134 CONCEPTION RATES OF IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED IN 6% GLYCEROL AND TRANSFERRED BY THE DIRECT METHOD

2007 ◽  
Vol 19 (1) ◽  
pp. 184
Author(s):  
N. Takada ◽  
S. Hayasaka ◽  
K. Chiba
Keyword(s):  
Ethylene Glycol ◽  
Conception Rate ◽  
Hatching Rate ◽  
Glycerol Solution ◽  
Direct Transfer ◽  
Bovine Embryos ◽  
Conception Rates ◽  
Chi Square Analysis

Ethylene glycol has been used as the standard cryoprotectant for direct transfer of bovine embryos due to its high permeability. But Merton et al. reported that cryoprotectivity of glycerol for bovine embryos was superior to that of ethylene glycol (2001 Theriogenology 55, 312 abst). We previously reported that nonsurgical transfer of in vivo-derived bovine embryos cryopreserved in a lower concentration (5%) of glycerol and thawed by stepwise method resulted in a 55.4% conception rate, whereas direct transfer without removal of cryoprotectant showed only a 45.1% conception rate (Takada et al. 2005 Jpn. J. Embryo Transfer 27, 59–64). In this experiment, survival and conception rates of in vitro-produced (IVP) bovine embryos cryopreserved in 6% glycerol solution (GLY) were compared to those of embryos cryopreserved in 10% ethylene glycol plus 0.1 M sucrose solution (EG). Cumulus–oocyte complexes were matured and fertilized according to Numabe et al. (2000 Theriogenology 54, 1409–1420). Presumed zygotes were cultured in mSOF supplemented with 5% calf serum (CS) and 0.25% linoleic acid albumin at 38.5�C under 5% CO2, 5% O2, 90% N2 for 7 days. At the expanded blastocyst stage, embryos were placed in GLY or EG in PBS supplemented with 20% CS for 15 min at room temperature and loaded into 0.25-mL straws. Straws were placed directly into an alcohol freezer. When the cryoprotectant was GLY, straws were seeded at -4.0�C, held for 10 min, cooled at 0.5�C min to -30.5�C, and then plunged into liquid nitrogen. When the cryoprotectant was EG, the seeding point was -7.5�C, and the plunging point was -34.0�C, but the rest of the protocol was the same as for GLY. In Exp. 1, thawing in both groups was done in a 30�C water bath, and the contents were directly rehydrated in PBS with 20% CS. Thawed embryos were cultured in mSOF with 5% CS for 24 h to assess embryo survival rate, based on the re-expansion of the blastcoele and on their hatching ability. In Exp. 2, embryos in both groups were thawed and transferred to synchronous recipients without removing the cryoprotectant. Data were analyzed using chi-square analysis. In Exp. 1, the developmental rates of post-thaw embryos were similar in GLY (46/52, 88.5%) and EG (45/52, 86.5%); however, the hatching rate was significantly higher (P < 0.05) in embryos cryopreserved in EG (26/52, 50.0%) than in GLY (15/52, 28.8%). In Exp. 2, the conception rates of embryos were similar in both groups, GLY (7/15, 46.7%) and EG (6/15, 40.0%). In conclusion, after direct rehydration of embryos, the developmental ability of IVP bovine embryos cryopreserved in EG was superior to that of embryos cryopreserved in GLY in vitro. However, conception rates in vivo were similar in both groups. These results suggest that a lower concentration of glycerol might be still useful as a cryoprotectant for direct transfer of IVP bovine embryos.

153 EFFECT OF DIFFERENT HOLDING AND TRANSPORT MEDIA ON CONCEPTION RATES FOLLOWING TRANSFER OF IN VIVO AND IN VITRO FERTILIZATION-DERIVED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 180
Author(s):  
C. A. Zanenga ◽  
C. M. Martins ◽  
N. C. Rodovalho ◽  
F. Aidar ◽  
J. F. Hasler ◽  
...  
Keyword(s):  
Animal Health ◽  
Conception Rate ◽  
Bovine Embryos ◽  
Chi Square ◽  
Mato Grosso ◽  
Conception Rates ◽  
Chi Square Test ◽  
Donor Cows

Two experiments were conducted to compare conception rates following embryo transfer (ET) of bovine embryos held and transported in Syngro® holding medium (Bioniche, Belleville, Ontario, Canada) with other 2 holding media: Emcare® (ICPbio, Auckland, New Zealand) for in vivo-derived embryos and HEPES-buffered synthetic oviduct fluid (H-SOF) for IVF-derived embryos. The first trial was performed in the period from October through December 2006 at the Curitiba farm in Poços de Caldas, Minas Gerais, Brazil. A total of 140 in vivo-derived embryos were produced from 20 Nelore donor cows and transferred fresh at the same farm. After each donor recovery, embryos were equally separated per stage (morula or blastocyst) and classification (grades 1, 2, and 3) into 2 Petri dishes, each containing either Syngro or Emcare. The embryos were held for an average of 3 h after recovery, loaded into 0.25-mL straws, and transferred fresh into recipients heifers, which were all previously synchronized with the same hormonal protocol treatment and presented a corpus luteum on the day of transference. Conception rate was checked at approximately 60 days of conception by rectal palpation. The chi-square test was used for statistical analysis. The conception rate of embryos maintained in Syngro was significantly higher than those in Emcare: 64.2% (43/67) v. 47.9% (35/73; P < 0.05). A second experiment was performed between September and December 2008 at Embriza Biotechnology Laboratory, Campo Grande, Mato Grosso do Sul, Brazil. A total of 1689 IVF-derived embryos (stage = 7, quality = 1), produced from Nelore donor cows, were randomly assigned to be held and transported in either Syngro (769) or H-SOF transport medium (920). Transportation time ranged from 1 to 9 h, and the recipient farms ranged from 100 to 1200 km in distance from the Embriza Laboratory. Crossbred recipient heifers (Bos taurus × Bos indicus) were synchronized with prostaglandin or vaginal progesterone device protocols. Pregnancy diagnosis was performed by ultrasonography approximately 60 days after ET. Statistical comparisons were performed using the chi-square test. Conception rates resulting from embryos transported in Syngro (45.1%, 347/769) and in H-SOF (42.0%, 386/920) were not different (P = 0.19). Financial support from Embriza Biotecnology, Tecnopec LTDA, and Bioniche Animal Health

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

scholarly journals 245RELATIVE ABUNDANCE OF HSP 70 MRNA IN BOVINE EMBRYOS PRODUCED IN VITRO USING DIFFERENT EMBRYO/VOLUME RATIOS IN CULTURE

2004 ◽  
Vol 16 (2) ◽  
pp. 243
Author(s):  
A.T.D. Oliveira ◽  
C. Gebert ◽  
R.F.F. Lopes ◽  
H. Niemann ◽  
J.L. Rodrigues
Keyword(s):  
Relative Abundance ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Gene Transcripts

In spite of in vitro embryo production systems having been greatly improved over recent years, employing a variety of culture conditions (media, protein sources, gas atmosphere, etc.), we still do not know much about the real necessity of embryos to develop under the same conditions as occur in vivo. These differences between in vivo and in vitro culture at preimplantation embryonic stages can produce deviations in gene expression and in normal fetal development (large offspring syndrome). Heat shock proteins (Hsp) are engaged in cell response to regulatory signals or perturbations in the microenviroment and can be used as a sensitive indicator of stress caused by suboptimal culture conditions (Wrenzycki et al., 2001Hum. Reprod. 16, 893–901). Hsp act as chaperones in facilitating protein folding and assembly and stabilize damaged proteins to prevent aggregation of fragments, thereby allowing repair or degradation. The aim of the present study was to investigate the effects of different embryo/volume ratios on bovine embryo development and the relative abundance of Hsp 70.1 gene transcripts. In this experiment, oocytes were isolated from slaugterhouse ovaries and matured, fertilized and cultured in groups of 5, 10, 20 or 30 per each drop of 100μL. The oocytes were matured in TCM 199 supplemented with 0.4% BSA. After maturation, oocytes were fertilized in TALP medium, using frozen/thawed sperm, selected using a percoll density gradient. The zygotes were cultured to the morula or Day 7 blastocyst stage employing SOF supplemented with 0.4 % BSA. Developmental check points were cleavage rate (Day 3pi), blastocyst formation (Day 8pi) and hatching (Day 11pi). A semi-quantitative RT-PCR assay was used to determine the relative levels of gene transcripts in single embryos at morula (Day 6) and blastocyst (Day 7) stages (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317). Data of cleavage, blastocyst formation and hatching rates were analyzed using chi-square test. Relative abundance (RA) of Hsp 70.1mRNA were compared in tested groups using ANOVA followed a Tukey test. Differences at P&lt;0.05 were considered significant. Results show that no significative difference in hatching rate per blastocyst produced was detected among the four groups. Cleavage rate and blastocyst formation were significantly higher in groups with 5, 10 and 20 embryos compared with drops containing 30 embryos. Hsp transcripts were detected in morula and blastocyst stages in all groups. In morula stage, no differences were observed in the RA of Hsp 70.1mRNA among groups with 5, 10, 20 and 30 embryos cultured per drop. However, in blastocyst stage, the RA was significantly increased in the group with 20 embryos per drop as compared to the group with 5 embryos. The results show that different embryo/volume ratios in culture influence not only cleavage rate, blastocyst formation and hatching rate, but also expression of Hsp 70.1 gene. Further studies changing other culture conditions and using in vivo-derived bovine embryos will aid in elucidating which culture systems are ideal to produce bovine embryos in vitro. This research was supported by CAPES/DAAD program and CNPq.

Vitrication of in vitro produced bovine embryos at different ages using one- and three-step addition of cryoprotective additives

1997 ◽  
Vol 9 (7) ◽  
pp. 741 ◽  
Author(s):  
S. Saha ◽  
T. Suzuki
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Different Ages ◽  
One Step ◽  
Vitrification Solution ◽  
Hatching Rates

The effect of embryo age on development and ratio of live : dead cells after vitrication and warming was examined. One-step and three-step addition of cryoprotectants in vitrification solution (40% ethylene glycol, 0·3 M trehalose and 12% polyvinylpyrrolidone) were compared usingin vitro produced (IVP) bovine blastocysts and expanded blastocysts. Rates of development and hatching were 74 ·2% and 41· 9% for Day 7, 57·8% and 23· 8% for Day 8, 33· 7% and 6·1% for Day 9 embryos with one-step addition. In three-step addition, those rates were 86·2% and 77·3% for Day 7, 72·3% and 39·0% for Day 8, 47·3% and 10·5% for Day 9 embryos. Day 7 embryos showed highest (P < 0·01) development and hatching rates with one exception. Hatching rate of Day 7 embryos with three-step addition was higher (P < 0·01) than with one-step addition. The ratio of live : dead cells differed between one-step (94%) and three-step (97%) additions for Day 7 embryos (P < 0· 05). The results indicate the higher resistance of younger IVP bovine embryos against vitrication and the potential for three-step addition of cryoprotectants to yield a higher survival rate after warming than with one-step addition.

106 EFFECT OF GLYCEROL AND ETHYLENE GLYCOL ON THE DEVELOPMENT OF IN VITRO BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 161
Author(s):  
A. C. Nicacio ◽  
R. Simões ◽  
M. A. Peres ◽  
J. S. A. Gonçalves ◽  
M. E. O. D'Ávila Assumpção ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Cell Monolayer ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Chi Square ◽  
Chi Square Test ◽  
Hatching Rates

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2�C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability. This work was supported by FAPESP 04/05335-1.

149 IN VITRO BOVINE EMBRYOS FROZEN BY DIRECT TRANSFER METHOD WITH ETHYLENE GLYCOL

2015 ◽  
Vol 27 (1) ◽  
pp. 166
Author(s):  
S. H. Kizil ◽  
M. Satilmis ◽  
N. Akyol ◽  
T. Karasahin
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Hatching Rate ◽  
Direct Transfer ◽  
Embryo Freezing ◽  
Frozen Embryos ◽  
Transfer Method ◽  
Phosphate Buffered Saline ◽  
In Vitro Survival

The objective of this study was to search for capability of freezing by ethylene glycol direct transfer method of in vitro-produced cattle embryos. Fifty-six in vitro-produced good-quality cow embryos were frozen by direct transfer method with ethylene glycol in this study. Cattle ovaries were collected from a slaughterhouse and oocytes were aspirated from follicles with 2 to 8 mm diameters. Then oocytes were let for maturation of 20 to 22 h in 100-μL microdroplets of TCM-199 with 0.1 mM β-mercaptoethanol and 20% FCS. After 5 to 6 h of fertilization in Bracket Oliphant (BO), they were cultured for 7 days in 100 µL of CR1aa medium with 5% FCS under 5% CO2, 98% relative humidity, and 38.5°C in a CO2 incubator. Embryos were equilibrated for 15 min in room temperature in 1.8 M ethylene glycol + 0.1 M sucrose in Dulbecco's phosphate buffered saline (D-PBS) supplemented with 20% FCS. Embryos were then loaded individually into a 0.25-mL straw and placed directly into a cooling chamber of a programmable freezer with methyl alcohol precooled to –7°C. After 2 min, the straw was seeded and maintained at –7°C for 8 min more. Then it was cooled to –30°C at 0.3°C min–1 before plunging into liquid nitrogen. The frozen embryos were thawed by allowing the straw to stand in air for 5 to 6 s and then immersing them in a 30°C water bath for 10 s. After thawing, embryos were transferred into TCM-199 + 0.1 mM β-mercaptoethanol + 20% FCS medium to check in vitro survival rates at 48 h post-thawing. The re-expansion and hatching rate of blastocysts was 64.28% (36 blastocysts). This result indicated that ethylene glycol can be used effectively for cow embryo freezing as a suitable cryoprotectant for direct transfer method.

Effect of Two-step Vitrification on Developmental Competence of in vitro and in vivo Produced Bovine Embryos

2010 ◽  
Vol 79 (9) ◽  
pp. S55-S61 ◽  
Author(s):  
Jaroslava Hlavicová ◽  
Miloslava Lopatářová ◽  
Svatopluk Čech
Keyword(s):  
Developmental Stages ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Holstein Friesian ◽  
Quality Grade ◽  
Friesian Cattle

The aim of this study was to establish the effect of two-step vitrification on survival rate of bovine embryos produced in vitro (method A) and in vivo (method B) from Holstein-Friesian cattle. The embryos suitable for vitrification were frozen by a two-step technique, using increasing concentrations of dimethyl sulphoxide (DMSO) and ethylene glycol (EG). After thawing, the quality grade and developmental stage of embryos was assessed. In vitro developmental competence of embryos of different quality grade obtained by method B (n = 82) was significantly higher (p < 0.001) compared to method A (n = 98). The best results were detected when we vitrified the embryos of the grade 1 quality; namely, the hatched blastocyst stage was reached by 6.9% (2/29) of embryos retrieved by method A and by 36.7% (11/30) of embryos retrieved by method B (p < 0.01). In the case of developmental competence of embryos at different developmental stages we reached significantly better results (p < 0.001) when we vitrified the embryos produced by method B (n = 84) in comparison with method A (n = 67). We noted a higher hatching rate at the stage of expanded blastocyst; namely, the hatched blastocyst stage was reached by 7.4% (2/27) of embryos produced by method A and by 30.8% (8/26) of embryos produced by method B (p < 0.05). In general, the hatched blastocyst stage was reached by 15.1% (50/331) of all thawed embryos retrieved by method A and B. In conclusion, when we applied two-step vitrification on the grade 1 quality embryos at the stage of expanded blastocyst produced in vitro or at the stage of morula produced in vivo we achieved the highest hatching rates.

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.

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