• Korean Journal of Poultry Science
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• v.45 no.4
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• pp.237-244
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• 2018

In this study, to increase the survival rate of frozen/thaw rooster semen, standard protocols of semen thawing procedures were tested by computer-assisted sperm assay (CASA). We tested 4 different thawing protocols for frozen semen, $5^{\circ}C$ for 2 min, $35^{\circ}C$ for 30 s, $54^{\circ}C$ for 13 s, and $70^{\circ}C$ for 7 s. The pooled semen from 5 to 8 Ogye rooster line was diluted in the HS-1 diluent and frozen in 8% methylacetamide (MA) in liquid nitrogen vapors. To determine standard thawing method, straws were plunged into different temperatures and times. The resulting motilities were recorded by the CASA system. The results of this study showed that the best viability of the spermatozoa was shown by exposure at $5^{\circ}C$ for 2 min. Moreover, the longevity test of thawed sperm at $5^{\circ}C$ for 2 min also supported the higher viability under low temperature preservation of $17^{\circ}C$ for 1 hr. Further research is needed to increase the motility of thawed rooster semen for field application. In addition, the in vivo tests for different rooster lines are also needed for the establishment of avian genetic resource bank.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.272-279
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• 2019

The study was conducted to evaluate the seminal attributes and cryobanking of Achhami (Bos indicus) bull semen. Of two Achhami bulls, 8 ejaculates from each bull were evaluated for seminal attributes. For semen freezing and cryo-banking, 4 ejaculates (having ≥2 mL semen volume, ≥75% of sperm motility and ≥1,000 × 10⁶ cells/mL of sperm concentration) from each bull were used. Semen samples were diluted in egg-yolk-tris-citrate extender using a two-step dilution protocol, and were frozen in liquid nitrogen (LN₂) vapour in a styrofoam box. The mean semen volume, colour, sperm mass activity, motility, viability, concentration, abnormal acrosome, midpiece and tail and, abnormal head of two Achhami bulls were 4.4 ± 0.5 mL vs. 2.5 ± 0.2 mL, 2.5 ± 0.1 vs. 2.4 ± 0.1, 3.5 ± 0.1 vs. 3.5 ± 0.1, 77.0 ± 1.1% vs. 78.3 ± 1.3%, 94.4 ± 0.5% vs. 91.0 ± 0.6%, 1137.7 ± 73.7 × 10⁶ cells/mL vs. 1060.0 ± 44.3 × 10⁶ cells/mL, 10.2 ± 0.5% vs. 10.3 ± 0.5% and 6.7 ± 0.5% vs. 8.2 ± 0.3%, respectively. The post-thawed sperm motility and viability were 53.0 ± 2.0% vs. 50.0 ± 0.0% and 80.2 ± 0.4% vs. 73.2 ± 0.7%, while evaluating by computer-assisted sperm analysis (CASA) system, the percentage of the progressive motility, fast motility, slow motility, local motility and immotile sperm were 75%, 68%, 7.4%, 16.6% and 8.6%, respectively. A total number of 620 doses semen straw were cryo-banked. Due to the acceptable post-thawed sperm motility and viability recorded, cryopreservation of Achhami semen is hereby recommended so as to preserve the Achhami breed. For further validation, the fertility will be observed from the produced frozen semen.

• Journal of Embryo Transfer
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• v.32 no.1
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• pp.1-8
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• 2017

Cryopreservation of germ cells from genetically proven animals could be a source of restoration tools from the risk of extinction or disappearance of wanted characteristics. Using frozen semen, the genetic gains of Korean native cattle have been increased greatly for 70 years. The preservation of genetic resources as a form of frozen semen straw has limited availability due to the numbers. To circumvent this weakness of frozen semen, we tested two re-freezing methods with different initial thawing temperatures using frozen Korean proven semen and rare breed semen from albino, black and chikso breeders. It has been known that human sperm could resist to cryo-damages by repeated freeze-thaw cycles, but not for Korean proven bulls number (KPN) or for rare breeds. Total 7 frozen semem from brindled(2), black(1), Korean Albino(2) and KPN(1) bulls were used for our research. After thawing straws under $5^{\circ}C/2min$ or $37^{\circ}C/40sec$ with low temperature water bath and thermo jug, spermatozoa were re-diluted with triladyl diluents after first thawing and re-frozen. Sperm motilities were compared between animals and treated groups after re-thawing. Mean values of motility and viability of refrozen/thawed sperm for expansion of the number of straws were significantly higher in $5^{\circ}C$ than in $37^{\circ}C$ (P < 0.05). However, the activity of viable sperm thawed at $5^{\circ}C$ was significantly decreased before refreezing. It is estimated that re-freezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa.

• Korean Journal of Veterinary Research
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• v.38 no.4
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• pp.882-897
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• 1998

This experiment was conducted to predict the effects of motional characteristics on the fertility of Korean native cattle(KNC) by using CASA technology and in vitro fertilization system. Twenty-six KNC frozen semen straws were obtained from Korean KNC improvement department, livestock improvement main division, national livestock cooperatives federation in Korea. Specimens were allowed to thaw at $37^{\circ}C$ for 30 sec in water bath. Semen analysis was performed on semen image analysis system(SIAS, Medical supply, Korea) adjusted to the gate settings and used the semen droplet ($5{\mu}l$) placed on Makler counting chamber(Sefi medical instrument, Israel) prewarmed at $37^{\circ}C$. The same person used the same micropipette to fill the Makler counting chamber. A total of 150 or more of sperms were analysed in each specimen by a single trained person by scanning at least 5 to 10 fields. The oocytes collection, in vitro maturation, IVF, in vitro culture and determination of the cleavage rate were performed by the technique, as described by Hwang et al (1997). Statistical analysis was done by linear regression with use of the Sigma plot program on a IBM personal computer. The cleavage rate in vitro fertilized oocyte was significantly correlated(P<0.05) with MOT, VCL, VSL, VAP, ALH, BCF and MAD, but not CON, LIN, STR, WOB, DNM, DNC and HYP in regressional analysis. The results show that some kinematic characteristics of frozen-thawed semen by CASA can be predict the fertility in in vitro model system.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.139-143
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• 2007

This study was performed to investigate the reproductive characteristics of the cloned Hanwoo bulls produced by SCNT. The semen ejaculated from the cloned bulls (C-38 and C-39) and normal Hanwoo bull was properly measured the volume, the number of sperm, and the viability of frozen-thawed sperm. The sperm activity was analyzed using computer assisted sperm analysis (CASA). To analyze fertilizing ability of the cloned bulls, in vitro fertilization and artificial insemination were performed using the frozen-thawed semen. There were no differences in semen volume, sperm concentration, and the viability of frozen-thawed sperm between cloned bulls and normal bull. The difference was statistically significant in total motility, curvilinear velocity (VCL), straight-line velocity (VSL), and average-path velocity (VAP) of both cloned bulls compared to those of normal Hanwoo bull, respectively (p<0.05). The cleavage and blastocyst development rate were not different between the groups. five cloned cows were artificially inseminated using the frozen-thawed semen of C-38, two of them became pregnant. Two second generation calves (one male and one female) were produced. Based on these results, the cloned Hanwoo bulls showed normal reproductive abilities of semen parameters and sperm activity to their comparators and produced cloned calves, although there are some individual differences on the parameters.

• Animal Bioscience
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• v.37 no.7
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• pp.1177-1184
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• 2024

Objective: Semen cryopreservation is an effective method of preserving genetic material, particularly in native chicken breeds facing a substantial decline. In this study, we evaluated the quality of frozen/thawed rooster semen treated with different concentrations of oral administrations of black ginger (Kaempferia parviflora: KP) extract and determined its fertility. Methods: Thirty-two Thai native roosters (Pradu Hang Dum, 42 weeks old) were used in this study. The treatments were classified into four groups according to the concentration of KP extract administered to the roosters: 0, 100, 150, and 200 mg/kg body weight. The quality of fresh semen was analyzed before cryopreservation. Post-thaw sperm quality and fertility potential were determined. Also, lipid peroxidation was determined. Results: The results showed that sperm concentration and movement increased in roosters treated with 200 mg/kg of KP extract (p<0.05). The malondialdehyde (MDA) in the roosters receiving 200 mg/kg KP extract was lower than that in the other but had an insignificant difference within the KP treatment groups (p>0.05). The highest MDA levels were observed in the control group (p<0.05). The percentage of motile sperm (total motility and progressive motility) after semen thawing was higher in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). MDA levels decreased significantly in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). Fertility and hatchability were greater in the KP150 and KP200 groups than in the KP100 and control groups (p<0.05). Conclusion: The optimal amount of KP extract influencing initial sperm quality was determined to be 200 mg/kg. However, 150 mg/kg was the optimal low dosage of KP extract administration that maintained sperm quality and fertility following semen cryopreservation.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.113-120
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• 2022

Sperm cryopreservation is a fundamental process for the long-term conservation of livestock genetic resources. Yet, the packaging method has been shown, among other factors, to affect the frozen-thawed (FT) sperm quality. This study aimed to develop a new mini-straw for sperm cryopreservation. In addition, the kinematic patterns, viability, acrosome integrity, and mitochondrial membrane potential (MMP) of boar spermatozoa frozen in the developed 0.25 mL straw, 0.25 mL (minitube, Germany), or 0.5 mL (IMV technologies, France) straws were assessed. Post-thaw kinematic parameters were not different (experiment 1: total motility (33.89%, 32.42%), progressive motility (19.13%, 19.09%), curvilinear velocity (42.32, 42.86), and average path velocity (33.40, 33.62) for minitube and the developed straws, respectively. Further, the viability (38.56%, 34.03%), acrosome integrity (53.38%, 48.88%), MMP (42.32%, 36.71%) of spermatozoa frozen using both straw were not differ statistically (p > 0.05). In experiment two, the quality parameters for semen frozen in the developed straw were compared with the 0.5 mL IMV straw. The total motility (41.26%, 39.1%), progressive motility (24.62%, 23.25%), curvilinear velocity (46.44, 48.25), and average path velocity (37.98, 39.12), respectively, for IMV and the developed straw, did not differ statistically. Additionally, there was no significant difference in the viability (39.60%, 33.17%), acrosome integrity (46.23%, 43.23%), and MMP (39.66, 32.51) for IMV and the developed straw, respectively. These results validate the safety and efficiency of the developed straw and highlight its great potential for clinical application. Moreover, both 0.25 mL and 0.5 mL straws fit the present protocol for cryopreservation of boar spermatozoa.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.97-102
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• 2013

The objective of this study was to develop of semen transport system for cryopreservation and fertility in bull sperm. The ejaculated semen were diluted with Triladyl containing 20% egg-yolk for transportation. Diluted semen was transported by three methods that there were wrapping tissue (Tissue), sinking under $30^{\circ}C$ water (Water) and sinking between warm water and air (Air) methods. Semen was transported within 2 hours in $0.3^{\circ}C$. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk. And frozen-thawed sperm were estimated with SYBR14/PI double stain for viability, FITC-PNA/PI double stain for acrosome reaction analysis and Rhodamine123 double stain for mitochondrial intact assessment. In results, live sperm (SYBR+/PI-) in Air treatment group ($43.3{\pm}4.7%$) was significantly (p<0.05) higher than other treatment groups (Tissue: $16.3{\pm}2.7%$ and Water: $27.5{\pm}3.1%$), dying sperm (SYBR+/PI+) in Air treatment group ($55.6{\pm}4.7%$) was significantly lower than other treatment groups (Tissue: $77.6{\pm}3.2%$ and Water: $67.6{\pm}3.3%$) (p<0.05). Acrosome reaction in Air treatment group ($0.2{\pm}0.1%$) within live sperm (PI negative region) was significantly (p<0.05) lower than other treatment groups (Tissue: $0.7{\pm}0.2%$ and Water: $0.5{\pm}0.1%$), the acrosome reaction in Air treatment group ($28.6{\pm}2.8%$) within all sperm also was significantly lower than other treatment groups (Tissue: $44.2{\pm}1.8%$ and Water: $36.2{\pm}2.0%$) (p<0.05). And mitochondrial intact in Air treatment group within live ($97.1{\pm}0.4%$) and all ($61.9{\pm}3.3%$) sperm were significantly higher than other treatment groups (Tissue: $85.2{\pm}3.3%$, Water: $87.8{\pm}2.9%$ within live sperm and Tissue: $49.28{\pm}3.7%$, Water: $42.0{\pm}3.1%$ within all sperm) (p<0.05). Therefore, we suggest that transportation by sinking method between warm water and air was beneficial to improvement of fertility in frozen-thawed in bull semen.

• Korean Journal of Animal Reproduction
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• v.12 no.2
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• pp.112-119
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• 1988

These studies were conducted to investigate the effects of culture temperature and time on the in vitro maturation and semen type and media on the in vitro fertilization of bovine follicular oocytes, and to asses in vitro fertilization rate of oocytes cultured by extraffollicular method following fertilization in vitro, or transfer into the pseudopregnant rabbit oviduct or uterus. The bovine oocytes recovered from follicles were cultrued for 18 hrs or 72hrs at 38$^{\circ}C$ with 5% CO2 in moist air. Flesh-diluted(2 folds) and frozen-thawed semen in 0.5ml straw from a fertile bull were used. In order to obtain capacitation of spermatozoa were treated with bovine follicular fluids(BFF) and Inophore A(IA). The results obtained were summarized as follows: 1. The oocytes were classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 62.0%, 12.0%, 17.2%, 5.9% and 3.0% of the total oocytes harvested, respectively. 2. The oocytes matured to metaphase II were significantly increased between 24-48hrs of incubation and at 37-39$^{\circ}C$ with 5% CO2 in moist air. 3. The in vitro fertilization rate following transferred into rabbit oviduct or uterus with bull semen and in vitro matured oocytes were higher ligation than non-ligation of oviduct or uterus. 4. The in vitro fertilization rate of oocytes matured in vitro were higher neat than frozen semen and treatment of IA than BFF on the capacitation of spermatozoa. 5. The effects of semen types and media on in vitro fertilization of oocytes matured in vitro were higher fertilization rate of neat than friozen semen, and media was not significant.