• Journal of Embryo Transfer
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• v.28 no.3
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• pp.297-302
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• 2013

Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of genetically elite populations. Castration, catastrophic injury, sudden death or any other event that makes semen collection or mating impossible may prematurely terminate a stallion reproduction. Stallion epididymal spermatozoa vary widely in the loss of progressive motility, acrosomal integrity, and viability during freezing and thawing. The objective of this work was to investigate the effect of (1) freezing package types on cryopreservation efficiency, (2) thawing temperatures (37, 56 or $70^{\circ}C$) on Computer Assisted Sperm Analysis (CASA) parameters and (3) post-thawing incubation time (0, 1, 2 or 4h) on castrated stallion epididymis. Post-thawed sperm motility ranged between 59.69% and 64.28% ($56^{\circ}C$ and $37^{\circ}C$) in various thawing temperatures. When stallion epididymis sperm was frozen, straw was better than freezing tube on VCL (Velocity of Curvilinear Line) and VAP (Velocity of Average Path) parameter. Higher percentage of motility was observed at $37^{\circ}C$ thawing temperature even though no significant difference was observed among various temperatures. The motility, VCL, ALH (Amplitude of Lateral Head displacement), VAP, BCF (Beat-Cross Frequency) and STR (Straightness index) parameter of post-thawed sperm were significantly decreased by increasing the incubation time for all thawing temperatures. The present study showed that type of freezing package (Straw vs. Freezing tube) was not significantly different on cryopreservation efficiency. Furthermore, stallion epididymal spermatozoa frozen-thawed at $37^{\circ}C$ for 1 min resulted the highest proportion of motility and velocity movement. In addition, motility and viability of frozen-thawed stallion epididymal spermatozoa were also decreased by incubation.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.12
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• pp.1716-1721
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• 2006

The present study was conducted to observe the effect of osmolality of glycerolated TEST-yolk glycerol extenders on post-thawing sperm kinematics of ram spermatozoa of the native Malpura breed maintained in a semi-arid tropical environment. Good quality semen obtained from adult rams was pooled, split and diluted to 1,000 million spermatozoa per ml in complete TEST-yolk-glycerol extenders of 900, 1,200, 1,500 and 1,800 mOsm/kg osmolality. Diluted semen samples were loaded in 0.25 ml straws and cooled down to $-125^{\circ}C$ freezing temperature at the rate of $-25^{\circ}C$ per minute under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at $50^{\circ}C$ in a water bath for 10 seconds and sperm kinematics of the frozen-thawed spermatozoa were assessed by a computer-assisted sperm analysis technique. Osmolality of diluent had no significant effect on post-thawing % motility, % rapid, % medium and % slow moving frozen-thawed spermatozoa but significantly (p< 0.05) affected the % linearity and % straightness. The post-thawing % motility and % rapid motile spermatozoa were highest in samples extended in diluent of 1,500 mOsm/kg osmolality and lowest in 900 mOsm/kg. The curvilinear velocity of spermatozoa was significantly (p<0.05) higher for samples extended in 1,800 mOsm/kg, compared to those in 900 and 1,200 mOsm/kg, but the effect was not significantly different to those extended in diluent of 1,500 mOsm/kg osmolality. The study indicated that ram spermatozoa could tolerate a wide osmolality range for dilution in the complete TEST-yolk-glycerol extender for their cryosurvival. The highest recovery of motile spermatozoa following thawing was achieved in samples extended in the TEST-yolk-glycerol diluent of 1,500 mOsm/kg osmolality.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.82-90
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• 2021

In the present study, we examined if deep uterine artificial insemination (DUAI) can improve the pregnancy rate of artificial insemination (AI) using epididymal spermatozoa (ES) in Hanwoo cattle. The estrus cycles of 88 Hanwoo cows were synchronized, and 17 cows were artificially inseminated using the DUAI method with ES, 20 cows were artificially inseminated via the uterine body (BUAI) method with ES, and as a control, 51 cows were inseminated by using the BUAI method with ejaculated spermatozoa from 1 proven bull after frozen thawing. The pregnancy rate of the DUAI method (58.8%) was higher than that of the BUAI method (25.0%, p = 0.0498). The motility of ES was examined immediately after thawing and after 3 and 6 h of incubation. The rapid progressive sperm motility of the control group was significantly higher than that of the ES group immediately after thawing and after 3 and 6 h of incubation (p < 0.05). The straight line velocity and average path velocity of the ES group after 6 h of incubation were significantly lower than those in the control group (p < 0.05). The linearity and amplitude of lateral head of ES were lower than those at 6 h (p < 0.05). The flagellar beat cross frequency and hyperactivation of ES were lower than the control spermatozoa immediately after thawing and at 3 h (p < 0.05). These motility parameters suggested that ES had a low motility and fertilization ability compared to the control spermatozoa. After frozen-thawing and 3 h of incubation, the percentage of live spermatozoa with intact acrosomes in the ES was significantly lower than that in ejaculated spermatozoa (p < 0.05). Our findings suggested that the DUAI method can overcome the low pregnancy rate of ES, despite the low motility, viability, and fertilization ability of ES.

• 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.

• 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).

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.147-157
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• 1998

This study was conducted to investigate the effects of in vitro fertilization, culture and embryo development according to in vitro maturation rate, protectant composition and equilibrium time after frozen /thawing of bovine immature oocytes. This results obtained in studies on the effect of different cryoprotectants on the viability, maturation and development of in vitro bovine oocytes were as follow: 1.The post-thawing of immature oocytes matured to metaphase II during culture time for 0 to 26 h, and those group (62~3%) were low than control group (76.7%). The optimal maturation time of frozen-thawed immature oocytes was at 24 h. 2.The viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants. The developmental competence of frozen4hawed oocytes was not affected by cryoprotectants. These results indicate that an optimal maturation time of frozen /thawed immature oocytes was at 24h. Furthermore the viability of cryopreserved immature oocytes was not affected by sort of cryoprotectants and developmental competence of frozen /thawed oocytes.

• Korean Journal of Animal Reproduction
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• v.8 no.1
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• pp.22-28
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• 1984

This experiment was carried out to investigate effects of DMSO or ethylene glycol as a cryopotectant and of freezing methods on survival rate of forozen-thawed rat 2-cell embryos by morphological observation. 2-cell embryos were recovered from oviducts of Sprague Dawley females mated with males of same strain on day 2 of pregnancy after inducing superovulation by intrapertioneal injection of PMSG and HCG. In slow freezing and thawing groups, embryos were frozen to -79$^{\circ}C$ or -196$^{\circ}C$ in a glass test tube containing 0.2ml PBI with 1.5M DMSO or 1.2M ethylene glycol at a rate of 0.3-1.0C/min. and thawed slowly. When samples were frozen to -79$^{\circ}C$, higher survival rate was obtained in the medium containing DMSO (43.9%) than ethylene glycol (41%). And similar result was obtained (32.5% in DMSO vs. 31.4% in ethylene glycol) when samples were frozen. In rapid freezing and thawing groups, embryos were frozen to -79 or -196$^{\circ}C$ in a glass test tube containing 0.2ml of PBI with 1.5M DMSO or 1.2M ethylene glycol by rapid cooling, and thawed rapidly. When samples were frozen to -79$^{\circ}C$, 1.5M DMSO (13.2%) was more effective than 1.2M ethylene glycol (6.1%). When the storage temperature was -196$^{\circ}C$, survival rates were 9.8% in 1.5M and 5.4% in 1.2M ethylene glycol.

• Korean Journal of Food Science and Technology
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• v.28 no.6
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• pp.1021-1025
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• 1996

The experiments were carried out to find how the processing of ascidian (Halocynthia roretzi) was affected by the frozen storage. The quality of ascidian which takes the shell-on was changed quickly in the frozen storage. The causes of the change were as follows: 1. the damage caused by the ice crystal in the muscle, 2. a lot of drips after thawing, 3. the discoloration of the muscle after thawing. On the contrary, the quality of ascidian which takes the shell-off was even better in color, texture, yield and drips after 60 days of the frozen storage. But the muscle was blackened after that.

• Journal of Food Hygiene and Safety
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• v.3 no.1
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• pp.19-26
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• 1988

Most of meat spoilage bacteria area Gram negative, which are very sensitive to freezing ; for instance , 90% of E. coli cells are killed or sub-lethally injured by freezing at -3$0^{\circ}C$, and the freeze-injury rate is dependent upon freezing rate. Since the injured bacterial cells are sensitive to selective agents, they fail to multiply in selective media. Injured bacterial cells are, however, capable of spontaneous repair at appropriate environmental and nutritional conditions . Enumeration of injured bacterial cells involves artificial induction of repair at these conditions. Cubic beef samples(3$\times$3$\times$3cm) were frozen at -6$0^{\circ}C$, -4$0^{\circ}C$, or -18$^{\circ}C$. The samples frozen at each temperature were thawed at 4$^{\circ}C$, 2$0^{\circ}C$, or by microwave . After these respective freezing an thawing treatments, the percentage of sub-lethally injured total coliforms out of total surviving ones was measured and compared. The results were as follows: 1. The interaction between freezing and thawing on injury rate was not significant. 2. The injury rates(as means of all three thawing treatments post-freezing) by freezing at -6$0^{\circ}C$, -4$0^{\circ}C$, or -18$^{\circ}C$ were 32.2$^{\circ}C$ and 19.2$^{\circ}C$ respectively . 3. The injury rates(as means of all three freezing treatments)by thawing at 4$^{\circ}C$, 2$0^{\circ}C$, or by microwave were 49.3%, 11.7% and 21.0% respectively. The highest injury rate was caused by freezing at -6$0^{\circ}C$ and subsequent thawing at 4$^{\circ}C$. However since the injury rates by freezing treatment were not significantly different, freezing at -18$^{\circ}C$ and subsequent thawing at 4$^{\circ}C$ can also be recommended , from an economic perspective.

• Magazine of the Korean Society of Agricultural Engineers
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• v.33 no.2
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• pp.112-119
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• 1991

Some experiments were carried out to investigate the effects of freezing and thawing on the strength and strain characteristics of alluvial silty clay under the different temperatures, loading and moisture conditions. The results were as follows; 1. The soil used was proved to be consisted of silty clay with honey-combed structure, and showed higher dilatancy, frost activity and lower stability in natural state. 2. Soil treated with freezing and thawing cycles showed lower compressive strength compared with the non treated, The strength decreased with incement of freezing and thawing cycles. It's shapes of stress-strain curves were flat and did not formulate a peak while the peak strength of higher moisture content soil decreased with the increment of moisture content. It's decrement ratio was most distinctly shown at the first one cycle of freezing and thawing. 3. The cohesion decreased due to freezing and thawing cycles but internal frcition angle was not changed. 4. The liquid limit decreased with increment of freezing and thawing cycles, and became almost constant after three cycles of freezing and thawing. 5. The strength under simple loading at failure mode was appeared to be higher compared with the cyclic loading after freezing and thawing but initial moisture content effect was not observed. 6. Ice lense was not observed within 50% of ice content ratio but observed over 100%. The higher the ice content ratio, the higher the peak strength. As a matter of fact, it seems that an optimum ice content ratio exists for plastic mode and the least compressive strength.