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.

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.

231 EFFECT OF DIFFERENT EQUINE CHORIONIC GONADOTROPIN CONCENTRATIONS ON IN VITRO-PRODUCED BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 263
Author(s):  
C. Decanine ◽  
E. M. Pioltine ◽  
I. P. Emanuelli ◽  
R. Z. Puelker ◽  
M. F. G. Nogueira
Keyword(s):  
Calf Serum ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Experimental Conditions ◽  
Essential Components ◽  
Chi Squared ◽  
Equine Chorionic Gonadotropin ◽  
And Control ◽  
Hatching Rates

In vitro maturation (IVM) is one of the most challenging steps in the in vitro production of bovine embryos. The IVM medium must provide the necessary conditions for the occurrence of nuclear and cytoplasmic maturation, close to the physiological conditions. The pituitary gonadotropins are essential components for generating competent oocytes; however, whether these hormones (pituitary, placental, or both) are essential and which concentrations should be used are still controversial. Our work aimed to compare the effect of different concentrations of the placental gonadotropin (eCG) in the IVM medium on the in vitro-produced bovine blastocysts. Cumulus–oocyte complexes (n = 1341, grades I and II), obtained from ovaries from an abattoir were selected and distributed into six groups: (1) eCG (4 IU mL–1; n = 192); (2) eCG (1.5 IU mL–1; n = 204); (3) eCG (1.3 IU mL–1; n = 203); (4) eCG (0.15 IU mL–1; n = 202); (5) eCG (0.015 IU mL–1; n = 199); (6) control: FSH (0.1 mg mL–1), LH (50 µg mL–1), and 10% of fetal calf serum (FCS; n = 341). Medium from groups 1 to 5 also contained LH (50 µg mL–1) and BSA (6 mg mL–1). The cumulus–oocyte complexes were matured in TCM-199 for 24 h and were IVF (Day 0) in TALP-IVF for 22 to 24 h. Viable spermatozoa were selected by Percoll gradient, and they were evaluated (motility and spermatozoa concentration) to provide the insemination concentration (106 spermatozoa mL–1). Presumptive zygotes were cultured in SOF medium supplemented with FCS (2.5%) and BSA (5 mg mL–1) in an incubator (38.3°C, 5% CO2, and maximum humidity). Embryo development was evaluated in terms of cleavage (Day 3), blastocyst (Day 7), and hatching rates (Day 10). Mean rates were analysed by chi-squared test and ANOVA, and significance was considered when P < 0.05. The results obtained from the different groups are shown in Table 1. Cleavage, blastocyst, and hatching rates were not different among groups. We conclude that, under our experimental conditions, even minimal concentrations of eCG (0.015 IU) may be a viable and effective alternative in the replacement of FSH for the IVM of bovine oocytes. Table 1.Cleavage, blastocyst, and hatching rates of the experimental groups (1, 2, 3, 4, and 5) and control group1 Fellowships and support by CAPES and FAPESP.

scholarly journals 113COMPARISON OF TWO ETHYLENE GLYCOL EQUILIBRATION TREATMENTS FOR THE QUICK FREEZING OF IN VITRO-PRODUCED BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 178
Author(s):  
A.C. Nicácio ◽  
R. Simões ◽  
C. Yamada ◽  
H.V.A. Caetano ◽  
M.R.B. Mello ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Granulosa Cell ◽  
Cell Monolayer ◽  
Slow Freezing ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Quick Freezing ◽  
Hatching Rates

The aim of this study was to compare two ethylene glycol (EG) equilibration procedures for the quick freezing of in vitro-produced bovine embryos. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries. COCs were matured in TCM199 containing 10% of bovine fetal serum, LH, FSH and E2, and fertilized. Presumptive zygotes were co-cultured in TCM199 with a granulosa cell monolayer, at 39°C in humidified atmosphere of 5% CO2 in air. Grade 1, expanded blastocysts (n=761) were selected 7 and 9 days after insemination and randomly distributed to one of eight treatment groups. In Equilibration Procedure 1, embryos were exposed to 10% EG for 5 min, and then to 17%, 22% or 28% EG for 60s (respectively referred to as EG 17, EG 22 and EG 28). In Equilibration Procedure 2, embryos were exposed to the same EG solutions as in Equilibration Procedure 1, but the period of exposure was 10min to 10% EG and 30 s to EG 17, EG 22 and EG 28. In Equilibration Procedure 3 (slow-freezing controls), embryos were exposed to 10% EG for either 5 or 10min and then cryopreserved by slow-freezing method at 1.2°C/min. In all treatment groups, EG solutions were prepared in PBS+0.2% BSA, and embryos were exposed to EG solutions at 22°C. Embryos were loaded into 0.25mL straws and heat-sealed. Straws were cooled in liquid nitrogen vapor for 2min, and then plunged into and stored in liquid nitrogen. Straws were thawed in room temperature air for 10s, and then in 25°C water for 20s. Thawed embryos were diluted by transferring them into 0.5ml of PBS+0.2% BSA+0.3M sucrose for 3min, and then 0.5mL of PBS+0.2% BSA for 3min. Embryos were co-cultured on granulosa cell monolayer in TCM199 and evaluated after 24h for blastocyst re-expansion (EXP), and again at 48, 72 and 96h for hatching (HAT). A total of 724 in vitro-produced bovine blastocysts were used as controls to determine hatching rates. The results are presented in the table. Embryos exposed to 10% EG for 10min (Equilibration Procedure 1) yielded significantly higher rates of blastocyst re-expansion and hatching when compared to embryos exposed for 5min (Equilibration Procedure 2, P&lt;0.05). These results suggest that quick freezing of in vitro-derived bovine embryos may be an alternative to vitrification; however, additional studies are needed to optimize cryopreservation protocols and increase post-thaw survival. This project was supported by FAPESP (01/11266-4) Table 1 Effect of equilibration procedure on in vitro re-expansion and hatching rates of embryos cryopreserved by slow and quick freezing methods

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

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&lt;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

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.

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.

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 Birth of a Live Cria After Transfer of a Vitrified-Warmed Alpaca (Vicugna pacos) Preimplantation Embryo

2020 ◽  
Vol 7 ◽  
Author(s):  
Jennifer C. Lutz ◽  
Susan L. Johnson ◽  
Kimberly J. Duprey ◽  
Paul J. Taylor ◽  
Henry William Vivanco-Mackie ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Preimplantation Embryos ◽  
Embryo Survival ◽  
Important Species ◽  
Vicugna Pacos ◽  
Improvement Programs ◽  
The One ◽  
Humidified Air

The alpaca (Vicugna pacos) is an important species for the production of fiber and food. Genetic improvement programs for alpacas have been hindered, however, by the lack of field-practical techniques for artificial insemination and embryo transfer. In particular, successful techniques for the cryopreservation of alpaca preimplantation embryos have not been reported previously. The objective of this study was to develop a field-practical and efficacious technique for cryopreservation of alpaca preimplantation embryos using a modification of a vitrification protocol originally devised for horses and adapted for dromedary camels. Four naturally cycling non-superovulated Huacaya females serving as embryo donors were mated to males of proven fertility. Donors received 30 μg of gonadorelin at the time of breeding, and embryos were non-surgically recovered 7 days after mating. Recovered embryos (n = 4) were placed individually through a series of three vitrification solutions at 20°C (VS1: 1.4 M glycerol; VS2: 1.4 M glycerol + 3.6 M ethylene glycol; VS3: 3.4 M glycerol + 4.6 M ethylene glycol) before loading into an open-pulled straw (OPS) and plunging directly into liquid nitrogen for storage. At warming, each individual embryo was sequentially placed through warming solutions (WS1: 0.5 M galactose at 37°C; WS2: 0.25 M galactose at 20°C), and warmed embryos were incubated at 37°C in 5% CO2 in humidified air for 20–22 h in 1 ml Syngro® holding medium supplemented with 10% (v/v) alpaca serum to perform an initial in vitro assessment of post-warming viability. Embryos whose diameter increased during culture (n = 2) were transferred individually into synchronous recipients, whereas embryos that did not grow (n = 2) were transferred together into a single recipient to perform an in vivo assessment of post-warming viability. Initial pregnancy detection was performed ultrasonographically 29 days post-transfer when fetal heartbeat could be detected, and one of three recipients was pregnant (25% embryo survival rate). On November 13, 2019, the one pregnant recipient delivered what is believed to be the world's first cria produced from a vitrified-warmed alpaca embryo.

scholarly journals 97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 199 ◽  
Author(s):  
B. Peachey ◽  
K. Hartwich ◽  
K. Cockrem ◽  
A. Marsh ◽  
A. Pugh ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Slow Freezing ◽  
High Survival ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Frozen Embryos ◽  
Morula Stage ◽  
Fresh Embryos

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P < 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P < 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

Production of in vitro bovine embryos supplemented with l-carnitine in different oxygen tensions and the relation to nitric oxide

Zygote ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 403-408
Author(s):  
Daniela Moraes Pereira ◽  
Christopher Junior Tavares Cardoso ◽  
Wilian Aparecido Leite da Silva ◽  
Mirela Brochado Souza-Cáceres ◽  
Mariana Santos ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
Calf Serum ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Low Oxygen ◽  
Cleavage Rate ◽  
Treatment Groups ◽  
Oxygen Tensions

SummaryThe aim of this study was to evaluate the production of bovine embryos in vitro when supplemented with l-carnitine for 24 h beginning on day 5 (d 5) under two different oxygen tensions (20% or 5%) and the relationship of nitric oxide (NO) in in vitro culture (IVC) medium to embryo development. Cumulus–oocyte complexes (COC; n = 837) were matured in vitro for 24 h and fertilization was performed for 18 h. Zygotes were cultured in vitro for 9 days after in vitro fertilization in synthetic oviductal fluid (SOF) medium with 5% fetal calf serum. At d 5 the plates were assigned to one of four treatment groups: high (20%) or low (5%) O2 tension either with or without the addition of 3.03 mM l-carnitine (High-Cont, High-Lcar, Low-Cont, Low-Lcar). The concentration of NO in the culture medium was evaluated on d 5, d 6 and d 9. On d 7, parts of the embryos were submitted for evaluation of intracellular lipid droplets. The cleavage rate was similar (P > 0.05) between high and low O2 tension and the blastocyst rate was similar in all conditions evaluated. The hatching rate was higher (P < 0.05) for Low-Cont. The NO concentration was higher at d 9 under low O2 tension (P < 0.1). The addition of 3.03 mM l-carnitine between d 5 and d 6 of IVC was not efficient in reducing cytoplasmic lipid content of bovine embryos. Additionally, IVC at a low oxygen tension without l-carnitine promoted better conditions for embryo development. A higher concentration of NO in medium was observed under low O2 tension.

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