• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.155-159
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• 2013

This study was carried out to investigate synthetic extender for semen cryopreservation of Jeju Native Black Bull. The semen was collected using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by Tris-Egg yolk extender and contrifuged in 1,500 rpm for 15 minutes. The supernatant was removed. The pellect was diluted to final sperm concentration of $2{\times}10^8/ml$ by doubling in every 30 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 4 hours at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm for 10 minutes. The height and duration affect the freezing speed by temperature. The frozen straw was plunged to $LN_2$. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. Frozen-thawed sperm were evaluated sperm viability, membrane integrity and acrosome integrity. Post-thawed sperm viability has been significantly higher (p<0.05) in fresh sperm ($93.27{\pm}1.62%$) than frozen-thawed sperm ($73.34{\pm}3.27%$). However, there were no significant differences between fresh and frozen-thawed dead cell rate ($7.35{\pm}2.63$ vs, $13.71{\pm}2.85$). In sperm motility, between Triladyl and AndroMed Extender, there was no significant different ($72.86{\pm}2.83$ vs, $81.47{\pm}2.48$), similarly, the dead cell rates was similar ($18.41{\pm}3.42%$ and $17.26{\pm}4.25$). The results of our study suggest that AndroMed to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Jeju Native Black bull semen.

• Korean Journal of Animal Reproduction
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• v.10 no.2
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• pp.182-187
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• 1986

As a fundamental study to increase the fertility and to modify the sex ratio in cattle, highly motile sperm were separated by bovine serum albumin gradients. The pregnancy rates of Korean native cow and Holstein cow, and the sex ratio between AI and natural mating were also investigated. The results obtained were as follows. 1. First service pregnancy rate of Korean native cow in artificial insemination was higher than that of Holstein. 2. At secondary sex ratio in artificial insemination and natural mating, male ratio in artifical insemination was slightly higher than that in natural mating. 3. The sperm separated from marketed frozen semen using 6%, 10% and 20% bovine serum albumin showed significantly high value in motility, percent of normal sperm and progressive motility as compared with control sperm.

• 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.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.4
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• pp.490-496
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• 2020

A styrofoam box is used as a simple and easy freezing method to preserve animal semen as a livestock genetic source. This study optimized the methods of freezing chikso brindled cattle semen. To test the freezing box, the motility of spermatozoa was compared between two box sizes (length×width×heigh) with the dimensions of 23.5×30.5×22.5 cm and 25.5×46.5×26.5 cm. The motility of thawed sperm from brindled Korean bulls was used to confirm the efficiency of the freezing boxes. The box having a larger inner space with larger horizontal and height measurements supported better motility after thawing (60.4±5.3% vs 67.2±3.1%) with 10 min of exposure time in liquid nitrogen vapor. The optimized freezing space is estimated to be an essential element for successful freezing results and the larger box could be used for production of more than 60 frozen semen straws. These properties are also helpful to optimize the cryopreservation techniques that would control the quality and quantity of semen straws according to different animal species.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.141-144
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• 2005

This experiment was to investigate whether the mitochondria function assessment can be used for the prediction of sperm fertility through examining the correlation between mitochondria fluoromicroscopic frequency of frozen-thawed eight Hanwoo bull semen using rhodamine123 (R123) and in vitro embryo development following fertilization. Individual sperm were stained in 5 ${\mu}g/mL$ R123-added calcium-free Sp-TALP for 30 min at 0 h, 6 h, 12 h and 24 h after thawing and examined their mid-piece under an epifluorescence microscope using 495 nm excitation filter (x1,000). Three replications were taken, and at least 300 sperm per individual were examined. When semen samples were separated into two groups (good and poor) by sperm motility and fluorescent frequencies at just after thawing, average fluorescent frequencies were remarkably reduced as time going (0 h; $53.29{\~}72.94\%$, 6 h; $21.40{\~}58.90\%$, 12 h; $8.26{\~}25.93\%$, 24 h; $1.00{\~}13.78\%$, irrespective of selected group, and there were no differences at 6 h or 12 h after thawing between selected groups but indicated significant difference at 24 h after thawing (p<0.05). In vitro fertilization rates in good and poor groups ranging $70.8{\~}77.8\%$ and $52.1{\~}84.5\%$, respectively, were not significantly different. However, in vitro development rates of the same groups ranging $25.7{\~}40.0\%$ and $12.9{\~}1.8\%$, respectively, were significant different (p<0.05). These results demonstrate that mitochondria fluoromicroscopic assessment of frozen-thawed bovine sperm may be used as a criterion to select more fertile sperm.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.12
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• pp.1614-1619
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• 2009

The present study was conducted to observe the effect of increased osmolality of basic tris extender supplemented with trehalose and sucrose on post-thawing quality (motility, progressive motility, viability, the rate of acrosome abnormality, total abnormality and membrane integrity) of Markhoz goat spermatozoa. Fresh semen samples were evaluated for motility and sperm concentration. Only semen samples with motility more than 70% and sperm concentration higher than $3{\times}10^{9}$ sperm/ml were used for cryopreservation. In Exp. 1, trehalose (50, 75 or 100 mM) and sucrose (40, 60 or 80 mM) were added to a basic tris diluent. Based on the results of experiment 1, the goal of Exp. 2 was to investigate the combinational effects of the highest and lowest concentrations ($T_{100}+S_{80}$ or $T_{50}+S_{40}$) of trehalose and sucrose. As the control, semen was diluted and frozen in the tris diluent without trehalose or sucrose. The results in Exp. 1 showed that all evaluated spermatozoa characteristics improved significantly after freezing and thawing (p<0.05) and at the same time the increase of trehalose and sucrose concentrations in basic extenders was seen, with the best results obtained for extenders containing 70 and 100 mM trehalose and 80 mM sucrose. Comparing these results with those of control diluents, the effects of supplementation were significantly (p<0.05) better. In Exp. 2, the results showed no significant differences (p>0.05) between $T_{100}+S_{80}$ and $T_{50}+S_{40}$ extenders, but the results of $T_{50}+S_{40}$were slightly better than obtained with $T_{100}+S_{80}$ diluents. Furthermore, the results of this experiment indicated that the sperm characteristics in the isotonic control extender were significantly (p<0.05) lower than examined extenders. In conclusion, the results of this study indicated that goat sperm can tolerate hypertonic trehalose and sucrose solutions better than isotonic control diluents in the freezing period. In particular, these positive effects have been shown for acrosome integrity, which is very important for the fertilization capacity of sperm. The data indicated that addition of trehalose plus sucrose to the freezing extender can be recommended for cryopreservation of goat spermatozoa, but more data is needed on pregnancy rate, acrosome reaction and IVF to ascertain the real effect.

• 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.

• Journal of Veterinary Clinics
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• v.17 no.2
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• pp.431-437
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• 2000

Evaluation of viability and capacitation of canine sperm is of great importance to deter- mine good condition for freezing canine semen and consequently to improve conception rate by arti-ficial insemination. Semen were collected from nine male dots which had been proved to be fertile in the post and the semen were treaded for freezing procedure. Semen were thawed at 37$^{\circ}C$ for 30 seconds. In this study, hypoosmotic swelling(HOS) test and chlortetracycline(CTC) test were per- formed to evaluate post-thaw viability and capacitated status of sperm, respectively. In HOS test far canine sperm, the highest percentage of curled sperm was shown at 60 mOsm. In HOS test for canine semen, there were considerably significant correlation between HOS values and sperm motil- ity(r=0.9064, p<0.01) and converse correlation between HOS values and sperm abnormality(r=- 0.6905, p<0.05). The sperm viability and HOS-values for chilled extended semen were significantly decreased from 0 to 72 hours during storage at 5$^{\circ}C$ (p<0.05). Of the media added to canine semen after thawing, the most capacitated sperm were shown in CCM(p<0.01), and then This Fructose Cit- rate(TFC) medium with calcium from 3 hours after incubation with media. It was concluded that HOS test is of great value to determine the viability and motility of canine sperm, whereas CTC test is usable to determine the capacitated status. Consequently, both tests were thought to be useful as the additional tests to standard semen analysis.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.51-59
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• 2003

This study was carried out to evaluate the effect of different freezing and thawing rates on the viability, motility and acrosomal changes of frozen canine spermatozoa. The ejaculated semen was extended with Tris-egg yolk buffer containing 8% glycerol and equilibrated for 60 min after cooled to 4$^{\circ}C$ for 58 min. The straws were cryopreserved gradually by slow-cooling at different distance(6, 10 and 17 cm, respectively) from the liquid nitrogen (L$N_2$) to achieve temperature rate of 3, 8.9 and 19$^{\circ}C$ /min. Thawing of the straws was performed in a water bath fur 2 min at 37$^{\circ}C$ and 55$^{\circ}C$ , respectively. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Concentration of the ejaculated fresh semen was normal range of 3.44 $\times$ 10$^{8}$ /ml. Freezing temperature were reduced to -110, -70 and -35$^{\circ}C$, as higher distance from liquid nitrogen, 6, 10 and 17 cm, respectively. Freezing at 3$^{\circ}C$/min in distance of 17 cm from liquid nitrogen yielded better motility, viability and rate of intact acrosome than 8.9 or 19$^{\circ}C$/min and the optimal thawing was 37$^{\circ}C$ for 2 min.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.91-99
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• 1986

This experiment was undertaken to examine the effects of HIS treatment on the motility and acrosome reaction of frozen bovine spermatozoa and to test their abilities to interact with zona-free hamster eggs in vitro. Also, in vitro results were compared with those of bull's fertility in AI. The frozen semen from four Holstein bulls were exposed to HIS-DM for 5 minutes after thawing and then preincubated for 60 minutes in DM prior to insemination. The hamster eggs were mounted, fixed and stained 6 hours after exposure to boving spermatozoa and examined under a phase-contrast microscope. 1. The sperm motility expressed as a mobility index dro, pp.d significantly from 60-75 to 12-24 after exposure to HIS-DM, but increased in 32 to 41 at insemination. Bull C showed a low motility index than those of the orher bulls. The percentage of acrosome reaction by staining procedure were increased by HIS-DM treatment but did not change during 7 hours incubation period in DM. 2. The overall percentage of hamster eggs interacting with bull spermatozoa was 56.3%, 58.3%, 66.6% and 70.0%, respectively. Although there was no significant difference among bulls in the penetration rate of spermatozoa into hamster eggs, high proportions of eggs interacted with spermatozoa from Bull C and D than those from Bull A and B. 3. The conception rates (60-90 day RP) resulting from AI were 62.5%, 67.5% and 70.9% for Bull A, B and C, respectively. These results were in good agreement with the invitro results that the proportions of bull sperm-egg interction were greater for Bull C than for Bull A and B.