35 EFFECT OF 6-DIMETHYLAMINOPURINE TREATMENT DURATION ON PRONUCLEAR FORMATION AND IN VIVO DEVELOPMENT OF CANINE CLONED EMBRYO

2015 ◽  
Vol 27 (1) ◽  
pp. 110
Author(s):  
H. J. Oh ◽  
G. A. Kim ◽  
M. J. Kim ◽  
Y. K. Jo ◽  
Y. B. Choi ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Calcium Ionophore ◽  
Brdu Incorporation ◽  
Surrogate Mothers ◽  
Donor Cells ◽  
Significant Difference ◽  
Implantation Rates ◽  
Artificial Activation ◽  
Pronuclear Formation

Artificial activation is an important step for successful somatic cell nuclear transfer (SCNT). In order to clone animals, diverse methods of activation have been studied to increase the developmental efficiency of cloned embryos. Here, we investigated the pronucleus formation and in vivo development of canine cloned embryos produced by different durations of 1.9 mM 6-dimethylaminopurine (DMAP) treatment. For canine SCNT, in vivo-matured oocytes were enucleated, microinjected into the perivitelline space with donor cells, and fused by electrical stimulation. For activation, the fused couplets were cultured for 4 min in 10 μM calcium ionophore, and then they were divided into 2 groups: (1) the 2DMAP group was cultured for 2 h in DMAP; (2) the 4DMAP group was cultured for 4 h in DMAP. Activated cloned embryos were subjected to 2 analyses: (1) observing the pronuclear formation by bromodeoxyuridine (BrdU) incorporation at 2 h, 4 h and 8 h post-activation (hpa), and (2) following fetus formation and pregnancy efficiency after embryo transfer into naturally synchronous recipients. Pregnancy diagnosis was performed by ultrasonography on Day 26 of embryo transfer. Data were analysed using Graph Prism software (GraphPad Software Inc., San Diego, CA, USA). All cloned embryos of the 2DMAP group showed BrdU incorporation at 2 hpa, whereas 4DMAP embryos showed 77.7% BrdU incorporation at 2 hpa (P < 0.05). Incorporation of BrdU was detected in all cloned embryos of both experimental groups after 4 hpa and 8 hpa. A total of 370 cloned embryos were transferred to 24 surrogate mothers (182 cloned embryos into 12 recipients in 2DMAP group and 188 cloned embryos into 12 recipients in 4DMAP group). There was no significant difference in pregnancy rate (2DMAP; 41.6% v. 4DMAP; 33.3%) or implantation rates (2DMAP; 4.9% v. 4DMAP; 3.7%) between the 2 groups. In conclusion, DMAP exposure for 2 h in activation completed pronucleus development of canine reconstructed embryos. However, none of the applied tested treatments resulted in increased implantation rates. This study was supported by RDA (#PJ008975022014), IPET (#311062–04–3SB010), Research Institute for Veterinary Science, Nestlé Purina PetCare and the BK21 plus program.

36 EFFECT OF ACTIVATION METHODS ON DNA SYNTHESIS AND DEVELOPMENT OF CANINE PARTHENOGENETIC EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 132 ◽  
Author(s):  
H. J. Oh ◽  
M. J. Kim ◽  
G. A. Kim ◽  
Y. K. Jo ◽  
J. Choi ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Nuclear Transfer ◽  
Combined Treatment ◽  
Calcium Ionophore ◽  
Implantation Rate ◽  
Limb Bud ◽  
Brdu Incorporation ◽  
Significant Difference ◽  
Pronuclear Formation

Artificial activation is an important step for successful somatic cell nuclear transfer. In mammals, different methods of parthenogenesis have been studied to increase the developmental efficiency of cloned embryos. In an attempt to improve the techniques of nuclear transfer in canine species, this study investigated the timing of DNA synthesis and in vivo development of canine parthenotes produced by different activation treatments. For canine parthenotes, in vivo matured oocytes were obtained by flushing (~72 h after ovulation) the oviducts of mixed breed bitches. Denuded oocytes were cultured for 4 min in 10 μM calcium ionophore, and then they were divided into 2 groups: (1) 2DMAP group was cultured for 2 h in 6-DMAP; (2) 4DMAP group was cultured for 4 h in 6-DMAP. The first experiment determined DNA synthesis of parthenotes by 1 h treatment with incorporation and immunofluorescent detection of thymidine analogue 5-bromo-2′-deoxyuridine-5′-triphosphate (BrdU). The primary antibody was mouse anti-BrdU (Sigma, St. Louis, MO, USA), and the secondary antibody was fluorescein (FITC)-conjugated affinity purified goat anti-mouse IgG (Jackson). In order to examine the pronuclear formation and the onset of DNA synthesis in experimental groups, parthenotes derived from 2DMAP and 4DMAP groups were observed by BrdU incorporation at 2, 4, and 12 h post-activation (hpa). Data were analysed using Graph Prism software (GraphPad, San Diego, CA, USa). The next experiment observed in vivo development as follows: parthenotes were surgically transferred to synchronized recipient female dogs. The implantation rate of parthenogenetic fetuses was observed in uterus of recipients on Day 26 of pregnancy. All parthenotes of the 2DMAP group showed BrdU incorporation at 2 hpa, whereas 4DMAP parthenotes showed 96% BrdU incorporation at 2 hpa. Incorporation of BrdU was detected in all parthenotes of both experimental groups after 4 hpa. A total of 98 parthenotes were transferred to 9 surrogate mothers (53 parthenotes into 5 recipients in 2DMAP group and 45 parthenotes into 4 recipients in 4DMAP group). There was no significant difference in pregnancy rate between the 2 groups (2DMAP: 60% v. 4DMAP: 50%), whereas the implantation rates were significantly higher in 2DMAP (24.5%) compared with 4DMAP (4.4%; P < 0.001). The recovered parthenotes were able to develop to the stages of limb-bud formation, but few parthenotes showed the small and degenerating formation. Regardless of treatment group, the implantation site of the fetuses indicated either one side or both of the uterus. In conclusion, the present results demonstrated that the protocols using combined treatment with 10 μM of calcium ionophore (4 min) followed by a 2-h culture with 1.9 mM of DMAP resulted in completing pronuclear formation and enhancing fetal formation. This result could be useful method for improving canine cloned embryos production. This study was supported by IPET (#311062–04–2-SB010), RNL Bio (#550–20130013), RDA (PJ008975022013), and Research Institute for Veterinary Science and Natural Balance.

42 DEVELOPMENT OF PIG EMBRYOS CLONED FROM DONOR CELLS TREATED WITH TRICHOSTATIN A

2008 ◽  
Vol 20 (1) ◽  
pp. 101 ◽  
Author(s):  
J. Li ◽  
Y. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
K. Villemoes ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Embryonic Genome ◽  
Donor Cells ◽  
Deacetylase Inhibitor ◽  
Vitro Development

Development to the blastocyst stage following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to a totipotent state. Reprogramming of the transferred somatic nuclei must be completed by the time normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, Enright et al. (2003 Biol. Reprod. 69, 896–901) reported that in vitro development of cloned cow embryos was improved by treatment of donor cells with a histone deacetylase inhibitor, TrichostatinA (TSA). So far, there are no reports available for adult pig fibroblast cells treated with TSA. The objective of this study was to investigate whether the development of handmade cloned embryos in pig could be improved by using TSA-treated donor cells. Adult pig fibroblast cells were treated with 100, 150, or 200 nm TSA for 24 h, compared to untreated controls, and were then used as donor cells. The cells were electrofused with handmade enucleated pig oocytes separately and were activated with calcium ionophore and cycloheximide. They were subsequently cultured in porcine zygote medium 3 (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) using the well of the well system (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Experiments were repeated 4 times and the data were analyzed with AVEDEV and t-test in Excel (Microsoft Excel 2007). The cleavage rates and the total cell numbers per blastocyst were similar between groups (P > 0.05), as shown in Table 1. However, the cloned blastocyst rate using donor cells treated with 100 nm TSA was higher than in the other groups (69.9 ± 4.7% v. 43.6 ± 4.3%, 43.1 ± 5.8%, or 46.6 ± 3.6%; P < 0.05), as shown in Table 1. These data suggest that proper TSA treatment for donor cells before somatic cloning improves the rate of development of porcine handmade cloned embryos to the blastocyst stage. Further research is needed to examine the in vivo development of embryos reconstructed with TSA-treated donor cells. Table 1. Developmental ability of cloned pig embryos derived fromTSA-treated donor cells

33 PROPAGATION OF ELITE LIFESAVER DOGS BY SOMATIC CELL NUCLEAR TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 164
Author(s):  
B. C. Lee ◽  
H. J. Oh ◽  
M. J. Kim ◽  
G. A. Kim ◽  
E. J. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Media ◽  
Calcium Ionophore ◽  
Defined Medium ◽  
German Shepherd ◽  
Surrogate Mothers ◽  
Working Dogs

Canine somatic cell nuclear transfer (cSCNT) has been used as a useful tool for propagation of elite working dogs. In 2009, 7 cloned dogs were successfully produced using somatic cells derived from the excellent drug-sniffing dog of Korea Customs Service. All cloned dogs perfectly performed drug detection in Incheon International Airport. The objective of the present study was to compare the efficiency of the 2 activation culture media to clone the retired Baekdu, a veteran rescue dog that performed lifesaving activities worldwide for 6 years in Korea National Emergency Management Agency (NEMA). Ear tissue was collected from a 10-year-old male German Shepherd and fibroblasts were cultured for cSCNT. The cells were injected into the perivitelline space of enucleated in vivo-matured dog oocytes, fused with electric stimulation using an electro cell fusion apparatus (Nepa Gene Co. Ltd.), and activated chemically. In the activation protocol, 2 different types of media were tested to investigate the effect of proteins with undefined functions. The first medium was a modified synthetic oviduct fluid (mSOF), which is a complex culture medium with BSA that includes undefined functions. The second medium was the porcine zygote medium (PZM-5), which is a chemically defined medium with polyvinyl alcohol (PVA). The fused couplets were activated by mSOF medium supplemented with 1.9 nM DMAP (SOF-DMAP), and PZM-5 supplemented with 1.9 nM DMAP (PZM-DMAP) for 4 h, followed by 4 min of calcium ionophore treatment. Then, reconstructed oocytes were transferred into the uterine tube of naturally estrus-synchronized surrogate dogs. In the PZM-DMAP group, a total of 56 activated cloned embryos were transferred into 3 female recipient dogs, and a total of 64 activated cloned embryos from the SOF-DMAP group were transferred into 4 female recipients. Pregnancy diagnosis was performed using a SONOACE 9900 (Medison, Seoul, Korea) ultrasound scanner with 7.0-MHz linear-array probe between 30 and 35 days after embryo transfer. As a result, pregnancy was detected in 1 out of 3 surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and 1 pregnancy (25%) was detected in 4 surrogate mothers receiving cloned embryos from the SOF-DMAP group. Two pregnant dogs each gave birth to 1 healthy cloned puppy by cesarean section. This study shows that existence of proteins with undefined functions in activation medium did not affect the dog cloning. In addition, the number of elite working dogs in diverse fields can be increased by the NT technique using donor cells derived from small tissue of elite working dogs. This study was supported by RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), IPET (no. 311062-04-1-SB010), Research Institute for Veterinary Science, and TS Corporation.

47 CRYOPRESERVATION OF BOVINE GERM CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE)

2017 ◽  
Vol 29 (1) ◽  
pp. 131
Author(s):  
T. Fujikawa ◽  
C. Kubota ◽  
T. Ando ◽  
S. Imamura ◽  
M. Tokumaru ◽  
...  
Keyword(s):  
Survival Rate ◽  
Germ Cell ◽  
Embryo Transfer ◽  
Conception Rate ◽  
Control Group ◽  
Vitro Fertilization ◽  
Polyamino Acid ◽  
Significant Difference

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34 v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P = 0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48 h and hatched rate until 72 h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v. CPLL: 31 v. 50), and recipients were diagnosed for pregnancy ultrasonically 50 days after embryo transfer. Conception rate of CPLL group was higher than control group (50.0% v. 29.0%; P = 0.063). In both experiments, the significant differences between control group and CPLL group were determined by chi-squared test. The effectiveness of CPLL in cells and embryos has been reported; however, there is no report using CPLL in bovine germ cells. In this research, CPLL improved the conception rate of AI and ET, probably due to its low toxicity and protection of the cell membrane. These results suggest that CPLL is available as a new cryoprotective material for bovine sperm and embryo in slow freezing methods.

scholarly journals comparison between Cleavage Stage versus Blastocyst Stage Embryo Transfer in an Egyptian Cohort Undergoing in vitro Fertilization: A possible Role for Laser Assisted Hatching

2011 ◽  
Vol 5 ◽  
pp. CMRH.S7735 ◽  
Author(s):  
Sherif F. Hendawy ◽  
TA Raafat
Keyword(s):  
Embryo Transfer ◽  
Intracytoplasmic Sperm Injection ◽  
Blastocyst Transfer ◽  
Assisted Hatching ◽  
Vitro Fertilization ◽  
Significant Difference ◽  
Implantation Rates ◽  
Group Ii ◽  
In Pregnancy

Background Extended in vitro embryo culture and blastocyst transfer have emerged as essential components of the advanced reproductive technology armamentarium, permitting selection of more advanced embryos considered best suited for transfer. Aim of study The aim of this study was to compare between cleavage stage and blastocyst stage embryo transfer in patients undergoing intracytoplasmic sperm injection, and to assess the role of assisted hatching technique in patients undergoing blastocyst transfer. Patients and methods This study was carried out on two groups. Group I: 110 patients who underwent 120 cycles of intracytoplasmic sperm injection with day 2-3 embryo transfer—for unexplained infertility or male factor within the previous 3 years. Their data obtained retrospectively from medical records. Group II: 46 age matched infertile female patients undergoing 51 intracytoplasmic sperm injection cycles for similar causes. Patients in Group II were further subdivided into 2 equal subgroups; Group Ila (23 patients), which had laser assisted hatching and Group IIb (23 patients), which did not have assisted hatching. All patients had an infertility workup including basal hormonal profile, pelvic ultrasound, hysterosalpingogram and/or laparoscope and semen analysis of the patient's partner. All patients underwent controlled ovarian hyperstimulation: Using long protocol of ovulation induction. Laser assisted hatching was done for blastocysts of 23 patients. Results Comparison between both groups as regards the reproductive outcome showed a significant difference in pregnancy and implantation rates, both being higher in group II ( P < 0.05) Comparison between both subgroups as regards the reproductive outcome showed a highly significant difference in pregnancy and implantation rates, both being higher in Group IIa ( P < 0.01). There was also a significantly higher rate of multiple pregnancies among Group IIa ( P < 0.05). Conclusion Blastocyst transfer is a successful and improved alternative for patients with multiple failed in vitro fertilization attempts, associated with a significant increase in pregnancy and implantation rates. Furthermore, laser assisted hatching increases implantation and clinical pregnancy rates.

190 EFFECT OF THE PROPERTIES OF CERVICAL MUCUS ON PREGNANCY RATES IN HOLSTEIN HEIFERS AND COWS AFTER EMBRYO TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 203
Author(s):  
T. Maekawa ◽  
S. Morita ◽  
O. Douchi ◽  
H. Koyama
Keyword(s):  
Embryo Transfer ◽  
Evaluation Method ◽  
Subjective Evaluation ◽  
Selection Criterion ◽  
Pregnancy Rates ◽  
Cervical Mucus ◽  
Chi Square ◽  
Significant Difference

Selection of animals as recipients of embryo transfer is an important procedure of embryo transfer on farms. Most animals are evaluated for their acceptability as recipients based on the quality of their corpus leteum (CL). However, since rectal palpation is a subjective evaluation method, a more objective method of assessing the suitability of the recipient is required. Cervical mucus may be able to be used to evaluate the condition of the uterus indirectly. The purpose of this study was to investigate the relationship between the properties of cervical mucus and pregnancy rates after embryo transfer in Holstein heifers and cows. Cervical mucus was collected using a swab off the ostium uteri externum and was stained with 5% Giemsa's solution for 20 min one day before embryo transfer. The stained cervical mucus were classified based on the type of staining pattern (Kitamura et al. 2003 Theriogenology 59, 307) into five groups: filiaceous (Type 1), taenia (Type 2), claustral (Type 3), nubecula (Type 4), or aqueous (Type 5). Proportions of the types of cervical mucus and pregnancy rates were analyzed by chi-square test. In Experiment 1, 113 heifers and 266 cows were examined for cervical mucus type. No significant difference was observed in the proportions of the types of cervical mucus between heifers and cows (heifers: 35.4%, 18.6%, 16.8%, 25.7%, and 3.5%; cows: 24.4%, 14.3%, 20.3%, 30.8%, and 10.2% for Types 1∼5, respectively). In Experiment 2, either a fresh or frozen-thawed embryo was implanted in vivo in 84 heifers and 163 cows 7 days after estrus. The heifers and cows were judged to have normal sized CLs (normal, 17 mm or more) and have no vaginal abnormalities such as cervical mucus contaminated with pus and urovagina as per vaginal examination. The proportions of acceptable Type 5 recipients was lower than that of Type 1 (P < 0.05). The pregnancy rates were 47.6% for heifers and 45.4% for cows (Table 1). The pregnancy rates of Types 1–3 (53.5%) were significantly higher than for Types 4 and 5 (29.9%) in the cows (P < 0.05). Although there was no statistically significant difference, the same tendency was observed in the heifers. Pregnancy was unsuccessful in Type 5 recipients, both heifers and cows. The total pregnancy rates of Types 1–3 were significantly higher than for Types 4 and 5 (53.5% vs. 29.9%, P < 0.001). These results suggest that cervical mucus type can serve as an objective selection criterion for embryo recipients. Further, embryo transfer should be avoided in Type 5 recipients. Table 1. Cervical mucus type and pregnancy rates (%) in dairy cattle

Microtubule organisation, pronuclear formation and embryonic development of mouse oocytes after intracytoplasmic sperm injection or parthenogenetic activation and then slow-freezing with 1,2-propanediol

2013 ◽  
Vol 25 (4) ◽  
pp. 609 ◽  
Author(s):  
Dun-Gao Li ◽  
Yan Zhu ◽  
Feng-Ying Xing ◽  
Shan-Gang Li ◽  
Xue-Jin Chen ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Survival Rates ◽  
Slow Freezing ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes ◽  
Significant Difference ◽  
Artificial Activation ◽  
Pronuclear Formation

The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation–activation, embryonic development of oocytes–zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5 h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze–thawing did not affect the later development of zygotes.

16 EFFECT OF DONOR CELL AND TRICHOSTATIN A ON DEVELOPMENT OF CLONED DAIRY CATTLE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 119
Author(s):  
Z. Mei-Ling ◽  
Z. Yun-Hai ◽  
T. Yong ◽  
L. Ya ◽  
C. Hong-Guo ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Donor Cell ◽  
Developmental Competence ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Different Origins ◽  
Vitro Development

The objective of present study was to investigate the effects of treatments to donor cells with fresh digestion (FD), cryopreservation/thawing (CT), trichostatin A (TSA) and durations of culture using TSA-CR1aa medium on in vitro development of dairy cow cloned embryos. In addition, some somatic cell cloned embryos were transferred to surrogates in heat to evaluate the in vivo developmental competence. The results (Table 1) showed that pretreatment of donor cells using TSA could significantly increase both cleavage and blastocyst rates of embryos (P < 0.05) compared with FD and CT group, whereas no significant difference was found between FD and CT group. When cloned embryos were subjected to TSA treatment in CR1aa for different times (0, 24, 48 and 60 h), the results showed that the blastocyst rate in the 60-h group was the highest (36.11 ± 1.78%) compared with the other groups (P < 0.05). Whereas the reconstructed embryos derived from donor cells treated with TSA for 24 h were continually cultured in TSA for different times (24, 48 and 60 h), the results showed that the blastocyst rate (37.39 ± 1.78%) in the 60-h group was significantly higher than that of the 24-h (25.48 ± 1.34%) group (P < 0.05). Finally, when the cloned embryos from different groups were respectively transferred to 40 natural oestrus recipients, no significant difference in terms of pregnancy rate among groups was found; however, a viable cloned calf was successfully obtained from TSA-treated donor cells and cloned embryo. Therefore, cloned embryos treated with optimized methods can develop to term. Table 1.Pregnancy results established from embryos of different origins

85 Elongation of trophoblastic vesicles between Days 15 and 18 in cattle

2019 ◽  
Vol 31 (1) ◽  
pp. 168
Author(s):  
C. Richard ◽  
S. Degrelle ◽  
V. Gelin ◽  
A. Neveux ◽  
P. Chavatte-Palmer ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Standard Error ◽  
Normal Growth ◽  
Elongation Rate ◽  
Significant Difference ◽  
The Difference ◽  
Different Origins ◽  
Similar Growth

Once formed, bovine blastocysts differentiate while growing exponentially from 150-200μm (Days 7 or 8) to 200-250mm (Days 17 to 18; Sandra et al. 2017 Annu. Rev. Anim. Biosci. 5, 205-228). Thus, the length of the conceptus doubles every day between Days 9 and 16 (Berg et al. 2010 Theriogenology 73, 250-260); however, this was observed on whole conceptuses. The objective of the current study was to test whether this elongation rate is similar when the embryonic disc has been excised. Six heifers were used to produce Day-15 conceptuses, either fully developed in vivo or developed in vivo for a week after embryo transfer of Day 8in vitro-produced blastocysts. Day 15 conceptuses were recovered, measured, and cut into pieces to produce trophoblastic vesicles (TV; Heyman et al. 1984 J. Reprod. Fertil. 70, 533-540) of 4±0.07 or 4.4±0.65mm long (mean±standard error of the means) for in vivo- or in vitro-produced TV, respectively. All TV were transferred into oestrus-synchronized recipients (5 heifers and 1 cow). Each female received 8-9 TV so that a total of 24in vivo-derived and 26in vitro-produced TV were transferred in utero for a period of 3 days. The TV originating from different Day-15 conceptuses were mixed at the time of transfer, so that each recipient received the TV from different origins (conceptus and donor cow). Transcervical collection was used at Day 15 and 18 for conceptus and TV recovery (Richard et al. 2015 Theriogenology 83, 1101-1109). At Day 18, TV elongation size was analysed (mean±standard error of the means) by unpaired t-test using GraphPad Prism software (GraphPad Inc., San Diego, CA, USA). At Day 15, conceptuses from the in vivo and in vitro groups displayed different sizes and length variabilities (24-32v. 2-24mm, respectively). At Day 18, TV recovery rate was 79% for the in vivo- v. 62% for the in vitro-derived group and mean elongation rate (over 3 days) was ×5.4 (minimum=2.5, maximum=10) v. ×7.6 (minimum=0.7, maximum=20.5), respectively. There was no significant difference for TV size between groups at Day 18 (21.75±2.24 mm v. 33.38±11.63mm, respectively). Altogether, the variability in length at Day 15 was previously reported, the difference in TV recovery between in vivo and in vitro groups reached 17% and was similar to the loss of 11% that occurs in the first week after classical embryo transfer. In opposition to studies where in vitro-produced conceptuses were shorter than in vivo-developed ones, in vivo and in vitro groups of TV likely followed similar growth. Whether this reflects a normal growth awaits further studies.

Establishment of a basic method for manipulating preantral follicles: effects of retrieval method on in vitro growth of preantral follicles and intrafollicular oocytes

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 109-116 ◽  
Author(s):  
S.T. Lee ◽  
M.H. Choi ◽  
S.P. Gong ◽  
J.Y. Han ◽  
J.M. Lim
Keyword(s):  
Oocyte Diameter ◽  
Enzymatic Method ◽  
Mechanical Method ◽  
Retrieval Method ◽  
Preantral Follicles ◽  
Significant Difference ◽  
Step Growth ◽  
Pronuclear Formation

SummaryThe aim of this study was to establish a basic manipulation protocol of preantral follicles for deriving developmentally competent oocytes. Primary, early and late secondary follicles retrieved from the ovaries of 14-day-old F1 (C57BL/6 × DBA2) female mice mechanically or enzymatically were cultured singly and in vitro growth of the follicles and maturation of intrafollicular oocytes were subsequently monitored. A mechanical method retrieved more (p < 0.0001) follicles (339 ± 48 vs. 202 ± 28) than an enzymatic method. However, the enzymatic method collected more singly isolated follicles that could be provided for subsequent culture (102 ± 26 vs. 202 ± 28). When an enzymatic method was employed, early and late secondary follicles required 9 and 6 days for reaching the maximal incidence of the pseudoantral stage. However, primary follicles were not possible to develop into the pseudoantral stage. The optimal duration of oocyte maturation from the onset of follicle culture was 7 days and 5–7 days for early and late secondary follicles, respectively. A general decrease in oocyte diameter (65.2–65.53 μm vs. 75 μm) and zona thickness (5.41–5.74 μm vs. 7.76 μm) was detected in in vitro-derived compared with in vivo-derived matured oocytes. Pronuclear formation was detected in 86–94% of mature oocytes after parthenogenetic activation and no significant difference was detected among groups. These results showed that preantral follicles retrieved by an enzymatic method underwent step-by-step growth in vitro, which could yield mature oocytes.

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