103A NEW PAPER CONTAINER FOR THE VITRIFICATION OF BOVINE EMBRYOS

Vitrification has been used to eliminate ice crystal formation during the cryopreservation of mammalian embryos. However, this method may introduce some problems such as loss of eggs during cryopreservation (EM grid) and damage to the zona pellucida. This study examined an alternative container (paper) for the vitrification of in vitro-produced bovine blastocysts. Bovine oocytes were aspirated from slaughterhouse ovaries and cultured in TCM-199 supplemented with 25mM NaHCO3, 10% (v:v) FBS, 0.22mM sodium pyruvate, 25mM gentamycin sulfate, 10μgmL−1 FSH (Follitropin V; Vetrepharm, Canada) and 1μgmL−1 estradiol-17β for 24h. Matured oocytes were co-cultured with sperm (1–106mL−1) treated by percoll gradient for 42–44h. Cleaved embryos were cultured in 50μL CR1aa medium containing 0.4% BSA for 5 days. Blastocysts were exposed to 5.5M ethylene glycol in CR1aa medium for 20s. The blastocyst suspensions were vitrified by one of three methods: 1) aspiration into a 0.25-mL plastic straw (10 embryos/straw), heat sealing and immediate plunging into LN2; 2) transfer of a (∼5μL) drop containing 10 blastocysts onto a EM grid and immediate plunging into LN2; or 3) transfer of a (∼5μL) drop containing 10 blastocysts onto a piece of weighing paper (5mm by 5mm; VWR, West Chester, PA, USA) and immediate plunging into LN2. Straws were thawed by holding in air for 10s and then transfer into 37°C water. The embryos were recovered from the straw and transferred into a solution of 0.5M sucrose in CR1aa at 25°C for 1min. EM grids and paper containers were warmed by transfer into 3mL of a solution of 0.5M sucrose in CR1aa medium at 25°C for 1min. Embryos were then diluted serially by transfer into 0.25 and then 0.125M sucrose solutions (1-min steps), and then rinsed and cultured in CR1aa medium supplemented with 10% FBS. After thawing, the recovery rates of embryos from EM grids, straws and paper containers were not significantly different (Table 1). Broken zonae pellucidae were observed after thawing of embryos recovered from straws and EM grids, but not from the paper container. The survival rates of blastocysts cryopreserved on EM grids and paper containers (respectively, 78.1 and 77.1%) were significantly higher (P<0.05) than that of straws (52.1%). The in vivo developmental potential of blastocysts vitrified on EM grids and paper containers was assessed by the transfer of, respectively, 102 and 3 thawed embryos into recipient cows. Pregnancy rates were, as anticipated, 28 and 67%. These results suggest that paper may be an inexpensive and useful container for the cryopreservation of mammalian embryos. Table 1 The viability of vitrifield-thawed bovine embryos using various containers

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Cryopreservation of in vitro-produced bovine embryos by vitrification: In pursuit of a simplified, standardized procedure that improves pregnancy rates to promote cattle industry use

Biotechnology in Animal Husbandry ◽

10.2298/bah2003251h ◽

2020 ◽

Vol 36 (3) ◽

pp. 251-270

Author(s):

Van Do ◽

Andrew Taylor-Robinson

Keyword(s):

Slow Rate ◽

Survival Rates ◽

Slow Freezing ◽

Embryo Cryopreservation ◽

Human Embryos ◽

Bovine Embryo ◽

Bovine Embryos ◽

Cattle Industry

The goal of cryopreservation is to retain the original stage of gametes and embryos after they have endured cooling and warming. Slow freezing is a standard method for in vivo-derived bovine embryo cryopreservation, threefifths of such embryos being frozen by this method globally. However, it is evident that slow freezing is not efficient for cryopreserving in vitro-produced bovine embryos. Hence, only one-third of in vitro-produced bovine embryos are cryopreserved. Vitrification is a preferred method for storage of human embryos; consequently, it has been explored as a novel means to store in vitro-produced bovine embryos, for which it shows considerable promise as an alternative to slow freezing. This is due to several reasons: vitrification is often less time-consuming than slow freezing; it does not need expensive slow rate freezing machines; and it has been proven to have comparatively higher survival rates. Yet, in the cattle industry vitrification continues to present shortcomings, such as possible toxicity of vitrification solutions and failure to standardize methods, which pose a challenge for its application to in vitro-produced bovine embryos. Therefore, determining the most suitable procedure is crucial to make vitrification more practical in commercial settings.

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

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab51 ◽

2014 ◽

Vol 26 (1) ◽

pp. 139 ◽

Cited By ~ 1

Author(s):

K. Tsuchiya ◽

A. Ideta ◽

Y. Nishimiya ◽

S. Tsuda ◽

Y. Aoyagi

Keyword(s):

Pregnancy Rate ◽

Storage Period ◽

Bovine Embryo ◽

Bovine Embryos ◽

Hypothermic Preservation ◽

Mammalian Embryos ◽

Simple Medium ◽

Hypothermic Storage

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

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88 EFFECT OF VITRIFICATION PROCEDURE ON SURVIVAL RATE OF BOVINE EMBRYOS PRODUCED IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab88 ◽

2011 ◽

Vol 23 (1) ◽

pp. 149

Author(s):

E. Y. Herrera ◽

C. de Frutos ◽

R. Laguna-Barraza ◽

A. Gutierrez-Adan ◽

D. Rizos

Keyword(s):

Survival Rate ◽

Survival Rates ◽

Calf Serum ◽

Basal Medium ◽

Bovine Embryos ◽

Oviduct Fluid ◽

Bovine Oocytes ◽

Cryopreservation Method ◽

Hatching Rates

Vitrification as a cryopreservation method has many advantages compared with slow freezing. Many variables in the vitrification process exists that influence the survival rates of vitrified oocytes and embryos. These include the cryoprotectants (type, concentration, and duration of exposure), the temperature of the vitrification solution at exposure, the device used for vitrification, and the quality and developmental stage of embryos. It is worthwhile to mention that vitrification protocols successfully used in bovine oocytes and embryos have been used also with human oocytes and embryos. Vitrification is relatively simple, requires no freezing equipment, and relies on the placement of the embryos in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed straws. The aim of the present study was to evaluate the efficiency of 4 different vitrification protocols on the survival rate of in vitro produced (IVP) bovine embryos. Blastocysts were produced by a standard IVP procedure following in vitro maturation, fertilization, and culture in synthetic oviduct fluid supplemented with 5% fetal calf serum (FCS). On Day 7 (Day of IVF = Day 0), a total of 297 blastocysts were vitrified using (i) the open pulled straw (OPS) in 20% DMSO and 20% ethylene glycol (EG) in a basal medium of TCM-199 with HEPES supplemented with 20% FCS; (ii) the modified OPS, in 20% DMSO, 20% EG, and 0.5 M sucrose in a basal medium of phosphate buffer saline (PBS) supplemented with 20% FCS; (iii) the cryoloop, in 15% DMSO, 15% EG, 10 mg mL–1 Ficoll 70, and 0.65 M sucrose in a basal medium of PBS supplemented with 20% FCS; and (iv) in 0.25 straws in 20% glycerol, 20% EG, 0.3 M sucrose, 3% polyethylene glycol, and 0.3 M xylose in a basal medium of PBS. After warming, embryos were placed in culture for additional 24 h. Re-expansion and hatching rates were measured at 2 and 24 h after warming. Data were analysed by 1-way ANOVA. At 2 h post-warming, the re-expansion of blastocysts vitrified with cryoloop was significantly higher compared with OPS, modified OPS, and the 0.25 straw methods (54.08 ± 15.53 v. 10.40 ± 3.00, 22.67 ± 9.20, and 8.82 ± 2.15, respectively; P ≤ 0.028). At 24 h post-warming, only embryos from cryoloop and modified OPS were still alive with a survival rate of embryos vitrified with cryoloop significantly higher than that of those vitrified with modified OPS (48.45 ± 17.56 v. 3.75 ± 3.75, respectively; P ≤ 0.007). Hatching rates at 24 h post-warming were not different between cryoloop and modified OPS groups (5.63 ± 4.40 and 1.25 ± 1.25, respectively). These results clearly demonstrate that embryo cryotolerance is affected by the method used for cryopreservation. Moreover, cryoloop vitrification was found to be more effective than OPS and 0.25 straw methods for the cryopreservation of bovine embryos.

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Potential survival rates to term for transferred in vitro and in vivo derived bovine embryos

Theriogenology ◽

10.1016/0093-691x(96)84706-6 ◽

1996 ◽

Vol 45 (1) ◽

pp. 233 ◽

Cited By ~ 7

Author(s):

W.H. McMillan

Keyword(s):

Survival Rates ◽

Bovine Embryos

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102VITRIFICATION OF IN VITRO-PRODUCED BOVINE AND OVINE EMBRYOS USING THE MINIMUM VOLUME COOLING CRYOTOP METHOD

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab102 ◽

2004 ◽

Vol 16 (2) ◽

pp. 172 ◽

Cited By ~ 8

Author(s):

J.M. Kelly ◽

D.O. Kleemann ◽

M. Kuwayama ◽

S.K. Walker

Keyword(s):

Sucrose Solution ◽

Survival Rates ◽

Minimum Volume ◽

Bovine Embryos ◽

Embryo Viability ◽

Embryo Survival ◽

Mammalian Embryos ◽

Fresh Embryos ◽

Hatching Rates

Considerable progress has been achieved in the cryopreservation of mammalian embryos. The use of vitrification minimizes chilling injuries by increasing cooling and warming rates. This study assesses the effect of vitrification using the minimum volume cooling (MVC) method (Kuwayama & Kato 2000 J. Assist. Reprod. Genet. 17, 477) on in vitro-produced bovine and ovine embryos. A total of 1756 ovine and 753 bovine cumulus-oocyte complexes were obtained from the abattoir and matured, fertilized (Day 0) and cultured in vitro (Walker et al., 1996 Biol. Reprod. 55, 703–708, Kelly et al., 1997 Theriogenology 47, 291). Overall cleavage rates were 93.7% and 80.5% respectively. Embryos were vitrified (OPS or MVC method) on Days 5 (morula, compact morula), 6 (expanded blastocyst, blastocyst, compact morula) or 7 (hatched and hatching blastocysts, expanded blastocyst, blastocyst). Embryos were equilibrated with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 3min and then exposed to 16.5% EG, 16.5% DMSO, 0.5M sucrose and 20% FCS for 30s. Embryos were loaded onto either an MVC plate (Cryotop, Kitazato Supply Co, Toyko, Japan) or open pulled straw (OPS) and plunged into liquid nitrogen. After 5 days, embryos were thawed directly into 1.25M sucrose solution at 38.5°C, followed by stepwise dilution of the cryoprotectants. Embryo survival was assessed by culture to Day 8 and compared to the development of non-vitrified control embryos (Table 1). Variables were assessed using procedure CATMOD in SAS. The Cryotop method yielded a significantly higher percentage of viable ovine embryos after thawing compared with OPS (P<0.0001); neither day nor treatment x day interaction was significant (P>0.05). A significant interaction between vitrification treatment and day (P<0.007) indicated that the percentage of hatched embryos peaked at Day 6 using the Cryotop method compared with Day 7 for OPS. Hatching rates for fresh and vitrified embryos were similar at Day 7 and were independent of treatment. With the Cryotop method, day of vitrification did not influence the percentage of Days 6 and 7 bovine embryos that hatched after thawing but, on each day, this figure was significantly higher (P<0.003 and P<0.0001, respectively) than that obtained with fresh embryos. To further assess embryo viability, 36 fresh, 52 OPS and 56 Cryotop vitrified Day-6 in vitro-produced ovine embryos were transferred to synchronized recipients. Survival rates to Day 13 were 29/33 (87.9%), 23/36 (63.9%) and 42/51 (82.4%), respectively (P<0.05). This study demonstrates that using the MVC Cryotop method, the viability of vitrified embryos, as assessed at Days 8 and 13, is similar to that obtained with fresh embryos. Table 1

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Developmental potential of bovine oocytes matured in vitro or in vivo

Theriogenology ◽

10.1016/0093-691x(86)90218-9 ◽

1986 ◽

Vol 25 (1) ◽

pp. 164 ◽

Cited By ~ 7

Author(s):

M.L. Leibried-Rutledge ◽

E.S. Critser ◽

W.H. Eyestone ◽

D.L. Northey ◽

N.L. First

Keyword(s):

Developmental Potential ◽

Bovine Oocytes

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Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.771996 ◽

2021 ◽

Vol 8 ◽

Author(s):

Cristina Cuello ◽

Cristina A. Martinez ◽

Josep M. Cambra ◽

Alejandro González-Plaza ◽

Inmaculada Parrilla ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Pathways ◽

Survival Rates ◽

Gene Expression Pattern ◽

Control Group ◽

High Survival ◽

Embryo Survival ◽

Developmental Potential

Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70–75%), the pregnancy loss is 5–15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip® Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted <0.05 and a fold change cut-off of ±1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 ± 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.

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119CRYOPRESERVATION OF BIOPSIED BOVINE EMBRYOS PRODUCED IN VITRO USING ARABINOGALACTAN AND 1.5M ETHYLENE GLYCOL

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab119 ◽

2004 ◽

Vol 16 (2) ◽

pp. 182

Author(s):

B. Shangguan ◽

N. Yang ◽

R. Vanderwal ◽

M.D. Darrow

Keyword(s):

Ethylene Glycol ◽

Survival Rates ◽

Field Trials ◽

Bovine Embryos ◽

Embryo Survival ◽

Ag Addition ◽

Freezing Medium ◽

Frozen Embryos

Arabinogalactan (AG) in combination with 1.5M ethylene glycol (EG) has been used successfully in cryopreserving biopsied in vivo bovine embryos (Darrow, 2002 Theriogenology 57(1), 531). This study was undertaken to investigate the efficiency of AG addition in a freezing medium (FM) to cryopreserve biopsied bovine embryos produced in vitro (IVP). Blastocysts of grade 1 were collected at Days 7 and 8 post-insemination. After biopsy with a small blade, embryos were transferred to CR1aa medium and cultured for 2 hours (h) before being frozen. In experiment 1, a group of unbiopsied embryos were handled in a manner similar to that used for the biopsied embryos. Embryos were frozen using either 1.5M EG+0.1M sucrose (EG+) (AB Technology, Pullman, WA, USA) or a FM containing 1.5M EG and different concentrations of AG (AG1, 2 and 3, courtesy of AB Technology). Embryos remained in FM for 10 (exp.1), 5 (exp.2), 5 and 10 (exp.3) or 5, 10, and 20 (exp.4) minutes before being loaded into a freezer and cooled down to −35°C at 0.3°C/min. Frozen embryos were thawed (35°C, 20 seconds) and cultured in CR1aa at 38.5°C for 3 days. Embryo survival rates (S%) were recorded at 24, 48 and 72h post-thawing. Data were compared with t-test or ANOVA procedures using SigmaStat 3.0. Results from exp.1 (Table) indicate that biopsied and unbiopsied embryos survived well in EG+ or AG2. While the biopsy procedure did not affect the post-thaw S% of embryos in either FM, no significant differences were observed between embryos frozen with EG+ and AG2 (P=0.055). Reducing or increasing AG concentration in FM by 2-fold (AG1 and 3, respectively) did not significantly affect the post-thaw S% at 24h (EG+, 80.0%, n=133; AG1, 83.3%, n=135; AG2, 71.4%, n=137 and AG3, 75.0%, n=135; P=0.217, exp.2). However, shortened exposure from 10 to 5 minutes to AG2 resulted in an improvement in S% at 24h, from 35.7% (n=80) to 61.4% (n=82, P<0.05; exp.3). When AG1 (=0.5×AG2) was used in the FM the S% at 24h after different exposure times was not significant (5 minutes, 77.8%, n=179; 10 and 20 minutes, 66.7%, n=179 and 183; P=0.472, exp.4). This study demonstrates that addition of AG to the FM effectively sustains the viability of biopsied IVP embryos during freezing and any potential harmful impact of AG on embryo survival can be minimized by reducing AG concentration or the time of embryo exposure to AG prior to freezing. Further studies are needed to determine optimal AG concentration. Currently, field trials are underway to evaluate the ability of AG medium to promote pregnancies from frozen, biopsied IVP embryos. Table 1 Post-thaw survival rates of biopsied IVP embryos frozen in ethylene glycol with sucrose (EG+) and a FM containing arabinogalactan (AG2). Data are means±SEM

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Influences of zinc on fertilisation and development of bovine oocytes in vitro

Zygote ◽

10.1017/s096719949900057x ◽

1999 ◽

Vol 7 (3) ◽

pp. 195-201 ◽

Cited By ~ 17

Author(s):

J.L. Stephenson ◽

B.G. Brackett

Keyword(s):

Sperm Motility ◽

Oocyte Maturation ◽

Follicular Fluid ◽

Bovine Embryos ◽

Sperm Preparation ◽

Morula Stage ◽

Zinc Concentrations ◽

Bovine Oocytes

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 μg added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 μg zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 μg added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 μg per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 μg zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 μg/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 μg ml of zinc in vitro than in vivo because a concentration of 3.0 μg/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 μg/ml of zinc; however, higher concentrations were inhibitory.

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