• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.239-249
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• 1999

The role of amino acids in culture media for IVF-ET was examined in a total of 76 cycles. Patients received clomiphene citrate (CC) followed by hMG or GnRH-a combined with gonadotropins (FSH/hMG) for controlled ovarian hyperstimulation. Severe male (<$4{\times}10^6$ motile sperm) or age factor (>39 y) patients were excluded in this study. Pregnancy was classified as clinical if a gestational sac or fetal cardiac activity was seen on ultrasound. No significant differences were found in age, duration of infertility, follicle size, the level of $E_2$ on the day of hCG injection, the mean number of oocytes retrieved, total motile sperm count, fertilization rate and the mean number of embryos transferred between bHTF (without amino acids) and mHTF (with amino acids) groups. However, total ampules of gonadotropins were higher (p<0.01) in mHTF group than bHTF group. Significantly (p<0.05) more clinical pregnancies were recorded in mHTF group (13/30) compared with bHTF group (9/46). The multiple pregnancy rates were 11.1% in bHTF group and 7.7% in mHTF group. There were one ectopic pregnancy in mHTF group and one heterotopic pregnancy in bHTF group. Abortion rates were 22.2% in bHTF group and 7.7% in mHTF, respectively. The ongoing pregnancy or livebirth rate was significantly (p<0.05) higher in mHTF group (12/30) than bHTF group (7/46). These results suggest that the addition of amino acids in culture media is essential for culture of zygotes in vitro and adjustment of energy substrates in phosphate-free culture media appears to be beneficial for human IVF-ET procedure.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.83-93
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• 1997

Although IVF-ET is widely applied in the treatment of couples with male factor infertility, it may fail in many infertile couples with normal semen parameters, and certain couples cannot be accepted for standard IVF-ET due to unfertilization or extremely low fertilization rate of oocytes. Recently, several procedures of microassisted fertilization (MAF) using micromanipulation have been introduced, and pregnancies and births have been obtained after partial zona dissection (PZD), subzonal insertion (SUZI), and intracytoplasmic sperm injection (ICSI). This clinical study was performed to develop and establish ICSI as an effective procedure of MAF in infertile couples who could not undergo standard IVF-ET repetitively because of failure in fertilization or extremely low fertilization rate of oocytes with the conventional fertilization technique in the previous IVF-ET cycles. From March, 1995 to May, 1996, 27 cycles of IVF-ET with ICSI in 19 infertile patients were included in study group, and the outcomes of ICSI were analyzed according to fertilization rate, cumulative embryo score (CES), and pregnancy rate. The number of oocytes retrieved after controlled ovarian hyperstimulation (COH) was $10.50{\pm}6.13$ in 30 previous cycles, and $10.57{\pm}5.53$ in 27 ICSI cycles. In ICSI cycles, the number of oocytes optimal for ICSI procedure was $7.89{\pm}4.30$, and the fertilization rate of $67.9{\pm}20.2%$ could be obtained after ICSI. The number of embryos transferred was $1.43{\pm}2.40$ in previous cycles, and $4.36{\pm}1.77$ with the mean CES of $41.8{\pm}27.4$ in ICSI cycles. In ICSI cycles, the overall pregnancy rate was 29.6% (8/27) per cycle and 42.1% (8/19) per patient with the clinical pregnancy rate of 22.2% (6/27) per cycle and 31.6% (6/19) per patient. In conclusion, MAF of human oocytes with ICSI is a promising fertilization method for IVF-ET patients, especially with the past history of failure in fertilization or low fertilization rate of oocytes in the previous IVF-ET cycles, and ICSI using micromanipulation procedures applied to human oocytes will provide a range of novel techniques which may dramatically improve the pregnancy rate in IVF-ET program and contribute much to effective management of infertile couples.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.4
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• pp.166-172
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• 2019

Objective: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts. Methods: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined. Results: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p< 0.05). Conclusion: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.217-222
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• 2008

The techniques of IVM, IVF and IVC of canine oocytes may provide useful information for gamete salvage programs and the conservation of endangered canidae. This investigation has been made to determine the efficiency of in vitro maturation (IVM) as a basic experiment to study the development of canine oocytes after in vitro fertilization (IVF). The rate of oocytes developing to the Mil stage was higher in the hormone treated group (10 IU/ml hCG+eCG, 14.7%, p<0.05) than in the control group (0 IU/ml hCG+eCG, 10.0%). The monospermy and pronuclear rates of canine oocytes were investigated after caffeine treatment on IVF. Canine oocytes were fertilized in the Fert-TALP medium supplemented with 0, 10, 20 or 30 mM caffeine (Fert I, Fert II, Fert III or Fert IV, respectively). The highest pronuclear formation rate was obtained in the Fert I for 24 h IVF (6.7%, 6/89). Therefore, it is believed that unlike in other mammals, caffeine in canine IVF does not increase the efficiency of fertilization rate, and is not an important factor.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.145-150
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• 2009

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) ${\sim}4$ h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to $6{\sim}8$ cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.7
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• pp.1024-1050
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• 2001

Avian spermatozoa are characterised by high concentrations of polyunsaturated fatty acids (PUFAs), in particular docosatetraenoic (DTA, 22:4n-6) and arachidonic (AA, 20:4n-6) acids. As a result they are vulnerable to lipid peroxidation, which is considered to be an important factor of male infertility. Antioxidant systems are expressed in spermatozoa and seminal plasma and build three major levels of antioxidant defense. The first level is based on the activity of superoxide dismutase (SOD) which is, in conjunction with glutathione peroxidase (GSH-Px), catalase and metal-binding proteins, responsible for prevention of free radical formation. The second level of defence is responsible for prevention and restriction of chain reaction propagation and includes chain-breaking antioxidants such as vitamin E, ascorbic acid, glutathione and some others. The third level of antioxidant defence deals with damaged molecules, repairing or removing them from the cell and includes specific enzymes such as lipases, proteases, DNA repair enzymes etc. In the review, profiles of PUFAs and the two first lines of antioxidant defence in avian spermatozoa are characterised. Dietary manipulation of the breeder's diet (PUFA, vitamin E and selenium) as an effective means of modulating fatty acid composition and antioxidant system is also considered. Antioxidant properties of seminal plasma and efficiencies of inclusion of antioxidants into semen diluents are also characterised.

• Korean Journal of Animal Reproduction
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• v.7 no.2
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• pp.8-26
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• 1983

Various early pregnancy diagnostic methods have been developed in order to improve the reproductive efficiency in cow, mare, mule, sow, sheep, goat, dog, cat, rabbit, buffalo, camel, elephant, monkey, deer, lion, coipus and guinea pig. These methods include abdominal swelling, abdominal palpation, esturs cylce detection, Lupin test, gonadotropin assay, colostrum injection test, sperm motility assessment, cervical mucus viscosity test, Kaber chromagens method, estrogen test, A Scheim-Zond다 test, spectrophotometric detection of estrogen in urine and feces, boric acid crystraline formation test in urine, oxytocin injection test, diamino-oxidase test, PMSG HA test, behaviour test, Simolus iodine detection test, detection of tryptophane in urine, x-ray method, Cuboni and Lunaas method, vaginal biopsy method, Friedmann Schneider diagnostic method, electrode method, barium chloride detection method, ECG, Doptone method, ultrasound method, ultrasound scanning method, LDH method, rectal palpation method, CL palpation method, radioautography, serum creatine test, serum globulin test, chlormadine method, CAP method, Medata Do, pp.ers method, body fluid test, Plasma oCS detection method, ERIA, LHRH method, negative latex cogulation test and oestrone sulphate detection method. The most reliable methods with high a, pp.icability to farm animals such as sheep, mare, sow and cow are rectal palpation, ultrasound method and hormonal assay in blood and milk. However, they require complicated laboratory works for the early diagnosis of pregnancy and in most cases, the simple and economical methods which are described up to now need a long period of time after conception. Generally, it is possible to detect pregnancy after one estrus cycle, even though it varies depending on the species of animals. For improvement of the reproductive efficiency, it is required to develop a more accurate, economical, simple and early detectable method. It is anticipated that the result of a study on the detection method of EPF(early pregnancy factor) would be a, pp.icable to various animals within 6 hours after conception.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.271-277
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• 2000

This study was conducted to establish the optimal culture conditions for in vitro production of bovine embryos derived from slaughter house ovaries. Cumulus-oocyte- complexes (COCs) collected by aspiration from follicles of 2~7 mm in diameter were matured in Ham's F-10 medium supplemented with 0.01 $\mu\textrm{g}$/m1 epidermal growth factor (EGF) at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. After 24 hrs of culture, the oocytes were co-cultured with epididymal sperm selected off by Percoll-density gradient in TALP medium for 24 hrs. The presumptive zygotes were cultured in HECM-6 medium for 3 d post-insemination, and followed by cultured in TCM199 medium until 7 to 10d post-insemination. The cultures were compared of their cleavage and development into later stage in culture medium by additions of different protein sources (PVA, BSA and BCS) and by different embryo density. The rates of cleavage and development rates into blastocyst were not significantly (P<0.05) different among the culture media containing with BSA (75.0% and 40.5%), BCS (76.7% and 38.0%) and PVA (72.5% and 42.2%), respectively. Significantly (P<0.05) higher blastocysts rates were obtained in culturing of 30 and 40 embryos in each 50$\mu$l droplets of culture medium than in 5, 10 and 20 embryos. These results indicate that the optimal density of embryos is 30~40 embryos in a 50$\mu$l droplet of culture medium. Furthermore there is no effect of different protein sources on early embryonic development.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.197-205
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• 1996

This study was conducted to examine the effect of sustained release recombinant bovine somatotropin(SR-rBST) with different dosage(0, 0.03mg/kg body weight/14 day, 0.06mg/kg body weight/14 day, 0.09mg/kg body weight/14 day) on the economical traits of the semen characteristics and sexual behaviors and blood chemical values with sixteen Holstein dairy young bulls in Dairy Cattle Improvement Center of National Livestock Co-operatives Federation. Sensual testings of collected semens of Holstein young bulls were not different among treatments (p>0.05). Ejaculated semen volumes of control, SR-rBST 0.03mg, 0.06mg and 0.09mg groups were 5.6ml, 5.6ml, 6.0ml and 6.7m, respectively, but the result of SR-rBST 0.09mg group significantly increased semen volume(P<0.05). In sperm concentration, SR-rBST 0.06mg and 0.09mg groups significantly increased the total number of sperms than the other groups(P<0.05). In otherwise, SR-rBST treatments did not affect on pH, osmotic pressure, anti forzen rate, abnormality and motility of collected semen during whole experimental period. Reaction time(RT), sexual aggressiveness(SA) and tactile stimulation(TS) were not different among treatments. The libido scores(LS) of control, 0.03mg, 0.06mg and 0.09mg groups were 68.0, 80.5, 73.9 and 80.8, respectively, and LS were significantly improved by SR-rBST administration(P<0.05). The effect of SR-rBST on scrotal circumference measurement that is important factor to determine the ability of semen production, was not different among treatments. These studies indicates that SR-rBST treatments favourably affect on semen quality and sexual libido in Holstein young bull.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.269-275
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• 1998

It has been reported that the $Ca^{2+}$-ATPase and the $Ca^{2+}-Na^+$ exchanger play an important role for the regulation of intracellular $Ca^{2+}$ in somatic cells, the $Ca^{2+}$-ATPase located in the plasma membrane helps the $Ca^{2+}$ concentration in maintain low $[Ca^{2+}]_i$. Roldan & Fleming reported that the spermatozoan $Ca^{2+}$-ATPase plays an important role in the capacitation and acrosome reaction. We used to assess $Ca^{2+}$ changes by chlortetracycline (CTC) patterns in the capacitation and acrosome reaction of human and hamster spermatozoa. In the present study applying quercetin which has been known as an ATPase antagonist, the enzymatic effect of $Ca^{2+}$-ATPase on capacitation and acrosome reaction was found to be remarkable: a significant increase of the transformation from the original type to the B type and the AR type of spermatozoa. This finding suggests that $Ca^{2+}$-ATPase play an important role in the efflux and the influx of the $Ca^{2+}$ which have been known to be an essential factor for the capacitation and acrosome reaction, and that the inhibitory action of the $Ca^{2+}$-ATPase might be a prerequsit step toward the capacitation and acrosome reaction. In conclusion, this study suggest the considerable evidence as follows: the increment of the intracellular $Ca^{2+}$ concentration occurred by controlling the slope of $Ca^{2+}$ concentration through $Ca^{2+}$-ATPase activites in both the intracellular and extracellulr fluid may be important procedures for the capacitation and the acrosome reaction, and finally for fertilization of the sperm and ovum.