scholarly journals 159 TWIN vs. SINGLE TRANSFER OF IVP HOLSTEIN HEIFER EMBRYOS TO BEEF RECIPIENTS

2005 ◽  
Vol 17 (2) ◽  
pp. 230 ◽  
Author(s):  
A. Fischer-Brown ◽  
G. Barquero ◽  
S. Clark ◽  
C. Ferguson ◽  
F. Ireland ◽  
...  
Keyword(s):  
Large Scale ◽  
Survival Rates ◽  
Fetal Loss ◽  
Cumulus Cells ◽  
Embryo Production ◽  
Chi Square ◽  
Embryo Survival ◽  
Production Scheme ◽  
Chi Square Analysis

Use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex. Here we evaluate a production scheme involving single and bilateral twin transfer of Holstein female embryos to beef cattle recipients. Holstein oocytes were fertilized with the X-bearing fraction of gender-sorted Holstein semen. Cumulus cells were removed with aid of a vortex or microfluidic device (μFD). Half of the vortexed embryos were cultured in KSOMaaBSA (control), as were all μFD embryos. The remaining vortexed embryos were cultured in control medium with 6% avian white yolk (WY). Embryo production and transfer occurred across five replicates. Cows (n = 475) were synchronized using an Ovsynch protocol. They were administered GnRH on Day −9, PGF on Day −2, and GnRH on Day 0. Half of the cows received a CIDR (1.38 g progesterone) with the 1st GnRH injection. The CIDR was removed at the time of PGF treatment. Day 7 Grade 1 blastocysts were transferred fresh 7 days after the 2nd GnRH injection. Control and WY embryos were transferred as ipsilateral singles and bilateral twins; μFD embryos were transferred singly. Pregnancy was diagnosed with ultrasound between 41–46 days and confirmed between 60–90 days; fetal sexing confirmed that 95% of fetuses were female. Effects on embryo survival were analyzed by logistic regression. Chi-square analysis was applied to survival rates. Replication affected embryo survival (P < 0.05). There was no effect of cumulus removal, medium, or CIDR use. Fetal loss between ultrasounds was greater for twin vs. single transfers (30% vs. 15%, respectively; P < 0.01). Probability of embryo survival was estimated to increase ∼0.006 with each increasing day postpartum. Five cases of hydrallantois were detected during the 5th month of gestation for 1 control twin, 1 WY single, and 3 WY twin transfers, originating from 3 replicates. On a production per transfer basis, the proportion of fetuses obtained for single and twin transfers was 30% and 55%, respectively (P < 0.001). Although there was greater embryonic loss for twin compared to single transfers, a higher percentage of cows receiving twins established and maintained pregnancy. Large-scale transfer of IVP Holstein heifer embryos to beef recipients is a feasible production scheme. Table 1. Embryo survival and pregnancy rates

22 Vitrification of bovine embryo using antifreeze polyamino acid

2019 ◽  
Vol 31 (1) ◽  
pp. 137
Author(s):  
T. Fujikawa ◽  
Y. Gen ◽  
S.-H. Hyon ◽  
C. Kubota
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Basal Medium ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Chi Square ◽  
Embryo Survival ◽  
Polyamino Acid

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound and a polyamino acid with a known functional resemblance to antifreeze proteins. We previously reported that CPLL is an effective cryoprotectant for bovine cells, sperm, and slow-frozen embryos. In this study, we investigated CPLL as a cryoprotectant for vitrified bovine embryos. We developed bovine embryos in vitro and vitrified them at the blastocyst stage. Embryos were equilibrated (3min) and vitrified (1min). Vitrified embryos were cryopreserved in LN (Cryotop® device; Kitazato Corp., Tokyo, Japan) for at least 1 week, thawed with a 0.3M sucrose warming solution, and then cultured in a basal medium (Gibco® medium 199, Grand Island, NY, USA; supplemented with 100µM 2-mercaptoethanol, 10% fetal bovine serum, and antibiotics) at 38.5°C in a humidified atmosphere (5% CO2, 5% O2, 90% N2). We evaluated the embryos morphologically for survival and hatched rate at 0, 24, 48, and 72h post-thawing. In control, the equilibration solution (ES) consisted of 7.5% (vol/vol) dimethyl sulfoxide (DMSO) and 7.5% (vol/vol) ethylene glycol, and the vitrification solution (VS) consisted of 16.5% (vol/vol) DMSO and 16.5% (vol/vol) ethylene glycol and 0.5M sucrose. In this study, CPLL was added to ES and VS at various concentrations instead of DMSO. The CPLL was added at 16.5, 11.0, 5.5, and 2.2% (wt/vol) to VS; respectively, these solutions were named P16.5, P11.0, P5.5, and P2.2. The ES was used 45% CPLL of VS each. Embryos underwent the above procedure concurrently, with testing replicated at least 3 times. We evaluated 88, 34, 38, 44, and 28 embryos with each solution (control, P16.5, P11.0, P5.5, and P2.2, respectively). Results were analysed statistically with a chi-square test and residual analysis, regarding P&lt;0.05 as significant. Survival rates were significantly greater in P11.0 at 24h post-thawing (55.7% v. 89.5%; P&lt;0.05) and in P11.0 and P5.5 at 48h post-thawing (47.7% v. 78.9% and 47.7% v. 79.5%, respectively; P&lt;0.05) relative to controls but showed no significant differences at 0h post-thawing. Hatched rates were significantly greater in P11.0 and P5.5 through 72h post-thawing relative to controls (44.7% v. 22.7% and 52.3% v. 22.7%, respectively; P&lt;0.05). The CPLL improved post-thawing embryo survival and hatched rates when applied during vitrification, thus demonstrating cryoprotective effectiveness. We conclude that CPLL acts as a low-toxicity cryoprotectant for vitrified bovine embryos, and our results are consistent with previous reports of protective CPLL effects for cells and cell membranes.

146 CO-CULTURE OF EARLY CLEAVAGE STAGE IVP EMBRYOS WITH BOVINE OVIDUCT EPITHELIAL CELLS DOES NOT IMPROVE EMBRYO DEVELOPMENT OR PREGNANCY RATES

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
R. C. Fry ◽  
K. L. Fry ◽  
W. Lan
Keyword(s):  
Embryo Development ◽  
Early Stage ◽  
Subsequent Pregnancy ◽  
Pregnancy Rates ◽  
Control Group ◽  
Becton Dickinson ◽  
Embryo Production ◽  
Chi Square ◽  
Chi Square Analysis

Pregnancy rates after the transfer of bovine IVP embryos are lower than that achieved after the transfer of MOET embryos. One reason may be that the relatively defined IVC (SOFaaBSA) culture system used in vitro is suboptimal for embryo development. We investigated whether the co-culture of early stage IVP embryos with bovine oviduct epithelial cell (BOEC) to Day 3 could provide some of the missing substrates and improve both embryo production and subsequent pregnancy rates after embryo transfer. COCs were collected by ovum pickup (OPU) from donor Brahman females, transported overnight at 38.5°C to the laboratory in HEPES-IVM media, then fertilized and cultured by our standard IVP methodology (Fry et al. 2003 Theriogenology 59, 446). Briefly, the IVC was carried out at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, 90% N2 in 4-well Nunc dishes in 500 μL of SOFaaBSA media overlayed by 500 μL of mineral oil. After 3 days of culture, the embryos were transferred to fresh IVC media and after 6 days placed in 5-mL Falcon tubes (Becton Dickinson Labware, Lincoln, NJ, USA) in fresh IVC media containing 2% FCS for overnight shipment. All grade 1 and 2 embryos were transferred to synchronized recipients on Day 7. Pregnancy diagnosis was between Days 50-90. In the BOEC treatment group, frozen aliquots of BOEC were thawed, seeded at 300,000 cells/mL, and grown for 2 days to 60-80% confluence in the Nunc wells in 500μL of DMEM/F12 media containing 10% FCS. On the first day of embryo culture, the media was removed and replaced by IVC media prior to the introduction of presumptive zygotes. After 3 days of co-culture, the embryos were transferred to fresh IVC media and thereafter cultured and transferred as for the Control group. In the Control group, 80 OPU sessions produced 1277 COCs (mean 16.0) of which 1064 (81.6%) cleaved producing 385 (33.7%) transferable embryos. Of the 337 embryos transferred to recipients (48 were vitrified), 141 (40.1%) resulted in pregnancies. In the BOEC group, 73 OPU sessions produced 1111 COCs (mean 15.2) of which 891 (80.2%) cleaved producing 388 (35%) transferable embryos that resulted in 161 (41.5%) pregnant recipients after transfer. Chi-square analysis showed no difference in either IVP embryo production or subsequent pregnancy rate between the Control group or the group where the IVP embryo was co-cultured for the first 3 days with BOEC.

255 SONIC HEDGEHOG PROMOTES IN VITRO OOCYTE MATURATION AND EMBRYO DEVELOPMENT IN GOATS

2015 ◽  
Vol 27 (1) ◽  
pp. 217 ◽  
Author(s):  
W. De-Chi ◽  
H. Jan-Chi ◽  
L. Neng-Wen ◽  
C. Hsin-I ◽  
C. Lih-Ren ◽  
...  
Keyword(s):  
Embryo Development ◽  
Sonic Hedgehog ◽  
Oocyte Maturation ◽  
Survival Rates ◽  
Cumulus Cells ◽  
Cell Surface Receptor ◽  
Control Group ◽  
Embryo Survival ◽  
Receptor System

The signalling of the Hh family peptides is mediated through a cell surface receptor system consisting of 2 proteins: patched (Ptc) and smoothened (Smo). In the absence of Hh ligand, the Hh receptor Ptc represses Smo, whereas in the presence of Hh, the suppression of Smo is lifted, leading to the activation of downstream transcriptional factors (Gli1, Gli2, and Gli3) in vertebrates. Previous studies have examined Sonic hedgehog (Shh) signalling pathways in developing and adult mouse ovaries and concluded that the Shh signalling pathway may be involved in granulosa cell proliferation and oocyte maturation. We investigated the effects of Shh protein on caprine oocyte maturation, embryo development, and embryo survival rate after transfer of vitrified/thawed in vitro-produced (IVP) embryos to recipients. Cumulus-oocyte complexes (COC) were collected by slicing ovarian follicles (1–5 mm in diameter). On average, 40 to 50 oocytes were randomly allocated to each well containing 500 μL of IVM medium and supplemented with 0 (control), 0.125, 0.25, 0.5, or 1.0 μg mL–1 recombinant mouse Shh protein. After 24 h of IVM, cumulus cells were partially removed. Oocytes were washed and transferred into a droplet of 80 μL of fertilization medium and were fertilized with frozen-thawed sperm for 18 h at 38.8°C. After IVF, presumptive zygotes were cultured on goat oviduct epithelial monolayers in M199 for 9 days. The 2 frozen-thawed selected embryos were transferred to one recipient. All data were subjected to ANOVA, using the general linear model procedure in SAS (version 9), followed by Tukey's test. Embryo survival rates were compared by using the chi-square test. The RT-PCR analyses showed that the expressions of Shh, SMO, Ptch1, and Gli1 were detected in whole ovaries, granulosa cells, COC, cumulus cells, oocytes, and oviduct epithelia except for Ptch1 in cumulus cells. Supplementation of Shh (0.25 or 0.5 μg mL–1) enhanced oocyte maturation as opposed to the control group (92.4%, n = 67 and 95.0%, n = 62 v. 86.2%, n = 64, respectively, P < 0.05). This effect could be reversed by the simultaneous addition of cyclopamine (0.5–1.0 μm), a Shh inhibitor. Similar to intact COC, denuded oocytes showed enhanced maturation (72.0%, n = 94 v. 60.5%, n = 126) with Shh supplementation. For subsequent embryo development, an improved blastocyst rate (P < 0.05) was 66.3 ± 10.9 (n = 135) when embryos were derived from the oocytes matured in the presence of 0.5 μg mL–1 Shh rather than 41.4 ± 12.9 (n = 137) of the control group. After embryo transfer, the kidding and embryo survival rates of vitrified embryos derived from the Shh-supplemented group were 56 (16 recipients) and 31% (48 embryos) higher than that 38 (16 recipients) and 15% (54 embryos) without Shh supplementation (P < 0.05). The present study suggests that Shh signalling is active in caprine ovaries during folliculogenesis and beneficial to oocyte maturation and subsequent embryo development to the blastocyst stage (in vitro) and to term.

scholarly journals 185ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS BY TRIPLE AND SINGLE TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 214
Author(s):  
A.M. van Wagtendonk-De Leeuw ◽  
A. Pugh ◽  
W. McMillan ◽  
J. Hepburn ◽  
B. Peachey ◽  
...  
Keyword(s):  
Survival Rates ◽  
Control Group ◽  
Chi Square ◽  
Embryo Survival ◽  
Actual Distribution ◽  
Holstein Friesian ◽  
Embryo Stage ◽  
Old Donor ◽  
Fresh Culture Medium

Factors that affect the viability of in vitro-produced (IVP) embryos are usually evaluated by comparing pregnancy rates of a treatment and a control group. The ‘er’ model of embryo survival (McMillan WH et al., 1998 Theriogenology 50, 1053–1070) utilizes twin embryo transfer to estimate embryo (‘e’) and recipient (‘r’) contributions to embryo survival, and allows the comparison of treatment effects without using a control group, when treatment is the only change in operations. Application of the model to data of contemporaneous single and twin transfer indicates that ‘e’ and ‘r’ are independent of the number of embryos transferred. Thus, twin transfers enable the efficient use of costly recipients while providing meaningful estimates of single embryo survival rates. The objective of this study was to assess the embryo survival rates of fresh IVP embryos of a newly established IVP lab by applying the model to triple transfers and comparing the expected embryo survival rates with those achieved for single transfers. Cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries of cows of unknown breeds or by ovum pick-up (OPU) from Holstein-Friesian 2- or 3-yr-old donor cows. COCs were matured in 500μL of TCM199+10% FCS (Life Technologies, Auckland, NZ), 10μgmL−1 FSH and LH (ICPBio, Auckland, NZ), 1μgmL−1 estradiol (Sigma, Auckland, NZ), 100μM cysteamine (Sigma) for 24h under 5% CO2 and then fertilized with 1×106 percoll-separated sperm mL−1 from a single bull (Tervit HR and Pugh PA, 2000 14th ICAR 18, 37(abst)). Twenty-four h after insemination, presumptive zygotes were transferred into 500μL mSOF (Pugh A et al., 2001 Theriogenology 55, 314 (abst)) and cultured for 4 days under humidified 5% CO2, 7% O2 and 88% N2. On Day 4, cleaved embryos were transferred into fresh culture medium and culture continued for a further 3 days under the same conditions. Embryo stage and grade were evaluated on Day 7 of culture. Grades 1, 2 and 3 (IETS manual, 2002) compact morulae and blastocysts produced from abattoir-derived COCs were transferred in triplets, while grades 1 and 2 compact morulae and blastocysts from OPU-derived COCs were transferred singly, in 0.25mL insemination straws into synchronized Holstein-Friesian heifers. Recipients received a CIDR (CIDR Cattle Insert, Pharmacia, Auckland, NZ) at Day −12 followed by a prostaglandin (Estroplan, Parnell Laboratories, Auckland, NZ ) injection at Day −6. CIDRs were removed at Day −2, followed by estrus at Day 0 (= day of IVF). Embryos were transferred on Day 7 and recipients received a CIDR after transfer (ET). CIDRs were removed at Day 19 to synchronize any returns. Two experienced practitioners performed all the transfers. Pregnancies (single transfers) and number of live fetuses (triple transfers) were confirmed at Days 60 and 42, respectively. Pregnancies were terminated between Days 62 and 65 by two prostaglandin injections 48h apart. A total of 76 single transfers resulted in 36 pregnancies (47.4%, binomial SD 5.7%). A total of 75 triple transfers (225 embryos) resulted in 98 viable fetuses (44%) and 58 pregnant recipients (77.3%). For triple transfers, the estimates for ‘e’ and ‘r’ were 0.50 and 0.89, respectively, with the product yielding an expected triple embryo survival rate of 44.1%. The actual distribution of 17, 23, 30 and 5 recipients carrying 0, 1, 2, or 3 fetuses, respectively, was not significantly different from the expected values of 16, 25, 25 and 8 estimated from the model (chi-square=2.49, NS). Estimates for ‘e’ and ‘r’ were not significantly different when combined single and triple data were included in the model (‘e’=0.55 and ‘r’=0.90), indicating that embryo survival is independent of the number of embryos transferred. Results indicate that multiple transfers do increase pregnancy rate (from 47.4 to 77.3%), but not embryo survival posttransfer (44.1 v. 47.4%). Although single ET was done with OPU-derived embryos and triple with slaughterhouse-derived embryos and results are not strictly comparable, the similarity of estimates for ‘e’ suggests that using the same in vitro-embryo assessment criteria resulted in embryos of similar intrinsic viability from the two sources. In the near future, we will perform triple transfers of cryopreserved IVP embryos and use the model to estimate embryo and recipient contributions to embryo survival of frozen IVP embryos, without using a fresh control. We will continue to build a dataset based on triple and single transfers to further assess the effect on embryo survival rates of triple and single transfers.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

scholarly journals 252 COMPARISON OF TWO SPERM SEPARATION PRODUCTS FOR USE IN BOVINE IVF

2005 ◽  
Vol 17 (2) ◽  
pp. 276 ◽  
Author(s):  
J. Pryor ◽  
S. Romo ◽  
D.D. Varner ◽  
K. Hinrichs ◽  
C.R. Looney
Keyword(s):  
Sperm Concentration ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Before And After ◽  
Chi Square Test ◽  
Group 2 ◽  
Live Sperm ◽  
Sperm Separation

In commercial bovine in vitro fertilization (IVF) companies, there is a continuous need to improve results. Efforts to maximize in vitro embryo production have included modifications in the use of sperm separation gradients. The development of commercially available sperm centrifugation gradients represents a new possibility of increasing the number of viable sperm that can be obtained from low concentration (fresh or frozen, sexed or unsexed) semen samples in order to improve the efficiency of the IVF system to make embryo production as efficient as possible. The objective of this study was to compare two different separation gradients, as follows: Group 1: Percoll (Sigma, St. Louis, MO, USA), in 45% and 90% gradients; Group 2: EquiPure (Nidacon, Gathenburg, Sweden), in top and bottom layers. Before and after separation, sperm were evaluated at 200× magnification for total motility, and then stained to assess viability at 400× with fast-green/eosin stain (Sigma). Sperm separation was performed using frozen/thawed semen from one bull. Semen was separated by centrifugation at 200g for 30 min in both density gradients. Results obtained from Groups 1 and 2 were compared by chi-square test. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 92 × 106, vs. 159 × 106 sperm/mL for EquiPure; P < 0.05) but resulted in higher motility (60% vs. 39%, respectively; P < 0.05) of separated sperm. Rates of live sperm cells were not significantly different between groups (69.5% vs. 70%, respectively; P > 0.1). These results indicate that the commercial separation medium EquiPure may be associated with higher sperm concentration levels but with lowered sperm motility when compared to Percoll for bovine sperm separation. However, Equipure provided similar percentages of live sperm when compared to Percoll, which is currently used in our laboratory.

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

scholarly journals 63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS

2005 ◽  
Vol 17 (2) ◽  
pp. 181 ◽  
Author(s):  
D. Sage ◽  
P. Hassel ◽  
B. Petersen ◽  
W. Mysegades ◽  
P. Westermann ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Media ◽  
Cumulus Cells ◽  
Cell Number ◽  
Foster Mother ◽  
Chi Square ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate ◽  
Parthenogenetic Embryos

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

scholarly journals 152 IN VITRO AND IN VIVO DEVELOPMENT OF BOVINE IVP EMBRYOS FOLLOWING SINGLE-CELL BIOPSY ON DAY 4

2005 ◽  
Vol 17 (2) ◽  
pp. 226
Author(s):  
K. Hartwich ◽  
B. Peachey ◽  
K. Cockrem ◽  
A. Marsh ◽  
A. Pugh
Keyword(s):  
Single Cell ◽  
Fetal Loss ◽  
Commercial Application ◽  
Embryo Survival ◽  
Continue Development ◽  
In Vitro Development ◽  
Fetal Survival ◽  
Cell Embryo ◽  
Vitro Development

Maximum advantage can be gained from gene discovery programs, by screening embryos carrying the desired genes(s) prior to immediate transfer. This requires an efficient and reliable genotyping system and a method for biopsy preparation that does not compromise subsequent embryo or fetal development. The present study examined the effect of removing a single-cell from the developing 8–16 cell embryo on its subsequent ability to continue development to at least the late morula stage in vitro and then survive following triple transfer to recipients. Abattoir-sourced ovaries were obtained and subjected to IVP as previously described (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). Briefly, oocytes were matured in TCM199 +10% FCS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL estradiol, and 100 μM cysteamine under 5% CO2 in air at 38.5°C for 24 h. Percoll-separated sperm (1 × 106/mL) were then co-incubated with the matured oocytes (Day 0) for 24 h with the presumptive zygotes further cultured in mSOF medium under 5%CO2, 7% O2, 88% N2. On Day 4 embryos with a minimum of 8 cells were selected and held at 38.5°C in HEPES-buffered SOF (HSOF) until biopsy at ambient temperature. Embryo biopsy was performed in HSOF medium + 5 μg/mL cytochalasin B. A single cell was removed using a 30 μm biopsy pipette. Both biopsied and control embryos were then further cultured in mSOF in individual wells prepared in a 1% agarose matrix (Peura TT 2003 Cloning Stem Cells 5, 13–24). Embryos were scored for grade and stage of development reached on Day 7, and Grades 1 and 2 blastocysts and expanded blastocysts were transferred to synchronized recipients (three embryos of the same stage and grade to each recipient; n = 50). Fetal number was determined on Day 35 and 62 of gestation. A model for embryo survival was fitted to the data (McMillan WH et al. 1998 Theriogenology 50, 1053–1070) in order to estimate embryo (“e”) and recipient (“r”) contributions to embryo survival. Values were then compared to those determined for control embryos, produced using identical IVP methods (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). A total of 358 control and 561 biopsied embryos were cultured. Removal of a single cell did not significantly affect in vitro development (60.1% vs. 56.0%; control vs. biopsy). Day 35 survival of biopsied embryos was 44.7% with calculated “e” and “r” values of 0.48 and 0.94, respectively, which did not differ from control values (44.1%; 0.50 and 0.89). However, by Day 62 fetal survival had significantly decreased with a concomitant drop in “e” but not “r” (30.0%; 0.32 and 0.94, respectively; control “e” and “r” were unchanged). In conclusion, single-cell biopsy of the 8–16-cell embryo does not affect in vitro development or embryo survival to Day 35. However, significant fetal loss occurs by Day 62 that may limit commercial application. Further work is required to elucidate the cause of and overcome fetal loss.

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