98 EIGHT-CELL STAGE DELINEATES CRYOTOLERANCE OF VITRIFIED RABBIT EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 208
Author(s):  
T.-A. Lin ◽  
C.-H. Chen ◽  
L.-Y. Sung ◽  
J.-C. Ju ◽  
E. Chen ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Cell Stage ◽  
General Linear Model Analysis ◽  
Developmental Rates ◽  
Humidified Air ◽  
Rabbit Embryos ◽  
Hatching Rates

A number of groups have successfully vitrified rabbit embryos at morula or blastocyst (BL) stage. For earlier stages (1- to 8-celled), however, there are very limited studies and the results are generally unsatisfactory. In this study, we examined the survival and developmental competence of rabbit embryos vitrified at different preimplantational stages. Sexually matured female New Zealand White (NZW) rabbits were superovulated with our standard protocols, followed by mating with NZW males. At 18h post-hCG treatment, viable fertilized embryos were collected and cultured in 2.5% FBS B2 medium (Laboratories CCD, Paris, France) in 5% CO2 with humidified air at 38.5°C. A total of 691 rabbit embryos at pronuclear, 2-celled, 4-celled, 8-celled, and morula/early blastocyst (BL; Day 3) stages were vitrified by the open pulled straw (OPS) method in HEPES buffered TCM-199 medium containing 20% fetal calf serum, ethylene glycol, and dimethyl sulfoxide. After stored in liquid nitrogen for at least 1 month, embryos were sequentially warmed, rehydrated, and washed before culture in B2 culture medium. Survival and developmental rates were analyzed by general linear model analysis (SPSS 11.0, SPSS Inc., Chicago, IL, USA). Embryos vitrified at 8-celled stage or beyond showed greater survival, and expanded BL and hatching rates than those at pronuclear, 2-celled and 4-celled embryos (Table 1). In particular, the expanded and hatched BL rates were significantly higher in the 8-celled group than those in 4-celled group, suggesting that the 8-celled is the threshold stage to tolerate the vitrification procedure in rabbit embryos. In addition, the rates of expanded and hatched blastocysts in morula/BL group were still significantly higher than those in the 8-celled group. Our results may provide the proper timing of cryopreserving fertilized, transgenic, and/or cloned rabbit embryos at early stages for biomedical research. Table 1.Survival and developmental competence of rabbit embryos vitrified at different stages after thawing This study was supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

42 Vitrification of invitro-produced feline embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 146
Author(s):  
D. Fuller ◽  
J. Herrick ◽  
J. Graham ◽  
J. Barfield
Keyword(s):  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Epididymal Sperm ◽  
Quality Grade ◽  
Developmental Rates ◽  
Species Specific

Preservation of feline embryos is useful in propagating endangered species, preserving valuable genetics, and supporting biomedical research. Although a wide variety of cryoprotectants (CP) and protocols are successfully used for vitrification of invitro-produced (IVP) embryos, there are often species-specific differences in viability of embryos post-warming. The purpose of this study was to evaluate the viability of IVP feline embryos after vitrification using two common CPs, propanediol (PrOH) or ethylene glycol (EG). Embryos were produced with oocytes and frozen-thawed epididymal sperm collected from local spay-neuter clinics using a published IVP protocol developed for producing domestic feline embryos. Day 7 early blastocysts (stage 5), blastocysts (stage 6), and expanded blastocysts (stage 7) were evaluated for quality (grade 1 or 2) and randomly assigned to one of three treatments: vitrification with PrOH (n=32), vitrification with EG (n=31), or control (n=47), which was allowed to continue in culture until Day 8. The vitrification protocol was as follows. The base medium for all vitrification media was a HEPES-buffered feline optimized culture medium (FOCMH). Embryos were placed in 0.5mL of equilibration medium (7.5% dimethyl sulfoxide, 7.5% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% fetal calf serum (FCS)) for 5min at room temperature. Individual embryos were then moved to 20-μL drops of vitrification medium (15% dimethyl sulfoxide, 15% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% FCS) at room temperature for 30s before being loaded onto Cryolock devices and plunged into liquid nitrogen. Warming was done using a 3-step process for all vitrified embryos. First, embryos were moved from liquid nitrogen directly to 0.5mL of 1M sucrose, 10% Ficoll, and 20% FCS at 37°C for 1min. Next, embryos were moved to 0.5mL of 0.5M sucrose, 10% Ficoll, and 20% FCS at 20°C for 3min. Finally, embryos were transferred to 0.5mL of FOCMH for 5min at 37°C. All warmed embryos were cultured in medium, optimized for feline embryos, with 5% FCS and evaluated for re-expansion of the blastocoele and progression in development at 24 and 48h. Results are from five replicates. Embryos vitrified in EG exhibited higher percentages of viable embryos 24h after warming (84%) than embryos vitrified in PrOH (59%; P<0.05). The continued embryonic growth of viable embryos after culture for 48h showed equivalent developmental rates, at 87, 96, and 100% for control, EG-treated, and PrOH-treated embryos, respectively (P>0.05). Results indicate EG is a more successful CP treatment for vitrification of feline embryos when evaluating viability 24h post-warming. We report a higher viability of embryos post-thaw than previous studies using the same CPs (Pope et al. 2012 Reprod. Domest. Anim. 47, 125). This may be due to the shorter exposure time to the CPs we used during the vitrification process. We conclude that EG and PrOH are effective CPs for Day 7 feline IVP embryos using this protocol. Further research is needed to increase treatment numbers and evaluate pregnancy rates from embryos transferred post-warming.

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 Delaying meiotic resumption during transportation of bovine cumulus–oocyte complexes: effects on development, apoptosis and caspases activity of in vitro-produced embryos

Zygote ◽  
2017 ◽  
Vol 25 (6) ◽  
pp. 740-750 ◽  
Author(s):  
Priscila Chediek Dall'Acqua ◽  
Beatriz Caetano da Silva Leão ◽  
Nathália Alves de Souza Rocha-Frigoni ◽  
Fernanda Patrícia Gottardi ◽  
Gisele Zoccal Mingoti
Keyword(s):  
Bovine Serum Albumin ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
The Other ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Control Groups ◽  
Meiotic Inhibitors

SummaryThis study examined the effects of meiosis inhibition during bovine oocyte transportation on developmental competence and quality of produced embryos. The transportation medium was supplemented with: 100 μM butyrolactone I (BL), 500 μM IBMX + 100 μM forskolin (mSPOM), 100 μM milrinone (MR) or follicular fluid (bFF), and was carried out in a portable incubator for 6 h. Next, oocytes were in vitro matured (IVM) for 18 h, without the meiotic inhibitors, with the exception of mSPOM group, in which was added 20 μM cilostamide. The three control groups were IVM with 10% fetal calf serum (FCS) (Control Lab FCS) or 0.6% bovine serum albumin (BSA) (Control Lab BSA) in a CO2 in air incubator or in the portable incubator with 0.6% BSA (Control Transp BSA). Higher cleavage rates (P < 0.05) were obtained in the Control Lab FCS group (84.5 ± 5.3%) compared with the other groups (59.6 ± 3.4% to 70.9 ± 2.3%). Embryonic development was higher (P < 0.05) in the Control Lab FCS group (39.8 ± 4.7%) than in the Control Transp BSA (22.7 ± 3.4%) and MR (21.6 ± 2.3%) groups. However, they were similar (P > 0.05) to the other groups (23.6 ± 3.3% to 28.8 ± 2.7%). The total number of blastomeres was higher (P < 0.05) in the Control Lab FCS group (85.2 ± 5.6) than in Control Lab BSA (53.6 ± 2.9), Control Transp BSA (55.5 ± 4.4), BL (58.2 ± 3.0), mSPOM (57.9 ± 4.9) and MR (59.2 ± 3.9), but all these treatments did not differ (P > 0.05) from bFF (67.7 ± 4.2). No differences (P > 0.05) were found in apoptosis by the activity of caspases (139.0 ± 3.2 to 152.4 ± 6.5, expressed in fluorescence intensity) as well as the percentage of TUNEL-positive cells (12.3 ± 2.0% to 15.7 ± 1.7%). In conclusion, the transportation of oocytes over 6 h with BL, mSPOM or bFF enabled the acquisition of developmental competence at similar rates to the Control Lab FCS group.

98 SUCCESSFUL PREGNANCIES FOLLOWING TRANSFER OF VITRIFIED PORCINE EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 157 ◽  
Author(s):  
K. Hiruma ◽  
H. Ueda ◽  
H. Saito ◽  
C. Tanaka ◽  
N. Maeda ◽  
...  
Keyword(s):  
Calf Serum ◽  
Developmental Competence ◽  
Penicillin G ◽  
Cell Stage ◽  
Epidermal Growth ◽  
Potassium Penicillin ◽  
The One ◽  
Parthenogenetic Embryos

To date only in vivo-produced embryos have successfully produced live piglets after cryopreservation. In this study, we aimed to produce piglets from vitrified embryos derived from in vitro matured (IVM) oocytes. Cumulus-oocyte complexes collected from ovaries obtained at a local slaughterhouse were matured for 44 to 45 h in NCSU23 MEDIUM supplemented with 0.6 mM cysteine, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 75 �g/mL potassium penicillin G, 50 �g/mL streptomycin sulfate, and 10 IU/mL eCG/ hCG. These IVM oocytes were either activated for parthenogenesis or in vitro-fertilized (IVF). For IVF, oocytes were incubated with 5 � 106/mL of cryopreserved epididymal sperm in PGM-tac medium (Yoshioka et al. 2003 Biol. Reprod. 69, 2092-2099) for 20 h. Embryos were treated for removal of cytoplasmic lipid droplets (delipation; Nagashima et al. 1995 Nature 374, 416) at the 4- to 8-cell stages, around 50 to 54 h after activation or insemination. After culture in NCSU23 for 15 h, they were vitrified by the minimum volume cooling (MVC) method. Embryos were equilibrated with equilibration solution containing 7.5% (v/v) ethylene glycol (EG), 7.5% (v/v) dimethylsulfoxide (DMSO), and 20% (v/v) calf serum for 4 min, followed by exposure to vitrification solution containing 15% EG, 15% DMSO, 0.5 M sucrose, and 20% calf serum. Embryos were then loaded onto a Cryotop (Kitazato Supply Co., Tokyo, Japan) and immediately plunged into liquid nitrogen. Vitrified embryos were examined for viability in vitro and in vivo after warming. Their in vitro developmental competence was compared to that of corresponding control (nonvitrified) embryos. Vitrified 4- to 8-cell stage embryos, both parthenogenetic and IVF, showed developmental competence into blastocysts comparable to that of control embryos (parthenogenetic: 46.8%, 36/77 vs. 51.7%, 31/60; IVF: 40.0%, 30/75 vs. 44.3%, 35/79). Of four surrogate gilts that received a total of 251 vitrified parthenogenetic embryos, three became pregnant and had 20 fetuses (8.0%, 22 to 23 days old). Three surrogates gilts that received 267 vitrified IVF embryos all became pregnant. Of those, the one that received 47 embryos was confirmed to have eight fetuses (17.0%, 22 days old) by autopsy. The other two were examined by ultrasonography at 56 and 95 days of gestation and found to be pregnant. These results suggest that porcine embryos derived from IVM oocytes have a potential to develop into live offspring after delipation and MVC vitrification. This study was supported by PROBRAIN.

242 PROMISING TWO-STEP EVALUATION SYSTEM FOR SELECTING HIGH DEVELOPMENTAL COMPETENCE IN VITRO FERTILIZED EMBRYOS IN CATTLE USING WELL-OF-THE-WELL DISH

2015 ◽  
Vol 27 (1) ◽  
pp. 210
Author(s):  
M. Taniai ◽  
M. Takayama ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Evaluation System ◽  
Evaluation Method ◽  
Calf Serum ◽  
Time Lapse ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test

Bovine IVF embryos are evaluated morphologically using light microscopy just before transfer. However, this evaluation method is subjective, and an objective method with more certainty is needed. Sugimura et al. (PLoS ONE 2012 7, e36627) reported a promising system for selecting healthy IVF bovine embryo by using time-lapse cinematography and 5 prognostic factors. This study was to investigate the efficacy of a 2-step evaluation system of IVF embryos using microscopy for selecting high developmental competence IVF embryos. Cumulus-oocyte complexes (COC) were collected by ovarian follicular aspiration (2 to 5 mm diameter) obtained from a local abattoir. The COC (n = 488) were matured in TCM-199 medium supplemented with 5% calf serum (CS) and 0.02 IU mL–1 of FSH at 38.5°C for 20 h in an atmosphere of 5% CO2 (20 COC 100 µL–1 droplets). After 10 h of gametes co-culture (5.0 × 106 sperm cells mL–1), the presumptive zygotes were cultured in 125 µL of CR1 aa medium supplemented with 5% CS in well of-the-well culture dishes (AS ONE, Japan; 25 zygotes well–1) at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days. Two-step evaluations of embryos were done at 27 and 55 h post-IVF (hpi). In the first step of evaluation, cleavage patterns at 27 hpi were categorized as mono-cell, 2-cell with even blastomeres and without fragments (normal cleavage), 2-cell with uneven blastomeres, and ≥3 blastomeres. During the second step of evaluation, embryos were classified by their number of blastomeres (2 to 5 cells, 6 to 8 cells, and >8 cells) and the absence or presence of multiple fragments (<20 or >20%) at 55 hpi. The data were analysed by chi-square test. The blastocyst rate (BL%) of embryos cleaved before 27 hpi (56.6%, n = 106) was higher (P < 0.01) than those of embryos cleaved after 27 hpi (37.0%, n = 235). A greater percentage (P < 0.05) of 2-cell embryos with normal cleavage (68.0%, n = 50) developed to blastocysts than from with =3 blastomeres at 27 hpi (40.6%, n = 32). Superior BL% (P < 0.01) was obtained from embryos categorized as 6- to 8-cell stage (58.6%, n = 140) and >8 cell stage (70.6%, n = 25) compared with those embryos at the 2- to 5-cell stage at 55 hpi (26.1%, n = 176). Embryos with no fragments (58.0%, n = 467) had higher BL% (P < 0.01) compared with those with <20% fragments (30.7%, n = 127) and having with >20% fragments (17.5%, n = 25) at 55 hpi. The highest of BL% was observed in embryos showing a normal cleavage to 2-cells with at 27 hpi and having >6 cells with no fragments at 55 hpi (95.2%, n = 21, P < 0.01). These results demonstrate that the 2-step evaluation system at 27 and 55 hpi using microscopy is an effective method for selecting IVF embryos with high developmental competence.

67 The growth and development of invivo-derived dromedary embryos during short-term incubation: Use of embryo holding medium and the effect of embryonic morphology

2021 ◽  
Vol 33 (2) ◽  
pp. 140
Author(s):  
B. Asadi ◽  
F. Seyedasgari
Keyword(s):  
Growth And Development ◽  
Culture Medium ◽  
Calf Serum ◽  
Daily Basis ◽  
Developmental Competence ◽  
Dromedary Camel ◽  
Short Term ◽  
Experimental Media ◽  
Further Development ◽  
Short Term Culture

Production of invivo embryos for transfer in dromedary camel is a well-established practice, whereas freezing of these embryos is still an ongoing challenge. A common approach in evaluation of freeze–thawing method is achieved by studying invitro development of frozen–thawed embryos. However, not much is known about the development pattern of fresh dromedary embryos during incubation. The objectives of this study were to evaluate the usefulness of commercial holding media for easy short-term culture of these embryos and to provide preliminary insights on the growth and development of hatched blastocysts with different shapes. Recovered hatched blastocysts from superovulated donors were graded as transferable and non-transferable. Embryos with significant folding or crinkliness were further categorized as collapsed, whereas those with a round or oval appearance were categorized as spherical. Culture was performed in 500-μL drops at 38.5°C, 5% O2, 0–6% CO2, and maximum humidity in groups of 2 to 4. The 4 experimental media included culture medium (CM; TCM-199, 10% fetal calf serum (FCS), 0.3mM sodium pyruvate, 2.2mg mL−1 sodium bicarbonate), serum-supplemented holding medium (SSH; Syngro+10% FCS), serum-free holding medium (Syngro) and V-Onestep (Vitromed). In experiment 1, a total of 36 embryos were assigned to 4 groups and further development of the embryos was monitored up to 96h by morphological evaluations, identifying static and degenerating embryos on daily basis. In experiment 2, a total of 16 spherical and 16 collapsed embryos were cultured in SSH and CM and two-thirds of the culture drop was replaced with fresh medium at 72h. The proportion of developing embryos and their size expansion was compared between treatments by Fisher’s test and Mann–Whitney U test, respectively. Statistically similar proportions of embryos continued to develop in all media within the first 48h despite a numeric advantage in CM group; at 72h, the proportion of growing embryos was significantly higher in CM (77.8%) and SSH (66.6%) compared with SFH (11.1%) and OneStep (22.2%) (P&lt;0.05). None of the embryos in SFH and only 1 embryo in the V-Onestep group survived beyond 72h, whereas 3/9 embryos in SSH and 7/9 embryos in CM continued to expand. In experiment 2, the proportion of spherical embryos that developed was higher compared with their collapsed counterparts (8/8 in both groups vs. 5/8 and 4/8 in CM and SSH, respectively) at 24h. However, remaining collapsed embryos grew and expanded at similar rates to spherical ones in each group (P&gt;0.05). Replacing the medium did not favour continuation of embryonic growth in SSH beyond 72 h; only 5/16 embryos survived to 96h compared with 12/16 in CM. In conclusion, serum-supplemented commercial holding preparations provide comparable results to culture medium for short-term incubation of invivo dromedary embryos. Natural collapsing of hatched blastocysts might be associated with lower developmental competence.

scholarly journals Stimulierung von Kulturen embryonaler Rattenzellen durch eine Proteinfraktion aus fötalem Kälberserum / Stimulation of Embryonic Rat Cells in Culture by a Protein Fraction Isolated from Fetal Calf Serum

1971 ◽  
Vol 26 (10) ◽  
pp. 1045-1048 ◽  
Author(s):  
Dieter F. Hülser ◽  
Werner Frank
Keyword(s):  
Cell Cycle ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Surface Membrane ◽  
Calf Serum ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Membrane Potential Difference ◽  
Stimulation Of

Normal embryonic rat cells incubated in serum-free medium accumulate in G1-phase of the cell cycle. On addition of a growth-stimulating protein isolated from fetal calf serum they are triggered to proceed through the cycle, and they resume DNA-synthesis 15 to 20 hours later. In this paper it is demonstrated that the surface membrane potential difference (PD) decreases immediately after changing serum-free medium against culture medium containing either calf serum or the isolated serum protein; the original PD is restored 2 to 3 hours later. Serumprotein without growthstimulating activity does not affect the PD.A permanent rat cell line which grows independently of serum also has been tested. The PD of these cells is not significantly influenced by calf serum.

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.

Developmental competence in vitro and in vivo of cryopreserved, hatched blastocysts from the dromedary camel (Camelus dromedarius)

2004 ◽  
Vol 16 (6) ◽  
pp. 605 ◽  
Author(s):  
J. A. Skidmore ◽  
M. Billah ◽  
N. M. Loskutoff
Keyword(s):  
Survival Rate ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Sodium Lactate ◽  
Developmental Competence ◽  
Penicillin G ◽  
Dromedary Camel ◽  
Dromedary Camels

The present paper describes experiments designed to investigate methods for cryopreserving embryos from dromedary camels. Because preliminary studies had shown ethanediol to be the best cryoprotectant to use for camel embryos, the current experiments were performed to determine the minimum exposure time to 1.5 m ethanediol required to achieve cryoprotection. The uteri of 30 donor camels were flushed non-surgically 8 days after mating. Embryos were recovered and 158 were assigned to one of three groups, which were exposed to 1.5 m ethanediol for either 10 min (n = 67), 5 min (n = 51) or 1 min (n = 40). Embryos were subsequently thawed and rehydrated by expelling either directly into holding medium (HM; HEPES-buffered Tyrode's medium containing sodium lactate and 3 mg mL−1 bovine serum albumin, 10% fetal calf serum, 100 IU mL−1 penicillin G, 100 μg mL−1 streptomycin and 25 μg mL−1 amphotercin B) or initially into HM containing 0.2 m sucrose for 5 or 10 min. The survival rate of all embryos immediately post-thawing, as judged by the morphological appearance of the embryos, was high (91%), but was greatly reduced after 2 h culture (59%). Ninety-two embryos were transferred to recipient camels resulting in 18 viable fetuses (1 min ethanediol exposure, n = 1/15; 5 min ethanediol exposure, n = 3/34; 10 min ethanediol exposure, n = 14/43). Of the embryos rehydrated directly in HM, six of 65 resulted in viable fetuses and those rehydrated initially in 0.2 m sucrose for 5 or 10 min resulted in nine of 47 and three of 46 fetuses respectively. From these experiments, we conclude that camel embryos can be cryopreserved using ethanediol as a cryoprotectant when the embryos are cooled slowly (to 33°C) before being plunged into liquid nitrogen for storage.

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