• Journal of Veterinary Clinics
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• v.34 no.2
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• pp.156-160
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• 2017

We investigated the effect of ethylene glycol and antioxidants such as taurine, hypotaurine and trehalose with extenders during cryopreservation of Korean Jeju Black Bull spermatozoa. The cryopreservation of freshly collected spermatozoa was conducted with four different conditions. As a control, spermatozoa were cryopreserved with Tris egg-yolk extenders added 5% ethylene glycol (EG). Taurine (20 mM), hypotaurine (20 mM) and trehalose (20 mM) were individually added into tris egg-yolk extenders with 5% EG. After thawing of frozen spermatozoa with four different conditions, sperm viability, motility, acrosomal integrity, and membrane integrity were investigated. The significant (p < 0.05) improvement of sperm viability showed in all antioxidant treated thawed spermatozoa (taurine; $68.1%{\pm}4.4$, hypotaurine; $69.2%{\pm}6.7$ and trehalose; $68.0%{\pm}4.4$) when compared to control ($63.4%{\pm}5.6$). Neither positive nor detrimental effects of three antioxidants were shown sperm motility after thawing. The results of hypo-osmotic swelling test showed that the membrane integrity of taurine, hypotaurine or trehalose treated thawed spermatozoa ($64.1%{\pm}5.4$, $61.5%{\pm}3.7$ and $59.0%{\pm}4.0$, respectively) had significantly (p < 0.05) higher rate of the swollen sperm compared to control ($53.7%{\pm}9.7$). Hypotaurine treated frozen-thawed spermatozoa had siginificantly higher (p < 0.05) F pattern ratio than taurine, trehalose and control treated frozen-thawed spermatozoa. Trehalose added frozen-thawed spermatozoa had significantly higher (p < 0.05) acrosome reaction pattern ratio than taurine and hypotaurine added frozen-thawd spermatozoa. In this study, we found that antioxidants such taurine, hypotaurine and trehalose treatments during cryopreservation process could reduce damage of spermatozoa of Korean Jeju Black Bull and improved sperm capability of fertilization.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.155-162
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• 2012

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY ($69.00%{\pm}4.18$; EG and $63.00%{\pm}9.75$; 7% G) than 8% LDL ($57.00%{\pm}5.70$; EG and $52.00%{\pm}4.47$;G). Treatment of 4% LDL + 5% EY-EG ($66.85%{\pm}5.06$) has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG ($64.65%{\pm}6.10$) among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.229-234
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• 2002

This study was carried out to investigate the general characteristics such as concentration sperm motility and abnormality of sperm on the whole epididymal semen(EWS), RSP-S(removed seminal plasma by saline) and RSP-T(removed seminal plasma by tris-buffer) semen and survival rates after freezing on motility of whole and RSP-S and RSP-T semen and extender containing 2~8% glycerol, and ability of frozen-thawed sperm to penetrate homologous oocytes. 1. The concentration, motility and abnormality of epididymal WES, RSP-S and RSP-T sperm were 4.25 $\pm$ 0.25($\times$10$^{6}$ Cells/$m\ell$), 3.85$\pm$0.20($\times$10$^{6}$ Cells/$m\ell$), 4.05 $\pm$ 0.28($\times$10$^{6}$ Cells/$m\ell$), 50.55 $\pm$ 2.75%, 67.25 $\pm$ 2.55%, 78.75 $\pm$ 3.55 and 9.45 $\pm$ 2.25%, 37.75 $\pm$ 2.10%, 24.25 $\pm$ 1.55%, respectively. 2. The survival rates of slow and rapid frozen epididymal RSP-S and RSP-T sperm were 35.00 $\pm$ 2.35%, 45.50 $\pm$ 2.15％ and 16.50 $\pm$ 3.55%, 22.55 $\pm$ 3.95%, respectively. The survival rate of epididymal WES and RSP-T sperm after freezing following dilution with tris-buffer containing 2~8% glycerol were 9.25 $\pm$ 1.55%~17.50 $\pm$ 2.50%. 3. The percentage of capacitated and acrosome-reacted sperm prier to culture for fresh and frozen -thawed epididymal RSP-T semen were 45.25 $\pm$ 5.75%, 7.06 $\pm$ 0.25%, 48.20 $\pm$ 6.80% and 13.00 $\pm$ 2.35%, 3.55 $\pm$ 0.85%, 15.50 $\pm$ 1.90%, respectively. The penetration rate the number of sperm per penetrated for fresh and frozen-thawed epididymal RSP-T sperm were 39.25 $\pm$ 4.72%, 34.24 $\pm$ 3.93% and 1.30 $\pm$ 0.33, 1.10 $\pm$ 0.50., respectively.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.71-76
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• 1996

This experiment was carried out to investigate the developmental ability of bovine embryos matured and fertilized in vitro to the gastrulation stage. The bovine oocytes were collected from 2∼5mm follicles, matured for 20∼24hrs in 5% CO2 incubator and then fertilized with frozen-thawed semen. On day 9 after IVF and after freezing and thawing the hatching abilities of expanding blastocysts were examined. Cleavage rate and production rate to expanding blastocysts were 59.7%(955/1604) and 20.7%(333/1604), respectively. Hatching rate of day-9 expanding blastocysts was 54%(40/74), that after freezing and thawing was 56%(79/141). Also, developmental ability of hatched blastocysts to the primitive streak stage was 26%(6/23).

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.6-10
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• 2001

No. Sperm can be sexed with 90% accuracy by flow cytometry/cell sorting. No. The current speed of sexing is about 5,000 live sperm of each sex per second, remarkably fast considering that each sperm is individually sexed. No. Although fast, sperm sexing is not fast enough to use standard numbers of sperm per AI dose. No. With well managed heifers, pregnancy rates with low doses of sexed, frozen sperm are 70-80% of those with unsexed sperm with normal sperm numbers. Pregnancy rates are lower in lactating dairy cows. No. Calves from sexed sperm appear to be normal. No. Sexed, frozen semen from a few bulls currently is available commercially in the United Kingdom, and likely will be available in several other countries in 2002, probably at a premium of US ＄30-50 per straw. (omitted)

• Development and Reproduction
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• v.2 no.1
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• pp.63-68
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• 1998

This study was done to find optimal cryopreservation medium and method to improve the post-thaw motility of human sperm. Thirty three semaen samples were included in the study. Of these, nineteen Samples showing normal semen profile were frozen using three cryprotectants (TYB, TYB+DTT and KS II)to compare post-thaw motility. Fourteen samples were frozen with vapor freezing and programmable freezing methods to compare post-thaw motility in correlation with freezing method. After 24 hrs of cryostorage , the vials were thawed and the post-thaw sperm motility was assessed by two kinds of computer-aededsperm analysis (CASA; SAIS and Hamilton Thorn). As a result, the post-thaw motility of the KS II group was higher than the TYB or TYB+DTT group in normal semen(34.8%, 28.3% and 23.0%, respectively). In the asthenospermia group, a significantly hither post-thaw motility was observed in the KS II (18.5%) compared to those of TYB or TYB+DTT group (13.6%, 10.0%, respectively). No difference was observed between vapor freezing group and computerized freezing group in normal semen (27.8%, 33.2%, respectively) and semen group showing asthenospermia (12.8%, 12.9%, respectively). In conclusion, our results indicate that KS II medium is reliable for cryopreservation of human sperm and vapor freezing and programmable freezing were equally effective in terms of the recovery of motile spermatozoa.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.307-314
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• 2020

In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozen-thawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 μm/s, 51.4 ± 1.3 and 47.1 ± 1.1 μm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezing-thawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.157-162
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• 2005

To find out the possible inefficiencies of artificial inseminators at rectovaginal insemination in cows, inseminators' skill were evaluated by controlling the semen thawing procedure adopted and by using the technique of dye deposition in the genital tract of slaughtered cows. This was followed by refreshment training for the inseminators. Thirty seven artificial insemination technicians regularly working in the government, cooperative and NGO (Non Government Organization) artificial insemination programmes at different places of Bangladesh were included in the study. Individual technicians were asked to thaw a semen straw and deposit dye in the genital tract of slaughtered cows following the procedures they would have adopted in their actual practices of insemination. The time and water temperature adopted by technicians were recorded and genital tract after sham artificial insemination was dissected to determine the site of dye deposition. Then, the inseminators took part in a three days intensive training program. The training program was ended up with the same tests for thawing frozen semen straw and dye deposition in the genital tract of slaughtered cows. At pre training evaluation, only $25\%\;and\;72\%\;(n=36)$ inseminators adopted co..ect thawing time and temperature, respectively. At post training evaluation, all inseminators thawed semen straws for proper time and temperature. At pretraining evaluation, $21(57\%),\;11 (30\%)\;and\;3(8\%)$ inseminators deposited dye at the body of uterus, in the vagina or in cervix, and into the horn of uterus, respectively. In $2(5\%)$ cases dye did not pass into the genital tract, instead back flowed through the space between the barrel of insemination gun and sheath. At post training evaluation, all inseminators successfully deposited dye in the body of uterus. Frequent evaluation of inseminators' skill and subsequent training would help improvement of the artificial insemination technicians' skill.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.183-189
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• 2020

The pregnancy rate in indigenous ewes inseminated with frozen-thawed Suffolk semen following natural and synchronized estrus was determined. The serum Progesterone and Estrogen concentration and vaginal electrical resistance (VER) of ewes at the time of Artificial Insemination (AI) were observed as successful pregnancy determinants. 21 healthy ewes were selected for this experiment during January-April, 2017. 10 ewes were inseminated in natural estrus. Whereas, 11 ewes were inseminated after estrus synchronization using intravaginal sponges containing 60 mg medroxyprogesterone acetate. Trans-cervical Al (TCAI) was performed in all ewes within 12-16 hours of observed heat. Prostaglandin E₁ analogue impregnated vaginal sponge was used for cervical relaxation 6-8 hours before insemination. Pregnancy was diagnosed through trans-abdominal ultrasonography after 40 days of AI. The pregnancy rate of ewes in synchronized estrus was higher (54.5%) than in natural estrus (30%). Higher serum Progesterone level (0.90 ± 0.02 ng/mL) and significantly (p < 0.001) lower VER (257.78 ± 10.11 ohm) were observed at the time of AI in ewes becoming pregnant. Results suggest that higher Progesterone concentration and lower VER could be considered as pregnancy indicators. Oestrous synchronization could be implemented to increase the pregnancy rate in ewes.

• Korean Journal of Veterinary Service
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• v.18 no.3
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• pp.1-12
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• 1995

To investigate effect of seeding on post-thaw motility and viability of canine spermatozoa, the semen from male dogs which had been proved to be fertile in the past were frozen and seeded during freezing process. Post-thaw motility and viability of canine sperm which were frozen and seeded were investigated according to different seeding temperatures of $-5^{\circ}C$, $-10^{\circ}C$, or $-l5^{\circ}C$ and also according to different concentration of glycerol of 2%, 5% and 10%. In addition, post-thaw motility of canine sperm frozen by direct freezing in a deep freezer or programmed freezing in a programmed cell freezer was investigated. Post-thaw motility of canine sperm was compared according to different seeding temperatures : The sperm seeded at $-5^{\circ}C$ showed considerably higher post-thaw motility than that of non-seeding, and that seeded at $-10^{\circ}C$, or $-l5^{\circ}C$, respectively, in 2% and 5% glycerol groups on both 2 and 7day after freezing(p<0.05). In 10% concentration of glycerol, the sperm seeded at each seeding temperature showed considerably higher post-thaw motility than that of non-seeding group on day 7 after freezing(p<0.01). Post-thaw viability of canine sperm was compared according to different seeding temperatures : The sperm seeded at $-5^{\circ}$ showed significantly higher post-thaw motility than that of non-seeding, and that seeded at $-10^{\circ}C$, or $-l5^{\circ}C$, in 5% and 10% glycerol groups on day 7 after freezing(p< 0.05). In comparison of post-thaw motility of canine sperm seeded according to different concentration of glycerol, 5% glycerol group and 10% glycerol group showed considerably higher post-thaw motility than 2% glycerol group without difference between those two groups in all seeding temperatures($-5^{\circ}C$, $-10^{\circ}C$ and $-l5^{\circ}C$) on day 2 and 7 after freezing(p<0.01). In comparison of post-thaw viability of canine sperm seeded according to different concentration of glycerol, 5% glycerol group and 10% glycerol group showed the same considerably higher post-thaw viability than 2% glycerol group on each thawing day(p<0.01). The canine sperm frozen and seeded by programmed freezing method showed considerably higher post-thaw motility than that frozen by direct freezing method in all different seeding temperatures($-5^{\circ}C$, $-10^{\circ}C$ and $-l5^{\circ}$). These results indicated that the higher post-thaw motility and viability was obtained in the spermatozoa seeded than that of non-seeding, that among different seeding temperatures of $-5^{\circ}C$, $-10^{\circ}C$ and $-l5^{\circ}C$, the sperm seeded at $-5^{\circ}C$ showed higher post-thaw motility and viability than the other temperatures, also among different concentrations fof glycerol of 2%, 5% and 10%, the sperm frozen and seeded in 5% and 10% concentration of glycerol showed higher post-thaw motility and viability than that in 2% of glycerol, and that the sperm frozen and seeded by programmed freezing method showed higher motility than that by direct freezing method.