• Animal Bioscience
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• v.36 no.11
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• pp.1647-1654
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• 2023

Objective: Aging roosters typically exhibit subfertility with decreasing semen quality, furthermore Thai native roosters reared in rural areas are raised for a longer duration than their usual lifespan. The present study therefore aimed to assess the effect of selenium supplementation as an antioxidative substance in diets to improve the semen cryopreservation of aged roosters. Methods: Semen samples were collected from young (n = 20) and aged (n = 20) Thai native roosters (Pradu Hang Dum) at 36 and 105 weeks of age when starting the experiment, respectively. They were fed diets either non-supplemented or supplemented with selenium (0.75 ppm). Fresh semen quality and lipid peroxidation of fresh semen was evaluated before cryopreservation using the traditional liquid nitrogen vapor method. Post-thaw sperm quality and fertility potential were determined. Results: Advancing age is unrelated to decreasing fresh semen quality (p>0.05). However, lipid peroxidation in rooster semen depended on age, and the malondialdehyde (MDA) concentration increased in aged roosters (p<0.05). Selenium supplementation in diets significantly decreased the MDA concentration and increased the sperm concentration (p<0.05). In contrast, cryopreserved semen was affected by advancing rooster age, and selenium influenced sperm quality (p<0.05). Younger roosters had higher post-thaw sperm quality and fertility potential than aged roosters (p<0.05). Likewise, diet selenium supplements improved post-thaw sperm quality and fertility compared with the non-supplement group. Conclusion: Rooster's age does not influence the rooster sperm quality of fresh semen, while sperm cryotolerance and fertility were greater in young roosters than in aged roosters. However, sperm of aged roosters could be improved by dietary selenium supplementation.

• Korean Journal of Poultry Science
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• v.30 no.2
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• pp.91-94
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• 2003

Artificial insemination of cryopreserved semen is not practiced in broiler breeder industry although its benefit has been recognized for years. Thus, the present study explored the possibility of artificial insemination with cryopreserved semen in Ross broiler breeders. Sixty Ross female broiler breeders were artificially inseminated either with fresh or with cryopreserved semen pooled from five male Ross broiler breeders. On.e hundred eighty eggs were collected from each semen treatment and hatched. Fertility and hatchability of fertilized eggs collected from hens artificially inseminated with cryopreserved semen were 68.0 and 47.1％, respectively, compared to 88.3 and 87.2％ with fresh semen. Both unfertilized eggs and dead embryos during hatch were significantly (P<0.05) higher in the cryopreserved semen­treated group by approximately 2.7­ and 3.2­fold than the fresh semen­treated counterparts. the moderately low fertility and markedly low hatchability of fertilized eggs obtained from cryopreserved semen observed in this study may be of practical interests in broiler breeder industry and deserves further study.

• Journal of Life Science
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• v.30 no.1
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• pp.77-81
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• 2020

The aim of this study was to overcome some of the limiting factors that the maxi cryopreservation straw of 5 ml presents in processing boar semen. Cryopreservation of semen samples was conducted in 0.5 ml and 5.0 ml straws at two freezing rates: -140℃ in 8 minutes and 30 seconds (FR-1) and -140℃ in 14 minutes (FR-2). The straws were then thawed and the semen parameters were compared by Computer Assisted Sperm Analysis, and sperm morphology and acrosome status were examined by Coomassie blue staining. The effects of different thawing temperatures and durations were also compared, namely 37℃ for 115 sec, 50℃ for 45 sec, or 70℃ for 25 sec. In general, the FR-1 group showed higher (p<0.05) sperm viability and motility than the FR-2 group in the 5.0 ml straws. Compared to other ranges, thawing at 50℃ for 45 sec showed the highest sperm viability and motility (68.4±3.6% and 69.5±2.2%, p<0.05), suggesting that thawing temperature should be adjusted concurrently with freezing rate. Sperm morphology and acrosome integrity did not significantly differ among the groups (p>0.05). The data obtained in this study suggest that improving the freezing-thawing protocol for one artificial insemination dose straws (5.0 ml) retains the sperm's parameters from 0.5 ml cryopreservation, and is more convenient to handle, which could result in enhanced reproductive performance.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.106-115
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• 2021

Sperm cryopreservation is an important method of assisted reproductive techniques and storing genetic resources. It plays a vital role in genetic improvement, livestock industrial preservation of endangered species, and clinical practice. Consequently, the cryopreservation technique is well organized through various studies, especially on Korean native cattle (Hanwoo). However, the cryopreservation technique of Korean native brindled cattle, which is one of the native cattle species in Korea, is not well organized. Therefore, it is necessary to develop a Supplementary Table technique for the cryopreservation of Korean native brindled cattle. For this purpose, it is important to first evaluate the quality of the currently produced cryopreserved sperm of Korean native brindled cattle. In this study, we randomly selected 72 individual Korean native brindled cattle semen samples collected from 8 different region research centers and used them to evaluate sperm functions. We focused on the quality evaluation of cryopreserved Korean native brindled cattle semen following the measurement of motion kinematics, capacitation status, intracellular ATP level, sperm motility, and cell viability. Then, the values of each of the eight groups were derived from various sperm parameters of nine individual samples, including sperm motility, kinematics, cellular motility, and intracellular ATP levels, which were used to compare and evaluate sperm function. Overall, differences in various sperm parameters were observed between most of the research centers. Particularly, the deviations of motility and motion kinematics were high according to the sample. Therefore, we suggest that it is necessary to develop a standard method for the cryopreservation of Korean native brindled cattle semen. We also suggest the need for sperm quality evaluation of the cryopreserved semen of Korean native brindled cattle before using artificial insemination to attain a high fertility rate.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.2
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• pp.67-75
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• 2019

Objective: Sperm cryopreservation has been widely used in assisted reproductive technology, as it offers great potential for the treatment of some types of male infertility. However, cryopreservation may result in changes in membrane lipid composition and acrosome status, as well as reductions in sperm motility and viability. This study aimed to evaluate sperm DNA fragmentation damage caused by conventional freezing using the sperm chromatin dispersion test. Methods: In total, 120 fresh human semen samples were frozen by conventional methods, using SpermFreeze Solution as a cryoprotectant. Routine semen analysis and a Halosperm test (using the Halosperm kit) were performed on each sample before freezing and after thawing. Semen parameters and sperm DNA fragmentation were compared between these groups. Results: There was a significant decrease in sperm progressive motility, viability, and normal morphology after conventional freezing (32.78%, 79.58%, and 3.87% vs. 16%, 55.99%, and 2.55%, respectively). The sperm head, midpiece, and tail defect rate increased slightly after freezing. Furthermore, the DNA fragmentation index (DFI) was significantly higher after thawing than before freezing (19.21% prior to freezing vs. 22.23% after thawing). Significant increases in the DFI after cryopreservation were observed in samples with both normal and abnormal motility and morphology, as well as in those with normal viability. Conclusion: Conventional freezing seems to damage some sperm parameters, in particular causing a reduction in sperm DNA integrity.

• 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.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.69-75
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• 2021

Cryopreservation is a widely-used efficient means of long-term sperm preservation. However, unlike other types of semen, cryopreserved boar semen has reduced fertility and the efforts continue to optimize post-thawing sperm recovery. In this study, we evaluated the effects of various washing solutions (Hulsen solution, lab-made DPBS and commercial DPBS) on post-thawing porcine sperm kinematics (CASA system), viability (SYBR-14/PI) and acrosome integrity (PSA/FITC). We also examined the effect of washing-centrifugation on frozen-thawed semen kinematics. The results indicate that type of washing solution and post-thawing centrifugation alters parameters linked to sperm quality (total motility, progressive motility, viability and acrosome integrity). Significantly higher (p < 0.05) motility and progressive motility were obtained when cryopreserved semen was processed with Hulsen solution. The post-thaw percentage of live and intact acrosomal sperm was significantly higher in group 1 (Hulsen solution) as compared to other groups. Following thawing-centrifugation, the results showed significantly higher motility and progressive motility in group 1 than other groups. However, the latter two DPBS groups did not differ statistically. Taken together, Frozen-thawed spermatozoa motility, acrosome integrity and viability can be affected by the type of washing solution used. Moreover, centrifugation of frozen-thawed semen has an unfavorable effect on total motility and progressive motility.

• 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.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.55-60
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• 2011

The objective of this study was carried out to evaluate the efficiency of sperm collection methods on the post-thaw viability of cat semen. The cat semen was collected by artificial virginal (AV) and electronic ejaculate (EE) methods. The composition of semen extender was consisted of Tris-buffer supplemented with 20% egg yolk and 1% P/S antibiotics in Ext I, and more added 8% glycerol, 1.0% Equex STM paste of total volume in Ext II. The collected semen was adjusted the concentration and then diluted in Ext I for optimal concentration. The diluted semen was cooling to $5^{\circ}C$ temperature in refrigerator for at least 2 hrs and then diluted stepwise with Ext II for at least 1 hrs. After an equilibration for 1 hrs, the cooled semen was packaged in 0.5 ml straw and then freezing on the $LN_2$ vapor over 5 cm above from $LN_2$ and then immersed directly in $LN_2$ for cryopreservation. The frozen semen was thawed in $38^{\circ}C$ water for 15 sec and then evaluated the motility, viability, and morphology. Post-thaw semen were calculated the motility by SMI (sperm motility index). The live-dead sperm was evaluated by Eosin-B and morphological evaluation was by Diff-quik kit staining. The post-thaw concentration ($89{\times}10^6$ /ml vs. $128{\times}10^6$ /ml), viability ($22.6{\pm}10.6%$ vs. $37.1{\pm}26.1%$), morphological normality ($27.0{\pm}50.2%$ vs. $45.6{\pm}123.0%$) of EE and AV groups were not significant different, but the post-thaw motility was significant lower in EE than that in AV group ($53.1{\pm}3.6$ vs. $73.6{\pm}5.7$) (p<0.05). In conclusion, semen collection methods did not significant different between EE and AV groups except of post-thaw motility and so both semen collection methods could be applied in feline semen collection methods.