• Journal of Aquaculture
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• v.19 no.1
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• pp.47-51
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• 2006

Experiments were performed to find out the physico-chemical properties of milt and sperm motility in various storage conditions using starry flounder (Platichthys stellatus). The average sperm concentration and spermatocrit in stripped milt were $6.00{\pm}0.98{\times}10^8/mL\;and\;72{\pm}5$, respectively. The osmolality and pH were $337{\pm}9mmol/kg$ and $7.7{\pm}0.1$, respectively. The sperm of starry flounder was preserved with artificial seminal plasma (ASP), Stein's solution (SS) and marine fish Ringer's solution (MFRS) at $0^{\circ}C,\;2^{\circ}C,\;and\;4^{\circ}C$. The most effective condition for cold storage was SS at $0^{\circ}C$, and the preserved sperm remained motile for 30 days.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.243-250
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• 1995

Frozen storage of the oocytes has been used in a few mammalian species including mouse, hamster, human and cattle. However, frozen4hawed oocvtes show different sperm penetration on the levels of the zona pellucida and the plasma memhrane when compared with fresh oocytes. To elucidate biological changes occurring during freezing and thawing, we examined the kinetics of sperm penetration into frozen-thawed hamster oocytes. Oocytes obtained from superovulated female golden hamsters were frozen-thawed in an autofreezer according to an established method. Fresh and frozen4hawed oocytes were fertilized in vitro with capacitated hamster spermatozoa after removing the zona pellucida. The oocytes were examined at 1, 2, 3 and 6 h postinsemination. Sperm penetration found to be 1 h delayed in frozen-thawed oocytes. Other parameters such as degree of polyspermy and decondensing sperm heads were not affected by freezing and thawing. The results suggest that freezing and thawing may cause changes in the egg membrane surface and subsequently which leads to delay in the sperm-egg fusion.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.7
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• pp.1068-1076
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• 2020

Objective: In the present study, we determined efficiency of incorporating caffeine, melatonin or omega-3 polyunsaturated fatty acid in the diluent on mitigating consequences of (a) liquid chilled- and (b) cryo-storage of ram spermatozoa. Methods: In the first experiment, ejaculates (n = 30) were collected from 5 adult rams and were pooled, diluted (1:10) with Tris-citric acid (base diluent) and were split into 4 aliquots assigned for: control (untreated), caffeine (0.1 mM), melatonin (0.3 mM) or omega-3 fatty acids (0.3 mM) (T₀). The diluted specimens were stored at 4℃ for 48 h, during which sperm physical and cytological properties were evaluated along with oxidative stress indices (T₂₄, T₄₈). In the second experiment, 15 ejaculates (3 per male) were pooled, diluted with glycerolized base diluent (4% glycerol, v/v) and were split corresponding to the same previous treatment groups before being processed for cryopreservation. Post-thaw physical and kinematic sperm properties were assessed by a computer-assisted sperm analysis system. Results: The results clarified superiority of both melatonin and omega-3 supplementation on maintaining (p<0.05) sperm properties, while reducing (p<0.05) lipid peroxidase reaction and enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in preservation medium, compared to caffeine either during liquid-chilled storage or cryopreservation of spermatozoa. Conclusion: Melatonin and omega-3 are regarded efficient alternatives to caffeine when processing ram spermatozoa for application of artificial insemination or in vitro fertilization.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.340-345
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• 2008

Oxidative stress is one of the major causes of failure of in vitro storage of boar semen. Reactive oxygen species (ROS) are one of the important mediators of oxidative stress during in vitro storage of boar semen. Our study examined the effects of taurine on sperm characteristic and on in vitro developmental embryos during in vitro storage of boar semen for 7 days. Semen was randomly aliquoted into 3 centrifuge tubes and treated with different concentrations of taurine (25-100 mM). The characteristics of boar sperm were analyzed for motility by light microscopy, viability by using a Makler counting chamber and membrane integrity by a hypoosmotic swelling test (HOST). The percentages of motile spermatozoa in taurine groups after 5 days were significantly higher compared to the control. Sperm viability in the control was lower than in taurine groups after 7 days irrespective of different taurine concentration. In the hyoosmotic swelling test (HOST), significantly higher results were obtained in taurine groups after 3 days. Also, the developmental rates of IVM/IVF porcine embryos from semen treated with pyruvate and taurine were significantly increased when compared with the control (p<0.05). These results indicate that supplementation of taurine as an antioxidant in boar semen extender can improve the semen quality.

• Korean Journal of Poultry Science
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• v.30 no.2
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• pp.129-134
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• 2003

This study was conducted to investigate the effects of liquid rooster semen on reproductive ability in chicken. Raw and diluted semens were stored at 5$^{\circ}C$ cold temperature for 6, 30, and 54 hours after semen collection. There was no statistically difference in sperm motility throughout the 6 hours period of storage among raw semen and diluted semen groups with skim milk glucose solution (SM), egg yolk glucose solution (EY), and saline. But there was decrease in those throughout the period of 30 and 54 hours of storage. Sperm motility and normal sperm for the period of 30 and 54 hours of storage were significantly better in SM and EY diluted groups (P<0.05). Fertilization rates of rooster semen diluted with SM were 90.77, 87.70, and 59.46% for 6, 30, and 54 hours stored groups, respectively, those proved to be higher in SM-diluted group than other groups.

• Journal of Veterinary Clinics
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• v.33 no.6
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• pp.356-361
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• 2016

During storage, boar spermatozoa undergo several changes including diminished motility and viability and accumulated reactive oxygen species (ROS). In this study, we investigated the effects of green tea extract (GTE) supplementation in the Sui Dil extender on the sperm motility, viability, ROS and lipid peroxidation (LPO) of long-term preserved boar semen at $17^{\circ}C$. A total number of eight boars were used for this experiment. Pooled ejaculates were diluted to $20{\times}10^6sperm/ml$ in the Sui Dil extender containing 0 (control), 1, 10, 100 or 500 mg/l GTE and were preserved at $17^{\circ}C$ for 24, 72, 120 and 168 h, respectively. At each storage time, sperm motility and viability were estimated by microscopic examination and the fluorescent double stain $Fertilight^{(R)}$, respectively. Sperm ROS level and LPO were assessed using the 2', 7'-dichlorodihydrofluorescein diacetate ($H_2DCFDA$)/propidium iodide (PI) and C11-BODIPY581/591/PI with flow cytometry, respectively. Compared to that of the 500 mg group, there were higher sperm motility and viability in the 1, 10 and 100 mg GTE groups during the preservation from 24 to 168 h (p < 0.05). The ROS levels of the 10 and 100 mg groups during the 168 h preservation were lower than those of the 0, 1 and 500 mg groups (p < 0.05). There were no significant differences in LPO regardless of the preservation period or the GTE concentration. In conclusion, the optimal concentrations (10 and 100 mg/l) of GTE that led to lower ROS levels may be useful for liquid boar sperm preservation at $17^{\circ}C$ for a period of 168 h.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1369-1373
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• 2004

The percentages of sperm motility and normal acrosome on the liquid boar semen diluted and preserved at $4^{\circ}C$ with lactose hydrate, egg yolk and N-acetyl-D-glucosamine (LEN) diluent were significant differences according to preservation day and incubation time, respectively. The sperm motility steadily declined from 96.9% at 0.5 h incubation to 78.8% at 6 h incubation at 1 day of preservation. However, the sperm motility rapidly declined after 4 day of preservation during incubation. The normal acrosome steadily declined from 93.3% at 0.5 h incubation to 73.8% at 6 h incubation at 1 day of preservation. However, the normal acrosome rapidly declined after 3 day of preservation during incubation. The rates of sperm penetration and polyspermy were higher in 5 and $10{\times}10^6$ sperm/ml than in 0.2 and $1{\times}10^6$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in $10{\times}10^6$ sperm/ml compared with other sperm concentrations. The rates of blastocysts from the cleaved oocytes (2-4 cell stage) were highest in $1{\times}10^6$sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at $4^{\circ}C$ could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend $1{\times}10^6$sperm/ml concentration for in vitro fertilization of pig oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.9 no.6
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• pp.705-709
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• 1996

A simplified dilutor for cock spermatozoa at ambient temperature was achieved by adjusting the 5% concentration of fructose in isotonic saline. Motility of cock spermatozoa was arrested completely for maximum 6 hrs without affection the survivability of spermatozoa by employing this sugar. To study the effect of high concentration of fructose on fertility, sperm were inseminated with or without fructose at different hrs. Fructose from semen samples was removed by centrifugation. High fertility obtained in the hens inseminated with fructose free sperm (washed). In addition, washed sperm maintained the 85.00% fertility for 6 hrs in winter season ($17-21^{\circ}C$) and 82.67% fertility for 3 hrs in summer season ($31-35^{\circ}C$). Whereas control groups showed 47.33 and 25.33% fertility in winter and summer season respectively. No significant difference was found in percent motility and live counts between the control and washed experimental groups during winter season. However, these measures differed significantly in summer. Washing of cock spermatozoa more than once, high speed centrifugation and more duration for centrifugation proved harmful to fertility. It may be concluded that fructose (5%) can be used as a motility or metabolic inhibitor of spermatozoa for short-term storage of cock semen at ambient temperatures.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.7
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• pp.901-905
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• 2000

An experiment was conducted to investigate the effect of caffeine, cAMP and cattle seminal plasma on preservation of semen at ultra low temperature ($-196{^{\circ}C}$). Each semen sample was divided into four parts equal in volume and sperm concentration; three were treated with caffeine, or cAMP, or cattle seminal plasma (CSP) and the fourth was kept as control. Sperm motility, abnormal spermatozoa, live-dead count and acrosomal damage were studied at different stages of freeze preservation viz.; just after dilution, at $5{^{\circ}C}$, at glycerolisation, before freezing, just after freezing, 24 hours of storage, and one week of storage. Sperm motility (58.39, 61.33, 52.00 and 50.39 per cent), non-eosinophilic spermatozoa (72.55, 69.98, 63.31 and 67.64 per cent), abnormal spermatozoa (5.71, 4.98, 8.04 and 5.66 per cent) and acrosomal damage (13.28, 13.33, 14.80 and 14.65 per cent) were observed in cAMP, caffeine, cattle seminal plasma and control, respectively, at every stage of freeze preservation. From this study it could be concluded that freezability of buffalo semen can be improved through the addition of caffeine followed by cAMP and cattle seminal plasma.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.11
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• pp.1561-1565
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• 2006

The objective of this study was to investigate the anti-oxidative effects of taurine on sperm characteristics for in vitro storage of boar semen. Semen was randomly divided into 10 groups in conical tubes and treated with different concentrations of taurine (25-100 mM) with or without $250{\mu}M$ $H_2O_2$. The percentage of motile spermatozoa in taurine groups after 6 and 9 h were significantly higher at >94% and 87%, respectively, compared to the control group ($85.1{\pm}0.5$ and $72.4{\pm}0.3$, p<0.05). The sperm motility in taurine with $H_2O_2$ after 6 h incubation was slightly decreased compared to the taurine alone treatment, but after 9 and 12 h incubation % sperm motility dropped sharply in taurine with $H_2O_2$ ($75.3{\pm}0.3$ and $69.6{\pm}2.9$, p<0.05). For 3, 9 and 12 h incubation, sperm viability in the control was lower than in taurine groups, irrespective of taurine concentration. In eosin Y and nigrosin staining (ENS), the sperm survival rates (%) for 6 h incubation were significantly higher in 25 mM ($76.0{\pm}0.6$) and 50 mM taurine groups ($78.0{\pm}0.7$), respectively. Sperm survival rates for 9 and 12 h incubation were higher in taurine groups (${\geq}48%$ in 9 h and ${\geq}42%$ in 12 h) compared to controls ($43.0{\pm}2.1$ and $31.0{\pm}0.6$, respectively). In the hyoosmotic swelling test (HOST), sperm membrane integrity was similar to the results of sperm survival. These experiments indicate that supplementation of taurine to the semen extender can increase the sperm characteristics(motility, viability, survival and membrane integrity).