• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1276-1281
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• 2010

The present study was conducted to investigate the effects of butylated hydroxytoluene (BHT) supplementation on diluted, cooled and frozen-thawed ram spermatozoa. After primary evaluation of collected ejaculates, only semen samples with motility of more than 70% and sperm concentration higher than $3{\times}10^3$ sperm/ml were used for cryopreservation. The selected semen samples were then pooled and diluted 1:4 with Tris Citrate Fructose Yolk (TCFY) extender supplemented with different concentrations of BHT (0.5, 10, 2.0 and 3.0 mM). As the control, semen was diluted and frozen in the diluent without BHT. Motility, progressive motility, viability, membranes and acrosome integrity were evaluated after dilution (part 1), cooling (part 2) and freezing and thawing (part 3). The results of the first part of the experiment showed that there were no significant difference between treatments in the motility, progressive motility, viability, membranes and acrosome integrity of spermatozoa, but the results with 2.0 mM BHT were slightly better than obtained with other levels of BHT and control extender. Significantly better results (p<0.05) were observed in the second part of the experiment for cooled spermatozoa characteristics, when extender was supplemented with 2.0 and 3.0 mM BHT. Furthermore, the results obtained in the third part of the experiment indicated that, after freezing and thawing, all evaluated semen characteristics were improved significantly (p<0.05) by increasing BHT levels, with the best results obtained for extender containing 2 mM BHT. Comparison of these results with those of control diluent, the effects of supplementation were significantly (p<0.01) better. However, the higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of spermatozoa compared to extender containing 2.0 mM BHT. In conclusion, the results obtained in this study showed that the semen quality of rams was improved when BHT was added to extender used before the freezing process.

• Animal Bioscience
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• v.34 no.2
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• pp.192-197
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• 2021

Objective: The present study evaluated the preservation of ram semen at 0℃ using soybean lecithin with a Tris-fructose extender. Methods: Semen was collected by artificial vagina ejaculation from six rams with proven fertility. High quality ejaculates were diluted by soybean lecithin (0.25%, 0.5%, 0.75%, 1.0%, 1.25%) using Tris-fructose extender and control (Tris-fructose egg yolk extender), respectively. The ejaculates were diluted to a concentration of 5×108 sperm/mL, followed by cooling to 0℃ in 90 min and maintaining the temperature for 12 days. The diluted semen samples were examined and recorded for sperm progressive motility, acrosome integrity at 0, 24, 72, 144, 216, 288 h, respectively. Two hundred and twenty-three ewes were inseminated for 216 h with optimal soybean lecithin concentrated semen or control via trans-cervical insemination. Results: The results showed that there were no differences in sperm progressive motility at 0, 24, 72, and 144 h (p>0.05). After 216 h, the sperm progressive motility in the control group and 0.5% concentration groups was significantly higher when compared to 0.25% concentration (p<0.05). The 0.5% concentration group demonstrated the highest survival rate and had no difference with the control group (p>0.05). At 216 h, the sperm progressive motility of all groups was still above 50%. The acrosome integrity of all groups was decreased with prolongation of storage time, but there was no difference at each time point (p>0.05). There was no significant difference in the lambing rate and pregnancy rate between the 0.5% concentration group and the control group (p>0.05). Conclusion: These results suggest that ram sperm is capable of fertilization after preservation at 0℃ with 0.5% of soybean lecithin in Tris-based extender substituted for egg yolk and produce normal offspring after insemination.

• Animal Bioscience
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• v.35 no.5
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• pp.670-676
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• 2022

Objective: This study was conducted to examine influence of skimmed milk-based extender (SM), INRA 96 extender and BotuSemen Gold extender on parameters of stallions' ejaculate during storage. Methods: In this study, 14 stallions between 4 and 20 years of age were monitored. Total and progressive motility, viability and morphology of sperm were evaluated at time intervals of 24, 48, and 72 hours after collection. Results: The total motility, progressive motility, and values of sperm with normal morphology were significantly higher in the INRA 96 and BotuSemen Gold extenders than in the SM (p<0.01). The sperm viability differed significantly in all extenders (p<0.01). The highest value of sperm viability was in INRA 96 (64.69%±0.67%) and lowest in SM (59.70%±0.81%). The highest differences occurred at 72 hours of storage. Values of total motility, progressive motility and sperm viability decreased over time (p<0.01). In case of sperm morphology there was no statistically significant decrease between 48- and 72-hour time intervals. Conclusion: It can be concluded that the extenders with a chemically defined composition have shown better indicators of insemination capabilities in ejaculates than the SM. The BotuSemen Gold extender is a suitable alternative to the INRA 96, when used within 48 hours; after 72 hours of storage, however, the INRA 96 showed a higher share of viable spermatozoa.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.1-8
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• 2005

This experiment was carried out to investigate the effects of saccharide in the lactose-egg yolk(LEY) extender for freezing of boar semen on the viability, normal acrosome, fertilizable of in vitro or in vivo oocyte after thawed. Normal acrosome post-thawed spermatozoa was higher when increasing of glucose concentration in LEY extender with 3 or $4\%$ glycerol, but viability was not significant. Viability of the post-thawed spermatozoa was higher when fructose or fructose and glucose were added to LEY extender with $3\%$ glycerol than glucose and sucrose or fructose, glucose and sucrose(P<0.05). Rate of normal acrosome of post thawed spermatozoa was higher when both fructose and glucose$(81.4{\pm}2.3\%)$ were added to the LEY extender than saccharide not added$(41.6\pm0.6\%)$ to it(P<0.001). The percentage of fertilization, cleavage and development to blastocyst of oocytes fertilized with post-thawed spermatozoa from freezing by LEY extender were $70.8\~80.7\%$, $44.6\~45.7$ and $13.6\~16.0\%$, respectively. Conception rate by artificial insemination with frozen boa. semen was higher$(83.1{\pm}0.3\%)$ than commercial frozen semen from SGI company$(50.0{\pm}0.1\%,\;P<0.05)$, but litter size were no significant differences between frozen by LEY extender$(9.4{\pm}1.7\~10.4{\pm}0.7head/sow)$ and SGI semen$(8.0{\pm}1.1 head/sow)$.

• 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.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.43-48
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• 2005

This experiment was carried out to investigate the extender, cooling rate and concentration of glycerol for freezing of boar semen. The result obtained were summarized as follows: 1. Optimal cooling rate was $0.17\~022^{\circ}C/min$ from 25 to $5^{\circ}C$ in LEY extender on the viability and normal acrosome after thawed. 2. The LEY extender was effective in protecting frozen boar semen from cold shock among the extenders(p<0.001, respectively). 3. The sperm viability and normal acrosome rates after thawing was showed greater in the 3 or $4\%$ of glycerol concentration than $2\%$ in LEY extender. 4. Viability of sperm was higher when both 15mM of fructose and 3 or $4\%$ glycerol were added to the LEY extender compared with other concentrations of fructose and glycerol were added it(p<0.001).

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.65-70
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• 2011

This study was conducted to investigate the survival rate of AndroMed and Tris-egg yolk extender for cryopreservation of Korean Native Bull Semen (Chick Cow). Semen was collected from a Korean Native Bull Semen over 3 year's old. The semen was diluted 1:1 by AndroMed and Tris-egg yolk extender. The pellet was diluted to final sperm concentration of $5{\times}10^7$ cell/ml by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hrs at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes. And then the frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. The survival rates was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender ($89.7{\pm}19.8$ vs. $73.4{\pm}11.2$). However, motility was no significant differences ($78.4{\pm}18.7$ vs. $67.9{\pm}14.6$). Survival rate in time of equilibration between visual and CASA program had higher in 2 h ($86.33{\pm}9.4$ vs. $92.32{\pm}12.4$) than in 5 h ($78.20{\pm}7.8$ vs. $88.28{\pm}13.1$) 15 h ($65.24{\pm}6.6$ vs. $76.48{\pm}17.3$) 20 h ($56.26{\pm}4.6$ vs. $67.73{\pm}18.4$). The development rates to cleavage was higher in Tris-egg yolk extender than AndroMed extender (82.2% vs. 81.7%). Similarly, the development rates to blastocyst was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender (42.3% vs. 29.6%). In conclusion, the obvious impact of this study will be its practical application to improve viability and fertilizing ability of cryopreserved spermatozoa used for in vitro fertilization (IVF) and AI, Which in turn will be beneficial to animal genetic resources conservation.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.1
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• pp.16-18
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• 2002

During the process of freezing, spermatozoa suffer cold shock which increases their susceptibility to lipid peroxidation which plays an important role in ageing of spermatozoa, shortening their life span and affecting the preservation of semen. An experiment was therefore conducted to study the effect of addition of natural antioxidants into semen diluents on the preservability of buffalo semen. Split semen samples were extended in milk egg yolk diluents fortified with vitamin E (MYE), vitamin C (MYC) and control group (MYO); Tris-egg yolk diluents fortified with vitamin E (TYE), vitamin C (TYC) and control group (TYO) and evaluated for their preservabilities at 4-7$^{\circ}C$ and $37^{\circ}C$. Overall least squares mean of percent motility observed after 0, 24, 48, 72 and 96 h of preservation at 4-7$^{\circ}C$ were 66.70, 54.00, 36.80, 21.90 and 12.50, respectively while the estimates for semen extended in MYE, MYC, MYO, TYE, TYC and TYO were 44.80, 42.70, 38.70, 36.00, 35.20 and 33.00 percent, respectively. The results showed that motility was significantly (p<0.01) affected by extender (extender-antioxidant combination) and preservation interval. Overall least squares mean percent motility observed after 0, 4, 8, 12 and 24 h of preservation at $37^{\circ}C$ were 68.50, 58.90, 45.00, 38.10 and 18.10 percent, respectively, while the estimates for semen extended in MYE, MYC, MYO, TYE, TYC and TYO were 48.20, 49.30, 46.80, 45.30, 42.30 and 42.50 percent, respectively. Extender and storage interval were found to be significantly (p<0.01) affecting spermatozoa motility on room temperature preservation. The results indicated that the incorporation of antioxidants, especially vitamin E, had beneficial effect on preservability of buffalo semen.

• Korean Journal of Veterinary Research
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• v.40 no.2
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• pp.403-414
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• 2000

To improve the semen production, the selenium(Se) and vitamin E(Vit. E) were administrated into Hanwoo young sire for intensification an antioxidant system and the taurine were supplemented into semen extender for improving the semen characteristics. The 16 heads ranging from twenty to thirty two months of age were randomly assigned to control group, Se-admistrated group(Se-group), Vit. E-administrated group(Vit. E-Group) and Se and Vit. E administrated group(Se and Vit. E-group). Se and Vit. E dministrated 3 times every 30 days by intramuscular injection. The administration of Se, Vit. E, and Se and Vit. E didn't affect on semen volume, sperm concentration, and total sperm number among all groups(p>0.05). Adiministration of Se improved sperm motility and viability. Motility of Se-group and control were 26.01% and 19.20%, respectively(p<0.05). Viability of Se-group(47.07%) was higher than control group(35.73%), Vit. E group(36.55%)(p<0.05). The administration of Se and Vit. E didn't affect sperm capacitation and acrosome reaction. The 100mM taurine supplement into semen extender increased the motility of frozen/thawed semen in the Vit. E-group(p<0.05) and had a beneficial effects on decreasing abnormality of frozen/thawed semen in all groups(p<0.05). These results indicate that Se administration improve sperm motility and viability, and the taurine supplement into semen extender decrease abnormality in Hanwoo young sire.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.4
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• pp.486-494
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• 2006

In order to protect the spermatozoa against cold shock, hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders for domestic animals. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). The effects of LDL on sperm quality of bull and northern pike (Esox lucius) after freezing-thawing have been reported, but no study has been made to evaluate the effect of LDL on boar sperm motility and other characteristics. The experiment was carried out to investigate the effect of LDL on the freezing of boar sperm in 0.25 ml straws. The aim was to evaluate the quality of boar spermatozoa cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Tris-citric acid-glucose (TCG) and Tris-citric acid-fructose (TCF), two basic freezing extenders containing egg yolk. Similarly, acrosome and plasma membrane integrity were also evaluated and compared to semen cryopreserved with TCG and TCF. Analysis of sperm quality after freeze-thaw showed that the motility, acrosome and plasma membrane integrity were improved with LDL in the extender, as compared to the TCG and TCF. The highest post-thaw integrity of acrosome and plasma membrane and motility were obtained with 9% LDL (w/v). Consequently, the optimum LDL concentration in the extender was 9%. It is also suggested that the concentration of LDL addition is important for the effect on boar sperm protection during freezing and thawing. The percentage of motile spermatozoa was significantly higher after freezing in 9% LDL than in TCG and TCF 54.4% versus 30.4% and 30.1% (p<0.05), respectively. The integrity of acrosome and plasma membrane were also significantly higher at 70.3% and 50.5% respectively with semen frozen in 9% LDL extender compared to TCG at 37.8% and 30.3% and TCF at 36.4% and 29.9%, respectively (p<0.05),. In conclusion, we propose that extender containing LDL extracted from hen egg yolk could be used as a cryoprotective media with a better efficiency than TCG and TCF. LDL improved boar semen quality, allowing better spermatozoa motility, acrosome and plasma membrane integrity after the freeze-thaw process. Furthermore, we found out that the extender with 9% LDL concentration significantly enhanced motility, acrosome and plasma membrane integrity of boar sperm after freezing and thawing.