• Journal of Yeungnam Medical Science
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• v.38 no.1
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• pp.53-59
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• 2021

Background: There are no guidelines for the optimal incubation time or temperature to improve pregnancy outcomes in testicular sperm extraction-intracytoplasmic sperm injection (TESE-ICSI) cycles. We aimed to evaluate whether a 24-hour in vitro culture of testicular spermatozoa affects pregnancy outcomes in TESE-ICSI cycles. Methods: This was a retrospective study of 83 TESE-ICSI cycles using testicular spermatozoa in 46 couples with male partners suffering from nonobstructive or obstructive azoospermia. Sperm retrieval was performed either on the oocyte retrieval (OR) day (65 cycles in 33 couples; group A) or on the day before OR (18 cycles in 13 couples; group B) followed by in vitro culture for 24 hours. The clinical characteristics and pregnancy outcomes, including the number of retrieved oocytes, fertilization rates, embryo transfer rates, implantation and clinical pregnancy rates, were compared between the two groups. Results: There were no differences in terms of clinical characteristics except for the levels of luteinizing hormone (LH) in males. Group B had higher LH levels than group A (4.56±1.24 IU/L vs. 3.67±1.07 IU/L, p= 0.017). Group B showed higher fertilization rate (72.4%±32.1% vs. 59.2%±21.7%, p=0.045), implantation rate (35.0%±34.1% vs. 14.0%±21.5%, p=0.010), pregnancy rate per cycle (80% vs. 39%, p=0.033), and clinical pregnancy rate per cycle (80% vs. 37.5%, p=0.024) than those of group A. Conclusion: Testicular sperm retrieval performed on the day before OR followed by in vitro culture can potentially improve pregnancy outcomes.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 1997.05a
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• pp.27-34
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• 1997

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours($38^{\circ}C$, 5% $CO_2$) in modified TCM-199 medium supplemented with 5% superovulated cow serum(SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3M methyl cellosolve(MC) <1.1M diethylene glycol(DEG), 1.8M ethylene glycol(EG), 1.6M propylene glycol(PG) and 1.1 M 1,3-butylene glycol(BG) solutions. They were then loaded into 0.25ml straws, placed into an alcohol bath freezer at $0^{\circ}C$, cooled from $0^{\circ}C$ to $-6^{\circ}C$ at $-1^{\circ}C$/minute, seeded, held for 10 minutes, and stored in liquid nitrogen. After thawing in $30^{\circ}C$ water, the embryos wee rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with a good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred non-surgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows : EG(50.0%), MC(53.6%), DEG(56.9%), PG(58.0%) and BG(11.5%). The survival rate of embryos cooled at -0.3^{\circ}C$ vs. $-0.5^{\circ}C$/minute was not significantly different(P<0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of $0.3^{\circ}C$/minute(64.6%), 31/48) than at $-0.5^{\circ}C$/minute(22.6%, 12/53). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows : MC(48%, 10/21); DEG(30%, 3/10); EG(74%, 20/27); and PG(40%, 4/10). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF Bovine embryos.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.195-203
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• 2018

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.19-24
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• 2013

This study assesses of efficiency of oocyte recovery and in vitro development for during the non breeding season in goat. Thirty-four matured goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen implanted CIDR for 10 days. Superovulation treatment of the goats received twice daily intramuscular injections of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotropin treated goats were injected with 10 mg $PGF_2{\alpha}$ on Day 8 and 400~600 IU hCG in the afternoon on Day 10. Oocytes were recovered by follicle aspiration or oviduct flushing at 35 to 40 h after hCG injection through mid-ventral incision. The in vivo matured oocytes were activated by ionomycin (5 min) and 6-DMAP (3.5~4 h). The activated oocytes were cultured in mSOF medium containing 0.8% BSA at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$, 5% $O_2$, 90% $N_2$ for 7~8 days. There was no significant difference in the mean number of CL and in vivo matured and follicular oocytes recovered. But, quality of I+II grade follicular oocytes was lower (p<0.05) in the prepubertal goat (25.0%) than the adults (52.4%). The same results were also observed in the cleavage and blastocyst rate of activated oocytes. The clavage and blastocyst rate from prepubertal derived oocytes were lower (p<0.05) in the prepubertal goat (54.5%, 23.3%) than the adult goat (86.8%, 46.6%). Considering overall these results, we suggest that maturation of donor goats is a major factor affecting recovered oocytes quality and in vitro development of activated goat oocytes. There was no significant difference in oocyte quality between seasonal treatments.

• Korean Journal of Animal Reproduction
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• v.9 no.2
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• pp.140-147
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• 1985

Seventy four Holstein heifers were randomly assigned over three trials to PRID-7+PG-6 and Synchromate B-9 regimens to synchronize estrus cycle for embryo transfer. Sexual behaviors; moounting, standing, orientation, chin-resting, sniffing, licking, rubbing and butting, vaginal swelling and mucus discharge were observed between 06-08, 12-14 and 18-20 h on 1st day and 00-02, 06-08, 12-14 and 18-20 h on 2nd day after removal of hormones. Synchromate-B treatment (81.6%) showed higher synchronized estrus rate than PRID treatment (77.8%) during observation period. Standing estrus was observed within 74 h after PG injection in PRID and within 52 h after removal of implant in Synchromate-B. About 68% of heifers in PRID and 74% of heifers in Synchromate-B showed standing estrus between 0-14 h on 2nd day after removal of the hormones. Synchromate-B resulted in a tighter synchrony of standing estrus than PRID. Incidence of average mounting and standing per head during observation period was 22.3 and 16.6 in PRID and 28.1 and 13.6 in Synchromate-B. The PRID showed peak in active mounting at 18-20 h on 1st day, however, the Synchromate-B showed at 0-2 h on 2nd day after removal of hormone. Active standing was shown between 18 h on 1st day to 20h on 2nd day in PRID, however, between 0-14 h on 2nd day after removal of hormone is Synchromate-B. There was slight difference in pattern of active mounting and standing during estrus between PRID and Synchromate-B. Conception rate of synchronized heifers transferred with fresh and frozen embryos by non-surgical and surgical methods was higher in synchromate-B (62.5%) than in PRID (38.5%). Chin-resting showed highest incidence among 6 sexual behavioral components in the both treatments. Synchromate-B showed higher incidence of chin-resting (16.6) than PRID (10.7). Synchromate-B group showed also higher incidence of orientation, sniffing and butting than PRID group. Synchromate-B resulted in more active sexual behaviors than PRID. The pattern of incidence of chin-resting, licking and butting was almost symmetrical in PRID with their peak values at 6-8 h on 2nd day, however in Synchromate-B chin-resting and sniffing was symmetrical with their peak values at 12-14 h on 2nd day after removal of hormone. There was tendency to increase vaginal swelling according to time passage of synchronized estrus in the both treatments. Incidence of mucus discharge in Synchromate-B was slightly higher than in PRID. Twenty to 40% was false negative in conception rate by tall painting before re-estrus day as judged by rectal palpation.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.147-152
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• 2011

This study was conducted to examine whether efficiency of oocyte production from superovulated prepubertal goats. Fifteen prepubertal and twenty adult goats, maintained in a pen under natural day length and fed hay ad libitum, were pretreated with progestagen implanted CIDR for 10 days. Superovulation treatment of the goats received twice daily intramuscular injection of a total of 70 mg FSH for 3 days from Day 8 of CIDR. All the gonadotrophin treated goats were injected with 10 mg $PGF_2{\alpha}$ on Day 8 and 400~600 IU hCG in the afternoon on Day 10. Oocytes were recovered by follicle aspiration or oviduct flushing at 35 to 40 h after hCG injection through mid-ventral incision. The in vivo matured oocytes was activated by ionomycin (5 min) and 6-DMAP (3.5~4 h). The activated oocytes were cultured in mSOF medium containing 0.8% BSA at 38.5$^{\circ}C$ in an atmosphere of 5% CO$_2$, 5% O$_2$, 90% N$_2$ for 7~8 days. There was no significant difference in the mean number of CL and in vivo matured and follicular oocytes recovered. But, quality of I + II grade follicular oocytes was lower (p<0.05) in the prepubertal goat (25.0%) than the adults (52.4%). The same results were also observed in the cleavage and blastocyst rate of activated oocytcs. The cleavage and blastocyst rate from prepubertal derived oocytes were lower (p<0.05) in the prepubertal goat (54.5%, 23.3%) than the adult goat (86.8%, 46.6%). Considering overall these results, we suggest that maturation of donor goats is a major factor affecting recovered oocytes quality and in vitro development of activated goat oocytes.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.199-205
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• 2007

The present study was carried out to assess the effect of superovulation response and efficiency of embryo production induced with injection of FSH dissolved in polyethylene glycol (PEG) in Hanwoo. Eighty-eight cows were divided into four groups. In group 1, cattle were intramuscularly treated with twice-daily administration of 50mg FSH for 4 days. Group 2 and 3 were subcutaneously single injection of 400mg and 200mg FSH dissolved in 30% PEG, respectively. Group 4 were subcutaneously single injection of 200 mg FSH dissolved in 30% PEG at 7 day after CIDR insertion. The number of corpus luteum (CL) in group 2 resulted in significantly (p<0.05) higher compared to group 1, 3 and 4 (18.5 vs. 11.2, 13.1 and 13.9, respectively). However, the number of total ova $(7.9{\sim}10.4)$, transferable embryos $(3.7{\sim}4.7)$, degenerate embryos $(1.9{\sim}3.5)$ and unfertilized ova $(1.8{\sim}2.7)$ did not differ among treatment groups. No difference was observed in pregnancy rate after transferring the recovered embryos among groups $(36.0{\sim}50.0%)$. In addition, blood progesterone concentrations at embryo recovery did not differ among all groups. In conclusion, although no differences were observed in the number of total ova, transferable embryos and pregnancy rate after transfer, a single injection of reduced dose of FSH (200mg FSH) at 7 day after CIDR insertion is more practical for superovulation treatments than frequent injection because of reduction of stress in Hanwoo and decreases of cost and laber.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.263-267
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• 2008

The purpose of this study was to determine toxic effect of sucrose and trehalose prior to cryopreservation on nuclear maturation and embryonic development in immature bovine oocytes. All cryoprotectant was prepared in tissue culture medium 199-HEPES (TCM 199-HEPES) with 10% fetal bovine serum (FBS). Immature oocytes were exposed to 1.2M ethylene glycol (EG) and 0.1M sucrose or 1.2M EG and 0.1M trehalose for 3 min and then were exposed to 3.2 M EG and 0.25 M sucrose or 3.2 M EG and 0.25 M trehalose for 1 min. Oocytes treated with cryoprotectants were exposed to 0.25 M sucrose or 0.25 M trehalose for 5 min and then 0.1 M sucrose or 0.1 M trehalose for 5 min. Depending on type of sugar added to cryopreservation solution, oocytes were allocated to sucrose group and trehalose group, respectively. Oocytes exposed to TCM 199-HEPES with 10% FBS were considered as control. Oocytes were cultured in TCM 199 supplemented with 10% FBS, 5 ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone, and $1\;{\mu}g/ml$ estradiol for 24 h in $39^{\circ}C$, 5% $CO_2$. Nuclear maturation was assessed by staining oocytes with 1% aceto-orcein. Oocytes were fertilized in vitro and were cultured in TCM 199 supplemented with 10% FBS, 5 mM sodium pyruvate, and antibiotics in $39^{\circ}C$, 5% $CO_2$. The rates of cleavage and blastocyst, and cell number in blastocyst were assessed. Metaphase II rates were not different among experimental groups regardless of type of sugar. The cleavage rate of trehalose group (73.3%) was significantly higher (p<0.05) than those of sucrose group (62.8%) and control group (60.8%). The blastocyst rate was significantly higher in trehalose group (p<0.05). Mean cell number in blastocyst were not different among experimental groups, although cell number of blastocyst in trehalose group was significantly higher on day 7 (p<0.05). In conclusion, sucrose and trehalose were not toxic to immature bovine oocytes prior to cryopreservation. In particular, trehalose was more effective on embryonic development.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.45-51
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• 2011

This study investigated potential relationship between fetal deaths and plasma progesterone concentrations of bitches. Serial ultrasonographic examinations were performed on small-pet 23 dogs from gestation day (GD) 15 through parturition. The dogs were 3 non-pregnant bitches, 9 spontaneous delivery bitches, 6 partial early embryonic death bitches, 2 whole early embryonic death bitches, and 3 aborted bitches. The late pregnancy (GD 51-54) appeared in 2 of the 3 aborted bitches and the hypoluteoidism appeared in 1 of the 3 aborted bitches. The plasma progesterone concentrations of partial early embryonic death bitches (n=6) showed no significant difference when compared with the spontaneous delivery bitches. We observed that plasma progesterone concentrations were dramatic decrease before the onset of embryonic death in whole early embryonic death bitches that plasma progesterone concentrations of aborted bitches at late pregnancy were significantly decreased when compared with those of spontaneous delivery bitches. The plasma progesterone concentrations of the hypoluteoidism bitch were lower than those of spontaneous delivery bitches. At the hypoluteoidism bitch, fetuses were resorbed in early pregnancy and aborted in late pregnancy. On the basis of the results, the diagnosis of partial early embryonic death could not be confirmed without ultrasonographic examination. The partial early embryonic death was considered a spontaneous phenomenon and uncorrelated with plasma progesterone concentration. However, aborted bitches and whole early embryonic death bitches were related to plasma progesterone concentrations and that of bitches gradually decreased before fetal death. These findings suggest that administration of progesterone may be a useful preventing agent against fetal death.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.177-182
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• 2017

This study was conducted to investigate the ovarian cycle changes of the mare according to the season. Twenty four Jeju crossbred horses(Thoroughbred ${\times}$ Jeju horse) raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA were used to identify follicles and corpus luteum with ultrasonography once a week(May 2016~June 2017). Blood samples of experimental horses were collected twice a week for analysis of P4 hormone levels. The mares were considered to have resumed ovarian cyclicity on the day of ovulation if they followed by regular ovarian cycles. Only 13 cases(61.9%) of the total 21cases showed normal ovarian cycle, and 8 cases (38.1%) showed delayed ovarian cycle. Three cases(16.7%) in October, 5 cases(27.8%) in November and 5 cases(27.8%) in December(27.8%) ceased the heat and the remaining 5 cases(27.8%) showed that the estrus was maintained in winter. Horses that stopped estrus ceased the heat until March of next year, and 27.8% were continued the heat during non-breeding season. Eleven cases(61.1%) of 18 cases in April and 2 cases(11.1%) of 18 cases in May returned the estrus.