heifers had cervical problems, and five heifers and 10 cows did not have a CL &gt;10mm. Overall, 7.1% of the recipients had two CL on the day of embryo transfer. There was no effect (P&gt;0.05) of treatment, embryo type (or interaction) or class of recipient on pregnancy rate (overall, 44.1%, 86/195; Table 1). Similarly, mean (±SD) CL diameter and luteal area did not differ (P&gt;0.05) among groups or between pregnant and open recipients (overall, 22.0±3.4mm and 352.0±108.7mm, respectively). However, recipients with a CL diameter ≥18mm tended (P&lt;0.1) to have a higher pregnancy rate (45.8 vs 25.0%). In a subset of 40 recipients examined ultrasonically on Day 12, 50% of those treated on Day 5 and 70% of those treated with pLH on Day 7 had two CL. In summary, overall pregnancy rate in GnRH-synchronized recipients receiving in vitro- or in vivo-produced embryos by nonsurgical transfer was 44.1%. Embryo survival to Day 35 was not affected by type of embryo or treatment with pLH 5 or 7 days after ovulation. Table 1 Pregnancy rate in recipients on Day 35 based on pLH treatment and embryo-type

90 THE EFFECT OF LIPID SEGREGATION WITH OR WITHOUT ZONA PELLUCIDA LASER DRILLING ON POST-THAW EMBRYO DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 125
Author(s):  
J. H. Pryor ◽  
C. R. Looney ◽  
S. Romo ◽  
D. C. Kraemer ◽  
C. R. Long
Keyword(s):  
Zona Pellucida ◽  
Developmental Stages ◽  
Uv Light ◽  
Statistical Significance ◽  
Cell Number ◽  
Live Cells ◽  
Assisted Hatching ◽  
Embryo Viability ◽  
And Control

High levels of lipid within in vitro-produced embryos during freezing can increase intracellular damage and lower production rates (Seidel 2006 Theriogenology 65, 228). The objective of this study was to determine if lipid segregation with or without laser-assisted hatching (LAH), or zona pellucida drilling of in vitro-fertilized (IVF) embryos would enhance in vitro survivability and development 24 h post-thaw. Three replicates utilizing 1179 bovine oocytes (BOMED, Madison, WI, USA) were fertilized with frozen/thawed Tuli bull semen and cultured in G1.3/G2.3 supplemented with 8 mg mL–1 BSA (Vitrolife, Englewood, CO, USA). On Day 6 of culture, grade 1 & 2 embryos were morphologically divided into 3 developmental stages: 32-cell (n = 78), compact morula (CM, n = 223), and blastocyst (n = 56). Each group was then randomly allocated to the following treatments prior to cryopreservation in 1.5 m ethylene glycol (Vigro Freeze Plus, Bioniche, Pullman, WA, USA): no treatment (control), 7.5 µg mL–1 cytochalasin B for 20 min (CB), or CB with centrifugation (16 000g) for 20 min (CBCF). All CB treatments were extended to include embryo freezing. Embryos were loaded in sterile straws, frozen at 0.5�C min–1 from –6�C to –32�C, and then plunged into LN2. Frozen embryos were air-thawed for 7 s and then thawed in 35�C H2O for 10 s before being assessed for survivability. Immediately post-thaw, one-half of the CBCF and control groups were subjected to LAH, using a single laser pulse at 90% laser power for 600 µs using the XY Clone� system (Hamilton Thorne Biosciences, Beverly, MA, USA), creating groups CBCFLAH and LAH, respectively. All thawed embryos were cultured in G2.3 for 24 h and evaluated morphologically to determine survivability and development. Live/dead staining was performed by using Hoechst 33342 (2.5 µg mL–1) and propidium iodide (5 µg mL–1) under UV light. All percentage data were transformed using arcsin square root function prior to analysis, and means were compared for statistical significance using Student's t-test. Due primarily to low numbers in embryos in stages other than CM, no differences among treatments were detected. For CM, treatment means ranged from 89.6 to 95.0% and from 69.6 to 82.6% for survival and development, respectively, and no treatment differences were observed. Within the CM stage, CBCFLAH was not different from LAH, CBCF, and control (77.0 v. 71.9, 68.8, and 68.3%, respectively; P > 0.05), but showed a significantly greater percentage of live cells than CB (77.0 v. 65.5%; P < 0.05). CBCFLAH and LAH exhibited a significantly greater number of both total and live cells than control (total cells: 69.4, 69.3, and 53.0; live cells: 56.4, 54.7, and 39.3, respectively; P < 0.05). These data indicate that LAH post-thaw alone or in combination with CBCF improves both total cell number and embryo viability following cryopreservation. Financial support was provided by a grant from TAMU-CONACYT (USA-Mexico) and OvaGenix.

136 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS SUBMITTED TO LASER ASSISTED HATCHING ON DAY 6 AND DAY 7 OF EMBRYO CULTURE

2009 ◽  
Vol 21 (1) ◽  
pp. 168
Author(s):  
C. B. Ponchirolli-Schneider ◽  
J. H. Pryor ◽  
C. R. Looney
Keyword(s):  
Embryo Culture ◽  
Uv Light ◽  
Laser System ◽  
Pulse Length ◽  
Assisted Hatching ◽  
In Vitro Production ◽  
Stage Of Development ◽  
Number Of Cells

In vitro production (IVP) of embryos is a valuable tool in bovine assisted reproduction. IVP embryos show lower pregnancy rates when compared to in vivo produced embryos. The inability of the IVP embryo to hatch from the zona pellucida (ZP) after embryo transfer is believed to be one contributing factor. The aim of this study was to compare the ability of IVP embryos to hatch and develop in vitro after being submitted to laser assisted hatching (LAH) at two different time periods of embryo culture: 144 h (Day 6) and 168 h (Day 7). In vitro maturated oocytes from slaughterhouse ovaries (Ovitra Biotechnologies, Amarillo, TX, USA) were fertilized with frozen/thawed semen from two different bulls (Day 0) and cultured in G1.5/G2.5 medium supplemented with 8 mg mL–1 of BSA (Vitrolife, Englewood, CA, USA) at 38.5°C, 5% CO2, 5% O2, 5% N2, in humidified atmosphere. On Day 6 post-fertilization, all viable embryos (n = 251), from three replicates, were washed in holding medium (Vigro Holding Plus, Bioniche, Pullman, WA, USA) and divided into four treatment groups: laser assisted hatching Day 6 and Day 7 (LAHD6 and LAHD7), control Day 6 and Day 7 (CD6 and CD7). The groups LAHD7 and CD7 were immediately placed in G2.5 and returned to the incubator until Day 7. Embryos from groups LAHD6 and CD6 were placed in G2.5 in separate wells of a four well dish and covered with 300 μL of mineral oil. Embryos from LAHD6 group had one third to one half of the external edge of the ZP exposed to a laser beam, using XY Clone® (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) laser system, with a pulse strength of 90% and a pulse length of 600 μs. The group CD6 was submitted to the same conditions, but did not receive the laser treatment. On Day 7, the experiment was repeated for embryos belonging to groups LAHD7 and CD7. Embryos from all groups were cultured in vitro and evaluated on Day 8 and Day 9 for stage of development. On Day 9, a random sample of embryos from each treatment group (n = 48) was stained with Hoechst 33342 (2.5 μg mL–1) and evaluated under UV light to determine the total number of cells. The number of hatched blastocysts was not different (chi-square, p > 0.05) among the groups on Day 9 of culture (LAHD6 = 49/66, CD6 = 38/61, LAHD7 = 42/59, CD7 = 47/65; 74, 62, 71 and 72%, respectively). However, on Day 8 of culture, LAHD6 showed a higher number (p < 0.05) of hatched blastocysts (40/66, 60%), when compared to group CD6 (26/61, 42%). There was no difference between groups LAHD7 (33/59, 55%) and CD7 (31/65, 47%) on Day 8. Comparison of the total number of cells showed no difference (Student’s t-test, p > 0.05) among the groups (LAHD6 = 154.8 ± 12.2, CD6 = 184.4 ± 19.5, LAHD7 = 170.4 ± 15.8, CD7 = 162.5 ± 14.6), indicating that LAH does not have a detrimental effect on mean cell production throughout embryo development in vitro. These data show that LAH on Day 6 of culture improves in vitro hatching rates on Day 8, while LAH on Day 7 shows no improvement on either Day 8 or 9. Kathy Bradley, Hamilton Thorne Biosciences, Inc.

Fertilizing capacity of epididymal and testicular sperm using intracytoplasmic sperm injection (ICSI)

1995 ◽  
Vol 7 (2) ◽  
pp. 281 ◽  
Author(s):  
SJ Silber ◽  
P Devroey ◽  
H Tournaye ◽  
Steirteghem AC Van
Keyword(s):  
Pregnancy Rate ◽  
Intracytoplasmic Sperm Injection ◽  
Pregnancy Rates ◽  
Obstructive Azoospermia ◽  
Motile Sperm ◽  
Epididymal Sperm ◽  
Testicular Sperm ◽  
Cleavage Rate

For men with uncorrectable obstructive azoospermia, their only hope of fathering a child is microsurgical epididymal sperm aspiration (MESA) combined with in vitro fertilization (IVF). In 1988, proximal epididymal sperm were demonstrated to have better motility than senescent sperm in the distal epididymis, and it was thought that retrieval of motile sperm from the proximal epididymis would yield reliable fertilization and pregnancy rates after conventional IVF. However, the results to date have been poor, and although a minority of patients achieved good fertilization rates with IVF, the vast majority (81%) had consistently poor or no fertilization and the pregnancy rate averaged only 9%. Recently, intracytoplasmic sperm injection (ICSI) has been successfully used to achieve fertilization and pregnancies for patients with extreme oligoasthenozoospermia. ICSI has therefore been applied to cases of obstructive azoospermia and, in this report, 67 MESA-IVF cases are compared with 72 MESA-ICSI cases. The principle that motile sperm from the proximal segments of the epididymis should be used for ICSI was followed, although in the most severe cases in which there was an absence of the epididymis (or absence of sperm in the epididymis), testicular sperm were obtained from macerated testicular biopsies. These sperm only exhibited a weak, twitching motion. In 72 consecutive MESA cases, ICSI resulted in fertilization and normal embryos for transfer in 90% of the cases, with an overall fertilization rate of 46%, a cleavage rate of 68%, and ongoing or delivered pregnancy rates of 46% per transfer and 42% per cycle. The pregnancy and take-home baby rates increased from 9% and 4.5% with IVF to 53% and 42% with ICSI. There were no differences between the results for fresh epididymal, frozen epididymal or testicular sperm, and the number of eggs collected did not affect the outcome. The results were also unaffected by the aetiology of the obstruction such as congenital absence of the vas deferens or failed vasoepididymostomy. The only significant factor which affected the pregnancy rate was female age. It is concluded that although complex mechanisms involving epididymal transport may be beneficial for conventional fertilization of human oocytes (in vivo or in vitro), none of these mechanisms are required for fertilization after ICSI. Given the excellent results with epididymal and testicular sperm, ICSI is obligatory for all future MESA patients. Finally, the use of ICSI with testicular sperm from men with non-obstructive azoospermia is also discussed.

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.

37 EFFECT OF EMBRYO STAGE ON PREGNANCY RATE FOLLOWING DIRECT TRANSFER OF BOVINE EMBRYOS FROZEN IN ETHYLENE GLYCOL

2012 ◽  
Vol 24 (1) ◽  
pp. 131 ◽  
Author(s):  
J. F. Hasler
Keyword(s):  
Ethylene Glycol ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
The United States ◽  
Pregnancy Rates ◽  
Bovine Embryos ◽  
Embryo Freezing ◽  
Percentage Points ◽  
Embryo Stage

Annually, more than 400 000 in vivo-recovered bovine embryos are officially reported by members of the Canadian and American Embryo Transfer Associations. Between 65 and 70% of these embryos are cryopreserved and more than 95% are frozen in ethylene glycol (EG). Statistics on factors affecting embryo freezing are difficult to obtain because many cattle breeders/farmers no longer report pregnancy rates back to embryo transfer (ET) practitioners. Concerns are often expressed as to the optimal stage at which to freeze bovine in vivo-derived embryos. This is a retrospective analysis of results from 5 commercial ET programs (1 in the United States, 3 in Canada and 1 in the Netherlands) for which pregnancy data relative to embryo stage at freezing were made available. Embryos representing 4 stages of development, as defined by the IETS (4 = late morula, 5 = early blastocyst, 6 = mid blastocyst and 7 = expanded blastocyst) are included in the data. The number of embryos thawed and transferred ranged from 3954 to 24 827 for the 5 programs, with a total of 72 828. Embryos were frozen in either 1.5 M EG or 1.5 M EG + 0.1 M sucrose and exposure time to cryoprotectant before cooling ranged from 4 to 40 min. Pregnancy rates are shown in Table 1. Although the pregnancy rate for stage 6 embryos was only 2.6 and 3.2 percentage points lower than stages 4 and 5, respectively, these differences were highly significant and pregnancy rates for stage 6 embryos were lower than those for stages 4 and 5 in 4 of the 5 ET programs. The small decreased survival of stage 6 embryos is probably only moderately important in a commercial context. However, the pregnancy rate of stage 7 embryos was lower than all other stages for the combined dataset as well as in all 5 ET programs, with the difference between stages 5 and 7 ranging from 6.5 to 16.4 percentage points. Clearly, stage 7 embryos survive freezing at a significantly lower rate than stages 4, 5 and 6 and neither time of exposure to EG nor inclusion of sucrose in the freezing medium provided an obvious improvement. Although bovine ET practitioners routinely attempt to collect embryos on day 7 post-oestrus, recovery of stage 7 embryos cannot always be avoided. Further investigation into factors contributing to the decreased survival of stage 7 embryos is warranted. Table 1.Effect of embryo stage on pregnancy rate of bovine embryos frozen in EG

355 COMPARISON OF Tn5 AND SLEEPING BEAUTY SYSTEMS IN BOVINE EMBRYOS AND IN OVINE OFFSPRING

2015 ◽  
Vol 27 (1) ◽  
pp. 265
Author(s):  
R. J. Bevacqua ◽  
R. Fernandéz Martín ◽  
A. Gibbons ◽  
D. Teixeira ◽  
N. G. Canel ◽  
...  
Keyword(s):  
Sleeping Beauty ◽  
Factor Ix ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Fluorescent Reporter ◽  
Human Factor Ix ◽  
Recombinant Human Factor Ix ◽  
Beta Lactoglobulin

Current techniques for the production of transgenic domestic animals remain inefficient. Only recently, DNA transposons resulted in improved efficiencies for mouse and pig transgenesis. In this work, we evaluated Tn5 and Sleeping Beauty systems for transgenesis in bovine and ovine species. First, both transposon systems were assessed in vitro in bovine embryos employing transposons carrying fluorescent reporter genes. In vitro-produced bovine zygotes were microinjected with either 1) a complex of Tn5:egfp transposon (20 ng μL–1) (protein: transgene with mosaic ends recognised by Tn5, in Mg+2 free medium), or 2) two plasmids carrying Sleeping Beauty 100X (pSB100X, 5 ng μL–1) and pT2/Venus transposon (10 ng μL–1). In vitro results for Tn5 transgenesis in bovine showed that blastocysts, Day 4 egfp embryos and egfp blastocysts rates for the group injected with Tn5:egfp did not differ from the group injected with the egfp transposon alone (73/145, 50%; 86/145, 59%; and 65/145, 45% v. 65/129, 50%; 87/129, 67%; and 57/129, 44%, respectively). For SB transgenesis, blastocysts, D4 Venus embryos, and Venus blastocysts rates did not differ between co-injection of pSB100X and pT2/Venus or injection with pT2/Venus alone (46/99, 46.5%; 64/99, 64.6%; and 33/99, 33.3% v. 41/83, 49.4%; 52/83, 62.7%; and 26/83, 31.3%, respectively). However, Venus intensity in blastocysts was markedly higher for the group co-injected with pSB100X and pT2/Venus respective to pT2/Venus alone. Both systems were assessed in vivo for the production of transgenic lambs employing a functional transposon (hrFIX, recombinant human factor IX driven by a Beta-lactoglobulin promoter). Laparoscopic artificial insemination of donor sheep was performed, and presumptive zygotes were flushed from the oviducts. The microinjections were done identically as described for the bovine embryos. A total of 24 presumptive zygotes were recovered and injected with the Tn5:hrFIX complex. Then, 21 zygotes were transferred to 5 synchronized ewes; one pregnancy of siblings was obtained, and one animal was born. Genomic DNA from skin, placenta, and blood was genotyped by PCR, but the hrFIX gene could not be detected. For the SB approach, 64 presumptive zygotes were recovered from 4 superovulated ewes, microinjected with the SB plasmids, and 21 of them were transferred to 7 oestrous synchronized recipients. The remaining zygotes were cultured in vitro and blastocysts (n = 7) were vitrified. Currently, 3 donor ewes are pregnant, one with siblings (4 total fetuses). Deliveries are expected by the end of August of this year. Our results indicate that both Tn5 and SB systems are capable of resulting in the production of transgene expressing embryos, and the presence of the transposases does not affect embryo viability. However, phenotyping of blastocyst stages does not seem to be predictive for stable transgene integration. The in vivo results will help to better address the suitability of Tn5 and SB approaches for the production of transgenic sheep.

363 FACTORS INFLUENCING PREGNANCY RATES FOLLOWING TRANSFER OF BOVINE IN VIVO EMBRYOS BIOPSIED FOR SEX DETERMINATION

2007 ◽  
Vol 19 (1) ◽  
pp. 297
Author(s):  
S. Li ◽  
W. Yu ◽  
J. Fu ◽  
Y. Bai ◽  
F. Jin ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Pregnancy Rates ◽  
Chi Square ◽  
Embryo Survival ◽  
Chi Square Test ◽  
First Time ◽  
Fresh Embryos

Data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 was analyzed to examine the effects of various factors (biopsy, freezing, sample size, embryo development and quality, in vitro culture, and recipient quality) on pregnancy rates of in vivo-biopsied embryos. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sexing determination using protocols and sexing kits supplied by AB Technology Ltd. Fresh embryos were implanted on the same day or frozen with AG freeze medium (AB Technology Ltd., Pullman, WA, USA) for later transfer. Recipients were synchronized with CIDA + PG protocols. Embryos were cultured in 6-well dishes containing 1.3 mL of holding medium (AB Technology Ltd.) in each well at room temperature (20–25�C) for examination of embryo survival in vitro. The chi-square test was used in statistic analysis. The implantation of fresh embryos after biopsy did not affect pregnancy rates (49.6%, 257/518) compared to that of non-biopsied fresh and frozen–thawed embryo groups (52.9%, 47/140 and 46.6%, 177/380, respectively). However, for biopsied embryos subjected to frozen and thawed procedures before implantation, particularly for those subjected to the removal of a larger biopsy, a reduced pregnancy rate was observed (41.8%, 297/710; P &lt; 0.01). Pregnancy rates among biopsied embryos at 3 different development stages (morula-early blastocyst, blastocyst, and expanded blastocyst) were not different. Similar results were found between embryo groups of grade 1 and 2. A significant decrease in pregnancy rate (0/10) was observed with embryos held in vitro for a longer period of time (&gt;5 h), suggesting detrimental effects of in vitro conditions on embryo survival. The highest pregnancy rate (68.0%) was observed in recipients synchronized for the first time before being implanted with biopsied embryos. Significant decreases in such rates were found in recipients synchronized for the second or third times or those with an abortion history at the first or second synchronization-implantation treatment (P &lt; 0.01). Better pregnancy rates (45.6%, 41/90; 46.1%, 76/165; and 45.5%, 5/11) were obtained for recipients implanted with biopsied embryos at Days 7.5, 8.0, and 8.5 post-heat detection, respectively, compared to 16% at Day 7 (3/18, P &lt; 0.05). It is concluded that mechanical treatment (cutting) does not reduce the survival of biopsied embryos; however, cryopreservation reduces their ability to survive in vivo. The analyses also suggest that holding embryos in vitro should not be longer than 5 h unless more favorable in vitro conditions can be provided. To achieve better results of implantation of biopsied embryos, embryo transfer should be performed during 7.5–8.5 days post-estrus, and the healthy recipients synchronized for the first time should be used.

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