scholarly journals 88 PREGNANCY RATES FOR IN VITRO AND IN VIVO PRODUCED OVINE EMBRYOS VITRIFIED USING THE MINIMUM VOLUME COOLING CRYOTOP METHOD

2005 ◽  
Vol 17 (2) ◽  
pp. 194
Author(s):  
J. Kelly ◽  
D. Kleemann ◽  
M. Kuwayama ◽  
S. Walker
Keyword(s):  
Pregnancy Rate ◽  
Sucrose Solution ◽  
Bovine Embryo ◽  
Minimum Volume ◽  
Embryo Viability ◽  
Embryo Survival ◽  
First Order ◽  
Fresh Embryos

Previously we reported that, using the minimum volume cooling (MVC) cryotop vitrification method, in vitro-produced ovine and bovine embryo survival after thawing was similiar to that of fresh embryos (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172). While survival of vitrified embryos after thawing can be indicative of embryo viability, this assessment does not always correlate with embryo survival after transfer. This study assesses the effect of vitrification using the MVC cryotop method on the survival after transfer of in vitro- and in vivo-produced ovine embryos. Fresh or vitrified Day 6 ovine embryos (expanded blastocysts, blastocysts, compact morulae) were used in this study. Ovine cumulus–oocyte complexes were obtained and matured, fertilized (Day 0), and cultured in vitro (Walker et al. 1996 Biol. Reprod. 55, 703–708). In vitro embryos for vitrification were produced and vitrified (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172) 10 days prior to the day of transfer. In vivo embryos were recovered from donor Merino ewes and vitrified 7 days prior to the day of transfer while fresh in vivo embryos were collected and transferred on the same day. Semen used for both in vivo and in vitro embryo production was from the same sire. On the day of transfer, vitrified embryos were thawed directly into 1.25 M sucrose solution, followed by stepwise dilution of the cryoprotectants. Embryos were transferred as singles into synchronized recipient ewes on a randomized basis. Fetal number was detected at Day 50. Variables were assessed using the CATMOD procedure in SAS. Pregnancy rate for in vivo-derived embryos was higher (P < 0.01) than for in vitro-derived embryos. Embryo treatment (fresh vs. vitrified) did not significantly affect pregnancy rate. Pregnancy rate for ewes detected (by vasectomized rams) in estrus within 48 h of progesterone pessary removal was higher (P < 0.05) than for both the 48–68 h and unmarked groups. The latter two groups did not differ significantly. None of the first-order interactions were significant (P > 0.05). This study demonstrates that ovine embryos (in vitro and in vivo) can be vitrified, thawed, and transferred without compromising embryo viability. However, the differences in pregnancy rate between the recipient groups warrant further investigation. The MVC cryotop method is a vitrification technique that can be adapted to routine field use. Table 1. Pregnancy rate of fresh and vitrified in vivo and in vitro ovine embryos after embryo transfer

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

2004 ◽  
Vol 16 (2) ◽  
pp. 172 ◽  
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 &amp; 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&lt;0.0001); neither day nor treatment x day interaction was significant (P&gt;0.05). A significant interaction between vitrification treatment and day (P&lt;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&lt;0.003 and P&lt;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&lt;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

363 FACTORS INFLUENCING PREGNANCY RATES FOLLOWING TRANSFER OF BOVINE IN VIVO EMBRYOS BIOPSIED FOR SEX DETERMINATION

2007 ◽  
Vol 19 (1) ◽  
pp. 297
Author(s):  
S. Li ◽  
W. Yu ◽  
J. Fu ◽  
Y. Bai ◽  
F. Jin ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Pregnancy Rates ◽  
Chi Square ◽  
Embryo Survival ◽  
Chi Square Test ◽  
First Time ◽  
Fresh Embryos

Data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 was analyzed to examine the effects of various factors (biopsy, freezing, sample size, embryo development and quality, in vitro culture, and recipient quality) on pregnancy rates of in vivo-biopsied embryos. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sexing determination using protocols and sexing kits supplied by AB Technology Ltd. Fresh embryos were implanted on the same day or frozen with AG freeze medium (AB Technology Ltd., Pullman, WA, USA) for later transfer. Recipients were synchronized with CIDA + PG protocols. Embryos were cultured in 6-well dishes containing 1.3 mL of holding medium (AB Technology Ltd.) in each well at room temperature (20–25�C) for examination of embryo survival in vitro. The chi-square test was used in statistic analysis. The implantation of fresh embryos after biopsy did not affect pregnancy rates (49.6%, 257/518) compared to that of non-biopsied fresh and frozen–thawed embryo groups (52.9%, 47/140 and 46.6%, 177/380, respectively). However, for biopsied embryos subjected to frozen and thawed procedures before implantation, particularly for those subjected to the removal of a larger biopsy, a reduced pregnancy rate was observed (41.8%, 297/710; P &lt; 0.01). Pregnancy rates among biopsied embryos at 3 different development stages (morula-early blastocyst, blastocyst, and expanded blastocyst) were not different. Similar results were found between embryo groups of grade 1 and 2. A significant decrease in pregnancy rate (0/10) was observed with embryos held in vitro for a longer period of time (&gt;5 h), suggesting detrimental effects of in vitro conditions on embryo survival. The highest pregnancy rate (68.0%) was observed in recipients synchronized for the first time before being implanted with biopsied embryos. Significant decreases in such rates were found in recipients synchronized for the second or third times or those with an abortion history at the first or second synchronization-implantation treatment (P &lt; 0.01). Better pregnancy rates (45.6%, 41/90; 46.1%, 76/165; and 45.5%, 5/11) were obtained for recipients implanted with biopsied embryos at Days 7.5, 8.0, and 8.5 post-heat detection, respectively, compared to 16% at Day 7 (3/18, P &lt; 0.05). It is concluded that mechanical treatment (cutting) does not reduce the survival of biopsied embryos; however, cryopreservation reduces their ability to survive in vivo. The analyses also suggest that holding embryos in vitro should not be longer than 5 h unless more favorable in vitro conditions can be provided. To achieve better results of implantation of biopsied embryos, embryo transfer should be performed during 7.5–8.5 days post-estrus, and the healthy recipients synchronized for the first time should be used.

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

Post-ovulation nutritional status in ewes does not influence early conceptus development in vivo or luteotrophic protein secretion in vitro.

1993 ◽  
Vol 1993 ◽  
pp. 58-58
Author(s):  
J.M. Wallace ◽  
R.P. Aitken ◽  
M.A. Cheyne
Keyword(s):  
Pregnancy Rate ◽  
Immunosuppressive Agents ◽  
Maternal Environment ◽  
Embryo Survival ◽  
Domestic Species ◽  
Progesterone Secretion ◽  
Conceptus Development ◽  
In Pregnancy

Overfeeding during early pregnancy compromises pregnancy establishment and /or embryo survival in a variety of domestic species including sheep, cattle and pigs (reviewed by Robinson, 1990). Embryo survival was reduced in recipient ewes receiving a high as opposed to a low plane of nutrition from embryo transfer on day 5 post-ovulation to day 60 of gestation (McKelvey & Robinson, 1988).Similarly high plane feeding for only 12 days starting on day 2 after mating significantly reduced pregnancy rates at day 60 ( Parr et al .,1987). Although not extensively monitored in either study, peripheral progesterone concentrations were inversely correlated with feed intake. Indeed, the reduction in pregnancy rate in high plane ewes in Parr's study was reversed by progesterone supplementation on days 8-14 after mating.The inhibition of luteolysis and maintenance of adequate progesterone secretion by the corpus luteum is central to the maternal recognition of pregnancy in sheep ( Bazer et al .,1991 ). Progesterone plays a major role in controlling maternal secretion of nutrients, growth factors , immunosuppressive agents .enzymes and steroids required for successful embryo development. It seems likely therefor that the mechanisms underlying nutritionally induced differences in pregnancy rate and embryo survival may operate via changes in progesterone levels which in turn alter the secretory dialogue between the conceptus and its maternal environment.

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

2014 ◽  
Vol 26 (1) ◽  
pp. 139 ◽  
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.

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 Effects of supplementation with heat shock cognate 17 KDA protein (HSC70) on in vitro bovine embryo viability

2021 ◽  
Author(s):  
Aimé Jazmín Garza Arredondo ◽  
Diana Elisa Zamora Ávila ◽  
Uziel Castillo Velásquez ◽  
Gustavo Moreno Degollado ◽  
José Fernando De La Torre Sánchez ◽  
...  
Keyword(s):  
Heat Shock ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Stage Of Development

Abstract Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.

229 OOCYTE RECOVERY, IN VITRO FERTILIZATION AND EMBRYO TRANSFER IN THE SERVAL (LEPTAILURUS SERVAL)

2006 ◽  
Vol 18 (2) ◽  
pp. 223 ◽  
Author(s):  
C. E. Pope ◽  
M. C. Gómez ◽  
A. Cole ◽  
C. Dumas ◽  
B. L. Dresser
Keyword(s):  
Embryo Transfer ◽  
Oocyte Retrieval ◽  
Sub Saharan Africa ◽  
Medium Size ◽  
Embryo Survival ◽  
Frozen Semen ◽  
Old Donor ◽  
Fresh Embryos

Servals are medium size (9 to 18 kg) spotted cats found in sub-Saharan Africa that are protected by CITES under Appendix II regulations. There are at least six sub-species, one of which is listed as Endangered by the U.S. Endangered Species Act. In vitro-derived embryos have been produced in at least one-half of the 36 species of nondomestic cats, and kittens have been born after embryo transfer in six species. In the present study we evaluated (1) ovarian response of servals to repeated exogenous gonadotropin stimulation, and (2) in vitro and in vivo developmental ability of in vitro-derived embryos. One two-year-old and one five-year-old female were treated six and three times, respectively, over a 3.5-year period, with a total of 20 or 25 IU of porcine FSH (i.m.; Sioux Biochem, Sioux City, IA, USA) administered daily over four days during interestrus. On Day 5, 15 IU of porcine LH (i.m.; Sioux Biochem) was given, and laparoscopic oocyte retrieval was performed 24 h later. A total of 234 preovulatory oocytes were recovered: 182 (mean = 30.3) from the two-year-old and 52 (mean = 17.3) from the five-year-old female. A total of 91 and 91 oocytes were recovered at retrievals 1 through 3 and 4 through 6, respectively, from the two-year-old donor. Eighty oocytes from the two-year-old donor were inseminated with cooled (24 h, 4°C) semen. Frozen semen from the same male was used to inseminate 102 oocytes from the two-year-old female and 52 oocytes from the five-year-old female. Overall, 136 embryos (58% cleavage frequency) were produced: 119 (65% cleavage frequency) from the two-year-old and 17 (33% cleavage frequency) from the five-year-old female. Cleavage frequency of oocytes from the two-year-old female inseminated with cooled or frozen semen was similar, 68% (54/80) and 64% (65/102), respectively. Embryos were cultured for 5 or 6 days before controlled rate cryopreservation or uterine transfer (Gómez et al. 2003 Theriogenology 60, 239–251). On Day 5, 66 early to mid-stage morulae were cryopreserved at a slow controlled rate. Sixty Day 5 and 18 Day 6 embryos were auto-transferred to a recipient (8 to 26/transfer) in a total of six surgical procedures, of which five were with fresh embryos (n = 70) and one was with cryopreserved embryos (n = 8). The sixth embryo transfer procedure (26 fresh embryos) resulted in the unassisted birth of a live male kitten on Day 77 of gestation. We have shown that in vitro-derived embryos can be generated in the serval and that oocyte retrieval rates and cleavage frequencies are comparable to those reported for other species of mid-sized nondomestic cats. The nominal incidence of pregnancy and frequency of embryo survival may be improved by transferring early cleavage staged embryos into the oviduct, as demonstrated in the African wildcat (Felis silvestris lybica; Gómez et al. 2004 Cloning and Stem Cells 6, 247–258). This work was partially funded by the Dan Heard Conservation Challenge Grant.

71 WILL DEHYDRATION BY EXPOSURE TO SUCROSE IMPROVE POST-VITRIFICATION SURVIVAL OF MURINE EMBRYOS VITRIFIED BY THE OPEN PULLED STRAW METHOD?

2011 ◽  
Vol 23 (1) ◽  
pp. 141
Author(s):  
M. El-Gayar ◽  
J. Reischl ◽  
M. Gauly ◽  
W. Holtz
Keyword(s):  
Sucrose Solution ◽  
Active Agent ◽  
Virgin Female ◽  
Control Group ◽  
Embryo Survival ◽  
Intact Mouse ◽  
Group 4 ◽  
Group 2

Vitrification of mammalian embryos comprises suspension in highly concentrated solutions of penetrating cryoprotectants. These cryoprotectants are known to be extremely cytotoxic to cells in an unfrozen state. In the present experiment, it was attempted to reduce the amount of cryoprotectants entering the cells to a minimum by at least partially dehydrating the blastomeres before exposing them to vitrification solutions. Sucrose is known to be a non-permeating osmotically active agent and is, therefore, suited to serve this purpose. It has been shown that concentrations of up to 1.1 M sucrose are well tolerated (Krag et al. 1985 Theriogenology 23, 199). Morphologically intact mouse blastocysts collected from superovulated 5- to 8-week-old virgin female NMRI mice were randomly allocated to 4 treatment groups (100 embryos/group). A control group (Group 1) was vitrified according to the slightly modified (El-Gayar et al. 2008 Cryobiology 57, 191–194) protocol of (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). The protocol involves exposure to 10% dimethyl-sulfoxide (Me2SO) + 10% ethylene glycol (EG) for 1 min; 20% Me2SO + 20% EG for 20 s; loading into straws and plunging directly into liquid nitrogen. In embryos of Group 2, the procedure mentioned above was preceded by exposure to a 1 M sucrose solution for 1 min. In Group 3, the procedure of Group 2 was followed; however, vitrification solution no. 1 (10% Me2SO + 10% EG) was omitted. In Group 4, sucrose was added to both vitrification solutions at a concentration of 0.2 mol with the 10% Me2SO + 10% EG-solution and of 0.4 mol with the 20% Me2SO + 20% EG-solution. After warming, embryos of all groups were cultured in vitro in microdrops of M16 medium for 48 h. Differences between means were tested for significance by the chi-square test. All embryos were recovered after warming. In the control group, 97% of the embryos had continued development to the expanded blastocyst stage and 89% had proceeded to hatch. The corresponding values for Groups 2, 3, and 4 were 81 and 64%; 60 and 29%, and 93 and 88%, respectively. Earlier studies have shown that in vitro hatching is closely correlated with in vivo survival (El-Gayar et al. 2008 Cryobiology 57, 191–194; Cryoletters 2010, in print). The differences between the control group and Groups 2 and 3 were significant (P < 0.01), whereas with Group 4 it was not (P > 0.05). Thus, exposure to sucrose before vitrification (Groups 2 and 3) compromised the viability of murine blastocysts rather than protecting them, whereas addition of sucrose to vitrification solutions did not. Because embryo survival after cryopreservation by the standard OPS procedure was exceptionally high, it was impossible to detect a potential improvement. This study was partly supported by a grant from the Egyptian government.

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