91 PRELIMINARY RESULTS OF SURGICAL EQUINE EMBRYO TRANSFER AFTER OPEN PULLED STRAW VITRIFICATION

2006 ◽  
Vol 18 (2) ◽  
pp. 154 ◽  
Author(s):  
G. Duchamp ◽  
F. Guignot ◽  
J. Grizelj ◽  
M. Vidament ◽  
P. Mermillod
Keyword(s):  
Calf Serum ◽  
Embryo Cryopreservation ◽  
Success Rates ◽  
Ringer Lactate ◽  
Embryo Survival ◽  
Preliminary Study ◽  
Higher Sensitivity ◽  
Conventional Freezing

In equine species, embryo cryopreservation is not as widely developed as in some other species. Slow freezing has been applied to equine embryos but with relatively low success rates. This higher sensitivity to conventional freezing procedures may be explained by the presence of a capsule surrounding the equine embryo that may impair cryoprotectant penetration. Recently, good in vitro embryo survival rate was obtained after open pulled straw (OPS) vitrification (Moussa et al. 2005 Theriogenology 64, 1619–1632). The aim of the present study was to evaluate in vivo survival of vitrified embryos five days after surgical transfer into Welsh pony mares. Morulae (M), early blastocysts (EB), and blastocysts (B) ranging from 140 to 320 μm in diameter were collected (n = 20) in a Ringer lactate solution on Day 6.75 after ovulation. Before vitrification, embryos were assessed morphologically and their size was measured (McKinnon and Squires 1988 J. Am. Vet. Med. Assoc. 192, 401–406). Then, embryos were vitrified using the OPS method described by Berthelot et al. (2001 Reprod. Nutr. Dev. 41, 267–272). Briefly, embryos were washed twice in HEMES-TCM-199 + 20% newborn calf serum (NBCS) for 1 min, equilibrated in HEPES-TCM-199 + 20% NBCS with 7.5% dimethyl sulfoxide (DMSO) + 7.5% ethylene glycol (EG) for 3 min, and then with 18% DMSO + 18% EG + 0.4 M sucrose for 45 s. One embryo was then loaded per straw. For transfer, four straws were quickly thawed (5 s in air) and the narrow end of the straw containing the embryo was immersed in HEPES-TCM-199 + 20% NBCS + PBS + 0.2 M sucrose. Five to 8 min after thawing, four embryos were surgically transferred into the cranial portion of the uterine horn in each of five pony mare recipients. Five days after transfer, embryos recovered by transcervical flushing of the uterus were classified as viable if morphology was normal, no dark inner cells were present, the capsule was intact, and the diameter was at least 1000 μm. The results are shown in the table. One recipient of vitrified embryos had an endometritis and no embryo was recovered. From the four other recipients, nine embryos were recovered out of 16 (56%) transferred, seven of which were viable (44%). The results of the present preliminary study demonstrating survival of equine embryos transferred after OPS vitrification is very encouraging. However, the results should be confirmed by birth of foals after transfer of OPS-vitrified embryos to recipients. Table

44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
K. Kaneyama ◽  
S. Kobayashi ◽  
S. Matoba ◽  
Y. Hashiyada ◽  
K. Imai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Nuclear Transplantation ◽  
Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 146-157 ◽  
Author(s):  
Daniela Martins Paschoal ◽  
Mateus José Sudano ◽  
Midyan Daroz Guastali ◽  
Rosiára Rosária Dias Maziero ◽  
Letícia Ferrari Crocomo ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Similar Rate ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Vitrification Solution ◽  
Oviductal Fluid

SummaryThe objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

162 IMPROVED EMBRYO SURVIVAL AND QUALITY WITH EMCARE II

2006 ◽  
Vol 18 (2) ◽  
pp. 189
Author(s):  
A. Harvey ◽  
M. Lane ◽  
J. Thompson
Keyword(s):  
Calf Serum ◽  
Cell Number ◽  
Tunel Staining ◽  
Embryo Survival ◽  
Improved Outcomes ◽  
Student’S T ◽  
The Impact ◽  
Student’S T Test

Collection of embryos exposes them to a number of stresses, including light, air, and changes in temperature. Improvement of holding media to reduce the impact of handling stresses on the embryo during in vivo collection and transfer is therefore beneficial to ensure maintenance of viability following transfer. The aim of this study was to compare the effect of holding IVP-derived blastocysts at 25°C in Emcare I (ECMI, Emcare, Dallas, TX, USA) with those held in Emcare II (ECMII), a proprietry formulation designed to reduce in vitro-induced stress. In vitro-produced bovine embryos were generated using standard protocols. Blastocysts were randomly allocated to either ECMI or ECMII (ICPBio, Aukland, New Zealand) on Day 7 and were held at 25°C for a period of 24 h, after which they were cultured in Cook Bovine Blast (Cook Australia, Brisbane, Australia) supplemented with 10% fetal calf serum for 48 h. At 24 and 48 h, embryos were scored for hatching, and a cohort removed for TUNEL staining at each time point. Differences were analyzed by Student's t-test. At both 24- and 48-h culture, hatching rates tended to be higher for embryos held in ECMII than in ECMI (Table 1). The level of apoptosis at 48 h was reduced in blastocysts held in ECMII (P = 0.06). Moreover, the total cell number of hatched blastocysts at 48 h was significantly increased (1.5-fold) in those held in ECMII (P = 0.01). Results suggest that the formulation of ECMII improves the ability of IVP bovine blastocysts to re-expand and hatch following an imposed stress (25°C for 24 h). Furthermore, ECMII improves overall embryo quality through a reduction in the percentage of cells undergoing apoptosis as well as through increased cell numbers, evident 48 h following cessation of the stress. We suggest that Emcare II reduces the impact of (or increases the embryo's tolerance to and recovery from) an imposed stress, which, although severe in the present study, may provide improved outcomes following embryo transfer in field situations. Table 1. Hatching and apoptosis of blastocysts held at 25°C for 24 h in Emcare I or Emcare II This work was supported with funding by ICPBio (NZ).

49 PRACTICAL APPLICATION OF THE HOLLOW FIBER VITRIFICATION METHOD FOR CRYOPRESERVATION OF MAMMALIAN EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 138 ◽  
Author(s):  
A. Uchikura ◽  
T. Wakayama ◽  
S. Wakayama ◽  
H. Matsunari ◽  
M. Maehara ◽  
...  
Keyword(s):  
High Performance ◽  
Sucrose Solution ◽  
Calf Serum ◽  
Hollow Fibre ◽  
Mouse Embryos ◽  
Embryo Cryopreservation ◽  
Cell Stage ◽  
Common Marmosets

We recently developed the hollow fibro vitrification (HFV) method, which is a novel, high-performance embryo cryopreservation method (Matsunari et al., 2012). In this study, we aimed to verify the applicability of the HFV method for cryopreserving various types of embryos; BDF1 mouse embryos at the 2-cell stage, porcine parthenogenetic morulae derived from in vitro-matured oocytes, bovine morulae produced by in vitro maturation/fertilization (LIAJ Animal Biotechnology Center, Tokyo, Japan), and in vivo-derived blastocysts of common marmosets were vitrified, and their survival was assessed by culture or transfer. The embryos were vitrified using 20 mM HEPES-buffered TCM-199 containing 20% calf serum as a base medium. Cellulose acetate hollow fibres (25 mm) containing 1 to 20 embryos were placed in an equilibration solution containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 5 to 7 min, followed by incubation for 1 min in vitrification solution containing 15% EG, 15% DMSO, and 0.5 M sucrose. The embryos were then vitrified by immersion in LN. The embryos were devitrified by immersing the hollow fibre in a 1 M sucrose solution at 38.5°C, which was followed by stepwise dilution of the cryoprotectants and washing. For a subset of the vitrified mouse embryos, rewarming in a non-ultra-rapid manner by melting a hollow fibre in air at room temperature for 5 s was tested. Embryo transfer was performed to assess the viability of the vitrified mouse embryos. For porcine embryos, vitrification in LN vapor (–150°C) was tested. Development of the vitrified mouse embryos to blastocysts was equal to that of the non-vitrified embryos [105/110 (95.5%) v. 109/110 (99.1%)]. Post-transfer development to fetuses was also equal between the vitrified and non-vitrified embryos [pregnancy rates: 4/4 v. 2/2; developmental rates: 55/80 (68.8%) v. 35/40 (87.5%)]. Non-ultra-rapid rewarming did not decrease the survival of the vitrified mouse embryos [blastocysts: 94/100 (94.0%); pregnancy: 4/4; fetuses: 55/80 (68.8%)]. Blastocyst formation was equivalent for vitrification of porcine embryos in LN vapor [27/34 (79.4%)], direct immersion into LN [28/35 (80.0%)], and the non-vitrified control [31/32 (96.9%)]. Vitrification of 191 bovine morulae resulted in 153 (80.1%) blastocysts. In preliminary experiments, survival of marmoset blastocysts was 100% (n = 6). These data demonstrate that the HFV method is (1) effective for embryos of various species and production methods; (2) effective even for porcine in vitro-derived morulae, which are highly cryosensitive; and (3) amenable to modifications such as non-ultra-rapid warming and cooling in LN vapor, increasing the potential applicability of the HFV method. For instance, vitrification in LN vapor may allow embryo cryopreservation with high hygienic standards. This study was supported by JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project.

92 DOES BLASTOCENTESIS AFFECT CRYOPRESERVATION SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS?

2017 ◽  
Vol 29 (1) ◽  
pp. 153
Author(s):  
D. A. Tutt ◽  
R. E. Lyons ◽  
M. K. Holland
Keyword(s):  
Human Error ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Statistical Testing ◽  
Embryo Cryopreservation ◽  
Embryo Viability ◽  
Embryo Survival ◽  
Biopsy Procedure

The cattle industry primarily employs embryo bisection in order to obtain genetic samples for pre-implantation screening and selection of embryos. Although practical and rapid, bisection is invasive and adversely affects embryo viability and cryopreservation. An alternative biopsy approach is to aspirate the blastocoele fluid (referred to as blastocentesis), which not only provides a genetic sample, but also has the potential to improve cryopreservation (Palini et al. 2013 Repro. Biomed. 26, 603–610). This study investigates blastocentesis as a low impact biopsy procedure to rapidly sample bovine blastocysts with limited effect on embryo cryopreservation survival. In vitro-produced embryos were selected at expanded blastocyst stage and placed in a 50-μL drop of holding media on an inverted microscope. The embryo was held using a glass holding pipette attached to a micromanipulator, oriented so that the inner cell mass was toward the bottom of the view. A 7-μm spiked intracytoplasmic sperm injection pipette attached to the other micro-manipulator was used to pierce the blastocoele cavity and aspirate the blastocoele fluid. Once removed, the aspirate was transferred into 4-μL TE buffer for later genetic analysis. Collapsed blastocysts were then vitrified in ~7 μL 16.5% ethylene glycol, 16.5% dimethyl sulfoxide in TCM-199 (Hanks salts) with 20% FCS and 0.5 M sucrose. Embryos were held for a minimum of 1 week and then thawed and assessed for survival. Post-cryopreservation embryo survival was measured as the proportion of embryos that re-expanded after 48 h in culture. One-way ANOVA was used for statistical testing. A total of 181 control (intact) and 182 blastocentesis embryos were vitrified over 6 replicates. In all but one replicate, non-biopsied control embryos had higher re-expansion rates. Overall, the re-expansion rate was significantly (P = 0.05) higher for control embryos (73.5%) than blastocentesis embryos (61.5%) (Table 1). Initial experiments would suggest embryo survival is affected by the biopsy procedure; however, because this was not the case with every replicate, this may be batch or technician/human error dependent. Further study is required to assess full effect of blastocoele fluid aspiration on embryo cryopreservation, particularly investigating effectiveness for in vivo-produced embryos and subsequent effect on pregnancy rates. Likewise, further investigation is required to assess whether the sample collected is sufficient to allow accuracy over a variety of genetic tests. More than 20 embryos can easily be sampled in an hour using this technique, making it a rapid and efficient process. Given the speed and compatibility with cryopreservation, this sampling procedure may offer an alternative to current techniques used for cattle embryo genetic assessment. Table 1. Post-thaw survival rates of in vitro-produced embryos vitrified after blastocentesis1

scholarly journals Developmental Plasticity in Response to Embryo Cryopreservation: The Importance of the Vitrification Device in Rabbits

Animals ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 804 ◽  
Author(s):  
Ximo Garcia-Dominguez ◽  
José Salvador Vicente ◽  
Francisco Marco-Jiménez
Keyword(s):  
Body Weight ◽  
Peripheral Blood ◽  
Developmental Plasticity ◽  
Blood Analysis ◽  
Embryo Cryopreservation ◽  
Lactation Performance ◽  
Embryo Survival ◽  
Daily Growth

In this study, we evaluated the effect of embryo vitrification using two different devices on adulthood phenotype in rabbits. In vitro development, prenatal embryo survival, body weight, growth performance, haematological and biochemical peripheral blood analysis, reproductive performance, and lactation performance traits were compared between the experimental groups. They derived from naturally-conceived embryos (NC), fresh-transferred embryos (FT), vitrified-transferred embryos using mini-straw (VTs), or vitrified-transferred embryos using Cryotop (VTc). Straw-vitrified embryos exhibited lower in vitro developmental rates and in vivo survival rates following embryo transfer compared to its Cryotop-vitrified counterparts. Moreover, the VTs group exhibited higher foetal losses than VTc, FT, and NC groups. Independently of the vitrification device, vitrified-transferred (VT) offspring showed a skewed sex ratio in favour of males, and an increased birth bodyweight. In contrast, postnatal daily growth was diminished in all ART (i.e., FT and VT) animals. In adulthood, significant differences in body weight between all groups was founded—all ART progenies weighed less than NC animals and, within ART, VT animals weighed less than FT. For VT groups, weight at adulthood was higher for the VTs group compared with the VTc group. Peripheral blood parameters ranged between common values. Moreover, no differences were found in the fertility rates between experimental groups. Furthermore, similar pregnancy rates, litter sizes, and the number of liveborns were observed, regardless of the experimental group. However, decreased milk yield occurred for VTc and FT animals compared to VTs and NC animals. A similar trend was observed for the milk composition of dry matter and fat. Concordantly, reduced body weight was found for suckling kits in the VTc and FT groups compared to VTs and NC animals. Our findings reveal that developmental changes after the embryo vitrification procedure could be associated with an exhibition of the embryonic developmental plasticity. Moreover, to our best knowledge, this study reports the first evidence demonstrating that the vitrification device used is not a trivial decision, providing valuable information about how the cooling–warming rates during vitrification can be partly responsible of the postnatal phenotypic variations.

49 SUCCESSFUL CRYOPRESERVATION USING LOW ETHYLENE GLYCOL CONCENTRATION FOR IN VITRO-PRODUCED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 132
Author(s):  
M. Takayama ◽  
S. Sato ◽  
Y. Nishimura ◽  
K. Imai ◽  
O. Dochi
Keyword(s):  
Ethylene Glycol ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Lower Survival Rate ◽  
Treatment Groups ◽  
Chi Squared ◽  
Hatching Rates

In vitro-produced (IVP) bovine embryos tend to have a lower survival rate after cryopreservation than in vivo embryos do. Therefore, the freezing medium (FM) and concentration of cryoprotectant are very important factors. This study was to investigate the effect of 1.2 M ethylene glycol (EG) with 0.1 M sucrose (SUC) on survival of IVP embryos after freezing. The COC were matured in 25 mM HEPES-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS) and 0.02 AU mL−1 FSH. Oocytes (20 to 25) were cultured in 100-μL droplets of maturation medium for 20 h. After 6 h of gamete co-culture (5 × 106 sperm/mL), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS for 9 days (fertilization = Day 0). Only the expanded blastocysts from Days 7 to 9 were used in this experiment and separated into 3 treatment groups. The first and second groups were frozen in Dulbecco’s phosphate-buffered saline (D-PBS) supplemented with 20% CS, 0.1 M SUC, and 1.2 or 1.5 M EG (groups 1.2 or 1.5 M EG), respectively. The third group was D-PBS supplemented with 20% fetal calf serum (FCS), 0.25 M SUC, and 1.4 M glycerol (group GLY). In each group, embryos were equilibrated with their FM for 10 min and loaded into 0.25-mL straws individually. These straws were placed into the cooling chamber of a programmable freezer precooled to −7°C. After 2 min, the straws were seeded and then held for a further 13 min at −7°C. Then, the straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. The cryopreserved embryos were thawed by allowing the straws to stand in air for 7 s and then warming them in a 30°C water bath for 20 s. The thawed embryos were washed twice using 38°C D-PBS supplemented with 20% FCS. Subsequently, they were immersed in the same medium, held at 38°C for 10 min, and then each embryo was cultured in 20-μL droplets of TCM199 supplemented with 20% FCS and 0.1 mM β-mercaptoethanol for 72 h. The rates of embryos developing to the re-expanded and hatching blastocyst stages were determined 72 h after thawing. All data were analysed by the chi-squared test with Yates’ correction. The re-expanded and hatching rates of frozen-thawed embryos after 72 h in culture were not significantly different between 1.2 M EG (n = 39: 71.8% and 69.2%), 1.5 M EG (n = 38: 76.3% and 63.2%), and 1.4 M GLY (n = 37: 75.7% and 64.9%) groups (P > 0.05). Survival and hatching rates according to embryo quality were also not significantly different between 1.2 M EG (good n = 18: 88.9% and 88.9%; fair n = 21: 57.1% and 52.4%), 1.5 M EG (good n = 19: 89.5% and 84.2%; fair n = 19: 63.2% and 42.1%), and 1.4 M GLY (good n = 18: 77.8% and 66.7%; fair n = 19: 73.7% and 63.2%) (P > 0.05). In conclusion, cryoprotectant type and concentration did not affect embryo survival or development after cryopreservation in this study. Therefore, the ethylene glycol concentration used for the cryopreservation of IVP embryos can be reduced.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

Preclinical Evaluation of a Bone-Marrow Autograft Culture Procedure for Generating Lymphokine-Activated Killer Cells in Vitro

1992 ◽  
Vol 3 (suppl b) ◽  
pp. 123-127 ◽  
Author(s):  
Hans-Georg Klingemann ◽  
Heather Deal ◽  
Dianne Reid ◽  
Connie J Eaves
Keyword(s):  
Bone Marrow ◽  
Calf Serum ◽  
Cell Activity ◽  
Hematopoietic Cells ◽  
Lak Cells ◽  
High Dose ◽  
Lymphokine Activated Killer Cells ◽  
Lak Cell

Despite the use of high dose chemoradiotherapy for the treatment of acute leukemia. relapse continues to be a major cause of death in patients given an autologous bone marrow transplant. Further augmentation of pretransplant chemotherapy causes life threatening toxicity to nonhematopoietic tissues and the effectiveness of currently available ex vivo purging methods in reducing the relapse rate is unclear. Recently, data from experimental models have suggested that bone marrow-derived lymphokine (IL-2)-activated killer (BM-LAK) cells might be used to eliminate residual leukemic cells both in vivo and in vitro. To evaluate this possibility clinically, a procedure was developed for culturing whole marrow harvests with IL-2 prior to use as autografts, and a number of variables examined that might affect either the generation of BM-LAK cells or the recovery of the primitive hematopoietic cells. The use of Dexter long term culture (LTC) conditions, which expose the cells to horse serum and hydrocortisone. supported LAK cell generation as effectively as fetal calf serum (FCS) -containing medium in seven-day cultures. Maintenance of BM-LAK cell activity after a further seven days of culture in the presence of IL-2 was also tested. As in the clinical setting. patients would receive IL-2 in vivo for an additional week immediately following infusion of the cultured marrow autograft. Generation ofBM-LAK activity was dependent on the presence of IL-2 and could be sustained by further incubation in medium containing IL-2. Primitive hematopoietic cells were quantitated by measuring the number of in vitro colony-forming progenitors produced after five weeks in secondary Dexter-type LTC. Maintenance of these 'LTC-initiating cells' was unaffected by lL-2 in the culture medium. These results suggest that LAK cells can be generated efficien tly in seven-day marrow autograft cultures containing IL-2 under conditions that allow the most primitive human hematopoietic cells currently detectable to be maintained.

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

Sign in / Sign up

Export Citation Format

Share Document