• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.213-217
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• 2013

The objective of this study was to investigate the effect of bacterial contamination on elapsed time after preservation on boar semen. Known numbers of Escherichia coli (E. coli) were inoculated to freshly ejaculated semen and sperm parameters such as viability, motility, agglutination, acrosome integrity and hypo-osmotic swelling test were performed during 7 days of liquid preservation. Semen samples were prepared using antibiotic free BTS extender and 4 different levels of E. coli were treated to semen with following concentrations; 3,000, 5,000, 7,000, 10,000 CFU/ml of sperms. Semen samples were preserved at $17^{\circ}C$ for 7 days in semen storage until analyzed. Aliquots were subjected to measure the sperm viability, motility and agglutination using computer assisted sperm analysis (CASA) system, acrosome integrity was performed using chlortetracycline (CTC) staining method and hypo-osmotic swelling test was performed using hypotonic solution from day 1 (day of semen collection) to 7. Detrimental effects on sperm motility and viability were observed 3 days after preservation at the level of 5,000 CFU/ml (p<0.05). Percentage of sperm abnormality was higher (p<0.05) in over 5,000 CFU/ml groups. Sperm agglutination rate was also significantly higher (p<0.05) in groups of 5,000 and 7,000 CFU/ml. The rate of acrosome reacted sperm was higher as preservation time goes in all the samples but the pattern was clearly higher among E. coli contaminated groups (p<0.05). The sperm membrane integrity in terms of hypo-osmotic test, E. coli affects little compared to other sperm parameters. The deleterious effects observed due to the bacterial contamination in semen suggest that importance of hygiene protocol to minimize the bacterial contamination during semen collection and processing.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.163-166
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• 2012

This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS(Beltsville Thawing Solution) extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22~24 hr incubation from counting agar plate in which sperm dilute to $10{\sim}10^6$ in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern F), characteristic of incapacitated acrosome-intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern AR), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 ($77.24{\pm}6.47$, p<0.001) and 7 days ($77.24{\pm}6.47$, p<0.001) after preservation compared to 1 ($15.71{\pm}7.18$) and 3 days($18.39{\pm}7.22$) after preservation, respectively. Sperm viability was significantly lower ($53.25{\pm}35.03$, p<0.0001) at 7 days after preservation. Morphological abnormality of sperm was lower (p<0.001) at 1 ($15.71{\pm}7.18$) and 3 ($18.39{\pm}7.22$) days compared to 5 ($21.84{\pm}7.91$) and 7 ($22.59{\pm}9.93$) days after preservation. Acrosomal integrity and capacitation rate (pattern F) were significantly lower (p<0.001) from 5 days after preservation. Based on the data we obtained from this study suggested that semen preserved more than 5 days without antibiotic would not recommend use for artificial insemination.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.2
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• pp.190-197
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• 2008

This study was designed to analyze boar sperm to compare motility, acrosome morphology, viability and ATP by various preservation methods between Duroc boar A with cold shock resistance sperm and Duroc boar B with cold shock sensitivity sperm. Semen volume, sperm concentration, motility and normal acrosome between Duroc boar A and B did not show any differences within 2 h after collection. There were no differences in sperm motility and normal acrosome between boar A and B at 1 day of preservation at $17^{\circ}C$ and $4^{\circ}C$, respectively. However, sperm motility and normal acrosome from 2 day of preservation at $17^{\circ}C$ and $4^{\circ}C$, respectively, were higher for boar A than boar B. The frozen-thawed sperm motility and normal acrosome were higher for boar A than boar B. The sperm viability and ATP concentration according to storage period of liquid semen at $17^{\circ}C$ and $4^{\circ}C$ were higher for boar A than boar B. Also, the sperm viability and ATP concentration of frozen-thawed semen were higher for boar A than boar B. In conclusion, we found out that the original quality of boar semen with cold shock resistance sperm played an important role.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.25-30
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• 1997

This study was done to find out the preservation possibility of liquid boar semen at variabel room temperature of 9 to 16$^{\circ}C$. The percentages of sperm motility and NAR acrosome were highest in B tschwiler extender compared to B tschwiler＋Hepes, Andro＋Hepes and Andro extenders. The extenders with Hepes buffer showed detrimental effect for preservation of liquid boar semen. The pH of ejaculated sperm-rich fraction was 7.5. The pH of B tschwiler＋Hepes, B tschwiler, Andro＋Hepes and Andro extenders was 6.9, 7.5, 7.1 and 8.1, respectively. The pH of liquid boar semen with B tschwiler＋Hepes, B tschwiler, Andro＋Hepes and Andro extenders was 6.6, 6.9, 6.7 and 6.9 at 1st day of storage, and 5.5, 5.7, 5.6 and 5.8 at 7th day of storage, respectively. Gilts and sows were inseminated twice with liquid boar semen stored at 9~16$^{\circ}C$ in B tschwiler extender for 3~4 days. Farrowing rate, litter size and average pig weight at birth between AI and natural service did not differ significantly in gilt and sow, respectively. However, sow showed higher farrowing rate and litter size compared to gilt both in AI and in natural service. As a result of this study, we found out that liquid boar semen can be stored for 5~7 days at uncontrolled room temperature of 9~16$^{\circ}C$ in B tschwiler extender.

• Korean Journal of Poultry Science
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• v.48 no.4
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• pp.185-191
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• 2021

Chicken spermatozoa have the ability to survive in low-temperature environments; however, the effects of low temperature on sperm motility and acrosome damage have not been studied in detail. The present study investigated semen longevity following dilution of rooster semen with Beltsville Poultry Semen Extender (BPSE) and Lake extender in preservation vessels (1.5 mL e-tube and 0.5 mL straw). Spermatozoa motility in the closed-type vessel (0.5 mL straw) was higher than that in the 1.5 mL e-tube on day 3 of preservation (68.6±3.1% vs. 22.1±5.7%). The motility of rooster semen diluted with BPSE in 0.5 mL straw was also higher than that of the Lake extender on day 3 of preservation (57.7±5.6% vs. 37.7±5.4%). Furthermore, acrosome intactness was higher in 0.5 mL straw than in the 1.5 mL e-tube, and the rate of acrosome cap damage increased with preservation days. The present study demonstrates that a closed 0.5-mL straw vessel could be used for low-temperature semen preservation, with an increased motility rate and acrosome integrity in fresh rooster semen.

• Journal of Embryo Transfer
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• v.21 no.4
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• pp.345-351
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• 2006

The objective of this study was to investigate the effects of preservation period of porcine liquid semen on bacterial contamination and in vitro production of embryo. Extended liquid semen was prepared by three mixture of boar's ejaculates from each farm without antibiotics, and were kept in $17^{\circ}C$ semen preservation incubator until use. Sperm motility was significantly (p<0.05) decreased as semen preservation time goes by (78.7$\pm$2.4% for 1 day vs. 71.1$\pm$2.4 and 64.8$\pm$2.4% for 3 and 5 days of presentation, respectively). Quantitative of bacteria in semen was significantly (p<0.05) higher in 5 days ($57.8\pm105.2\times10^4$ Cfu) compared to 0 and 3 days ($32.1\pm76.8\times10^4$ and $26.9\pm46.6\times10^4$ Cfu, respectively) of preservation. In terms of development of in vitro fertilization of porcine embryos inseminated by preserved semen, the rate of normal fertilization (2PN) was significantly (p<0.05) decreased in 5 days (56.0$\pm$2.6%) compared to 1 and 3 days (66.0$\pm$2.7 and 64.0$\pm$2.7%, respectively) of preservation. Cleavage rate was also significantly (p<0.05) affected by preservation period (75.0$\pm$4% for 1 day, 70.0$\pm$0.3 and 71.0$\pm$0.3% for 3 and 5 days, respectively). The in vitro developmental rate of blastocyst stage embryo was significantly (p<0.05) affected by semen preservation period (15.0$\pm$1.0% for 1 day vs. 11.0$\pm$0.9 and 8.0$\pm$0.9% for 3 and 5 days, respectively). It is concluded that more than 3 days of liquid semen preservation without antibiotics increased the quantity of bacteria resulted in detrimental effect on sperm motility and decreased both normal insemination rate and the developmental rate of blastocyst stage embryo.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.479-483
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• 2011

The objective of this study was to determine the effects of E. coli isolated from porcine semen on sperm viability, motility, and semen pH. Semen samples were prepared using commercial extender, $Seminark^{Pro}$ (Noahbio Tech, Korea) that did not contain antibiotics. And 4 different levels of E. coli were artificially innoculated to semen with following concentrations; 4,000 of sperms with 1 of E. coli (T1), 400 with 1 (T2), 40 with 1 (T3), and 4 with 1 (T4). Semen samples were preserved at $17^{\circ}C$ for 5 days in semen storage box until analyzed by flowcytometer. Aliquots were subjected to measure the sperm viability (Live/$Dead^{(R)}$ stain), motility (mitochondrial function), and semen acidity (pH) from day 0 (day of semen collection) to day 5. Sperm motility and viability were significantly decreased (p<0.05) on day 0 (4 hrs after preservation at $17^{\circ}C$) in T3 and T4 compared to control groups and were significantly decreased (p<0.05) in all groups from day 3. Sample pH was acidic in T3 (6.90~6.86) and T4 (6.86~6.65) from day 3 to day 5 (p<0.05). On the other hand, sample pH was maintained 7.0~7.1 in control, T1, and T2 during the experimental period. Sperm motility and viability were significantly decreased from day 0 to day 5 compared to control in samples contaminated with E. coli above a value of 40:1 ($20{\times}10^6$ sperm cells/ml : $5{\times}10^5$ cfu/ml). Even on day 1 in T4 and on day 3 in T3, semen pH was acidic probably due to the acidification of dead spermatozoa. These results suggest that E. coli contamination has a concentration-dependent detrimental effect on extended porcine semen quality.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.3
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• pp.196-201
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• 2022

Objective: This prospective consecutive study investigated the variation in sperm DNA fragmentation (SDF) in multiple semen samples from patients with cancer. Methods: Eighty-one patients with various cancers underwent multiple semen collections on 3 consecutive days for sperm cryopreservation prior to cancer treatment. A commercial Halosperm kit was used to measure SDF. Within- and between-subject coefficients of variation were estimated via random-effects analysis of variance to assess the consistency of semen parameters and SDF. Intraclass correlation coefficients (ICCs) were calculated to assess the magnitude of the between-subject component of variance relative to the total variance. Results: The volume of semen in the day-2 and day-3 samples was significantly lower compared with the day-1 sample. Most parameters showed high ICC values, suggesting that within-subject fluctuations were small relative to the between-subject variability. The highest ICC values were identified for the SDF (ICC, 0.68; 95% confidence interval [CI], 0.45-0.84) and semen volume (ICC, 0.67; 95% CI, 0.45-0.84). Conclusion: Our findings showed that repeated ejaculates from patients with cancer had stable SDF levels.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.31-39
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• 2013

Alpha-tocopherol as an antioxidant acts in preservation of chilled semen by preserving cell membrane damage from lipid peroxidation. Optimum concentrations of ${\alpha}$-tocopherol in egg yolk-citrate (EYC) extender need to be studied in crossbred bull's semen. Different concentrations of ${\alpha}$-tocopherol viz. 0, 1, 2, 4 and 6mg per ml of extender were used. Semen was collected once a week from four bulls used to regular collection, aged 4 to 7 years, weighing 320 to 450 kg, and with body condition score 4 to 4.5 and scrotal circumference 23 to 32 cm. Semen was evaluated routinely and sperm morphology was viewed under light microscope at ${\times}1,000$ magnification after fixing with buffered formal saline. Over 90% had normal head, acrosome, mid-piece and tail. Semen was diluted with egg-yolk-citrate extender to produce $15{\times}10^6$ spermatozoa/ml and 0, 1, 2, 4 and 6 mg/ml ${\alpha}$-tocopherol were added. The semens amples were kept at $8^{\circ}C$. Sperm motility and viability were examined daily up to 5 days under light microscopy at ${\times}200$ magnification. Sperm viability was acceptable (${\geq}40%$) up to the $4^{th}$ day with all concentrations of ${\alpha}$-tocopherol and up to the $5^{th}$ day with 2 mg/ml ${\alpha}$-tocopherol. Sperm motility was acceptable (${\geq}40%$) up to the $3^{rd}$ day irrespective of ${\alpha}$-tocopherol concentration, and up to the $4^{th}$ day with 2 mg/ml ${\alpha}$-tocopherol. It is suggested that the lifespan of chilled semen may be extended up to 4 days by adding 2mg/ml ${\alpha}$-tocopherol.

• Korean Journal of Animal Reproduction
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• v.4 no.1
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• pp.20-27
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• 1980

This experiment was to study semen properties of Charolais for the a, pp.ication ot artificial insemination. The result obtained were summarized as follows: 1. In the preservation of liquid semen for 6 days, the survival rates of Charolais semen averaged 57.14% in skim milk solution and 58.17% in tris buffer solution. There were not differences. 2. Recovery of semen after thawing was vigorous in the semen that was diluted and frozen in 48 hrs. 3. The real rates of survival sperm for Charolais averaged 83% after living sperm was diluted and stained for 6 days. 4. Methylene blue reduction test diluted semen was fresh when it was diluted within 48 hrs. 5. If the diluted semen was preserved below 5$^{\circ}C$ in Charolais, the pH decreased by 0.2 in a day. 6. Diluted semen was more resistant to the cold shock than fresh semen. 7. In resistance against hot shock, sperm was almost dead in 20 minutes in 46.5$^{\circ}C$ in diluted semen, while it was dead in 30 minutes in 42.5$^{\circ}C$ in diluted semen. 8. In examination of morphological changes of sperm acrosome for 6 days, normal sperm in skim milk solution and tris buffer solution was 80% and 76.97% respectively, swelling sperm 12.8% and 15.27%, deficient sperm 0.6% and 0.97% abnormal staining 3.07% and 5.25%, immature sperm 0.28%, and 0.23%, whereas other abnormal sperm was 1.28% and 1.42%.