• Asian-Australasian Journal of Animal Sciences
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• v.14 no.2
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• pp.184-188
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• 2001

In a summer study during May to July, involving 12 young Murrah buffalo bulls at forty months of average age, the effects of multiple shower vs single shower body cooling and vitamin A, D and E supplementation on the sexual behaviour, semen quality and freezability were investigated. The animals were divided into two groups (6 animals in each group) and housed in a half-walled shed with proper spacing, the feeding management being identical. The bulls in the control group were given a single shower at 1000 h, whereas the experimental bulls were given four showers at 10,12,14 and 16 h. In addition, the experimental bulls were given vitamin A, D and E injections at fifteen day intervals. The sexual behaviour of bulls was observed in terms of reaction time, sexual aggressiveness and ejaculatory thrust. Semen quality of all the bulls was assessed in terms of volume, mass activity, live-dead sperm and sperm concentration, sperm motility and morphology, and acrosomal abnormality. The sexual behaviour did not vary significantly between the groups, whereas semen quality differed significantly for volume, per cent live sperms, total sperms per ejaculate and total live sperm per ejaculate between groups. It can be concluded that sexual behaviour was not influenced by the thermal comfort treatment coupled with periodic vitamin A, D and E injections. But the treatments improved most of the seminal traits in the experimental group of bulls. However, benefit of treatment was not reflected in the freezability traits of the semen.

• 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.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.311-323
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• 1994

This study was conducted to observe seasonal and individual changes in semen characteristics and sperm freezability, and sperm penetration into zona-free hamster eggs in Korean native goats. Buck response and change in semen characteristics to electrical stimulations was evaluated for four seasons throughout 2 years and percentage of motile sperm and normal apical ridge acrosome was investigated after equilibration and thawing for 4 seasons with 5 bucks. Sperm penetration rate was evaluated for 4 bucks. 1. Probe insertion at depth of 7cm and repeated stimulation for 3 sec was more effective(P<0.05) in buck response and semen collection than those of other conditions. 2. Semen characteristics from electrojaculation was signficantly(P<0.005) higher in spring and fall for semen volume, in spring and summer for sperm concentration and in fall for sperm motility than those in other seasons, respectively. However, there were no differences in total sperm among seasons. 3. Buck response to electrical stimulation showed significant difference(P<0.05) among individuals in all 3 seasons except winter. Significant individual difference in semen volume was only in spring and summer, but there was no indivudual difference in sperm concentration and total sperm in all season. 4. Washing of semen before freezing treatment was greatly(P<0.05) beneficial to sperm motility after thawing, no matter whether ejaculates exhibit egg yolk coagulation or not. 5. Sperm motility after glycerol equilibration was significantly(P<0.05) low in summer semen and motility after thawing was greatly(P<0.05) higher in winter semen than in other seasons. Freezability of unwashed sperm was significantly difference among bucks, but a yearly freezability of washed sperm after chilling and thawing were no differences among bucks and percentage of normal apical ridge acrosome were not different among seasons and bucks. 6. There was no significant difference in sperm motility after thawing between egg yolk levels in summer, although 20% level gave more higher motility than 5% level. 7. In summer, 3.2% glycerol and 3-h equilibration gave greatest percentage(P<0.05) of sperm motility and normal apical ridge acrosome after thawing. 8. Sperm penetration rate into zona-free hamster eggs was not different between bucks and seasons. Overall, it is concluded that to obtain maximum sperm output and successive semen freezing by electrojaculation method, buck selection with good response in all season could be basically considered and that seasonal effect on sperm freezability was more greater than that of individual bucks.

• 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.36 no.10
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• pp.1530-1535
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• 2023

Objective: Semen cryopreservation result in decreased sperm parameters and fertilization ability. Sericin exhibits antioxidant activity by reducing lipid peroxidation resulting from free radicals, which can potentially improve cryopreservation outcomes. The present study aimed to examine the efficacy of various sericin concentrations supplemented with a rooster semen-freezing extender on post-thaw semen quality and fertilizing ability of sperm after cryopreservation. Methods: Semen samples were collected from 40 roosters (5 reps), then were pooled, and divided into four groups by the levels of sericin supplementation (0%, 0.25%, 0.50%, and 0.75%) in a freezing extender. Semen suspensions were loaded in medium straw (0.5 mL) and cryopreserved with the traditional liquid nitrogen vapor method. Post-thawed semen was evaluated for sperm motility, sperm viability, and lipid peroxidation. Also, the fertility test was determined. Results: The results showed that supplementation of the freezing extender with 0.50% to 0.75% sericin resulted in greater total motility and progressive motility and lower malondialdehyde levels than the other groups after cryopreservation (p<0.05). However, the viability of 0.75% decreased compared with the value of 0.50% sericin supplementation (p<0.05). Moreover, the fertility and hatchability of total eggs were significantly higher in the 0.50% sericin group than in the other groups (p<0.05). Conclusion: In conclusion, 0.50% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved rooster semen.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.147-154
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• 2012

This study was designed to determine whether low-density lipoproteins (LDL) from egg yolk and taurine, hypotaurine and trehalose as antioxidant in extender improve the freezability and fertility of Korean Jeju Black Bull semen. The semen was cryopreserved with tris egg yolk extenders containing 7% glycerol and treated 4% LDL, 20 mM taurine, hypotaurine and trehalose. Frozen-thawed sperm were evaluated motility, viability, membrane, and acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender only as control. Frozen-thawed semen evaluation cleary indicated that the addition of LDL and LDL-antioxidants (taurine, hypotaurine and trehalose) combination were significantly improved (p<0.05) the viability (%; with staining test using eosin-Y) compared to control spermatozoa. Also, in membrane integrity (%; with supravital hypo-osmotic swelling test), not only LDL-antioxiants combination but also LDL were significantly increased (p<0.05) the swelled sperm using HOST compared to control. Sperm acrosome integrity state was classified by CTC (chlortetracycline) staining test. F pattern was significantly increased in LDL-antioxidant combination than control (p<0.05) and B pattern was not significantly differences among all treatments and control. However, AR pattern was significantly decreased in LDL-antioxidants combination than control (p<0.05). Pronucleus formation and sperm penetration index (SFI) were significantly increased in LDL and LDL-antioxidants combination than control (p<0.05). Especially, LDL-taurine significantly improved pronucleus fomation and SFI than LDL (p<0.05). It was concluded that LDL and LDL-antioxidants in extender improved the freezability and fertility of Korean Jeju Black bull spermatozoa.

• 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.3 no.1
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• pp.30-35
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• 1979

A, B and C dilutors were used to make Ka (A plus B (1 : 1)) and Na (B plus C(1 : 1)) dilutors in this experiment. Three aliqots of semen were respectivly diluted 1 : 1 and 1 : 2 (semen: dilutor) with Ka, Na and C dilutors and stored at 5$^{\circ}C$ for 7 days in order to study their livability during storage. Fertility was checked for the diluted semen with Ka, Na and C dilutors. Whole semen and extended semen with Na dilutos with and without DMSO were cold shocked at various temperatures for 10 min. Effects of different 1st and 2nd dilution with A, B, C and Na dilutors and of vessels on freezability of spermatozoa were investigtigated. 1. Extended semen 1 : 2 with Na and C dilutors showed highest live sperm index during storage for 7 days at 5$^{\circ}C$. 2. The components of Na dilutor per 100$m\ell$ were skim milk 2.5g, trisaminomethane 0.54g, citric acid 0.265g, glucose 2.835g, fructose 1.5g, sodium lauryl sulfate, 0.08g, penicillin 0.06g, streptomycin 0.075g, and egg yolk 10$m\ell$. 3. Fertility of diluted semen was higher than that of whole semen. Ka dilutor showed higher fertility than Na and C dilutors, and there was no difference in the fertility between Na and C dilutors. 4. Na dilutor with DMSO showed slightly higher livability than Na dilutor without DMSO during storage for 7 days at 5$^{\circ}C$. 5. Cold shock at 1$0^{\circ}C$ for 10 min. decreased greatly the sperm livalility of whole semen but not of extended semen with Na dilutor. Addition of DMSO to Na dilutor has no effect in prevention of cold shock. 6. The extended semen with C. C dilutor (1st and 2nd dilution with C and C dilutor) showed higher post-thawing sperm livability than A.A and Na. B dilutors. Na. B dilution shwed higher post-thawing sperm livability than A.A dilution. There was no difference in the post-thawing livability between semen in 1$m\ell$ straw and 10$m\ell$ aluminium package.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.199-206
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• 2012

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. This study was designed to determine effect of glycerol and ethylene glycol as cryoprotectant in extender on improve the freezability of Jeju horse semen. The semen was cryopreserved with glucose-EDTA extender containing each 5% glycerol, 5% ethylene glycol, 8% glycerol or 8% ethylene glycol, respectively. Post-thawed sperm were evaluated motility, viability, Membrane integrity and acrosome integrity. Post-thawed sperm motility were not significantly differences among treatments. However, sperm viability were significantly higher (p<0.05) in 8% glycerol ($39.85%{\pm}11.41$) than in 5% glycerol treatment ($18.08%{\pm}1.61$). In membrane integrity, swelling sperm ratio was significantly higher (p<0.05) in 8% glycerol ($34.12%{\pm}11.02$) than other treatments. In the percentage of capacitated sperm assessed by CTC staining, F pattern was significantly higher in 8% ethylene glycol than 5% glycerol and 5% ethylene glycol (p<0.05). B pattern ratio was significantly increased in 5% ethylene glycol compared with 8% glcerol and 8% ethylene glycol (p<0.05). Moreover, 8% ethylene glycol treatment was significantly decreased AR pattern ratio compared with other treatments (p<0.05). It is concluded that treatment of 8% glycerol was improved the sperm viability and 8% ethylene glycol was improved the sperm ascrosome integrity after thawing. However, they were not significantly difference between 8% glycerol and 8% ethylene glycol on post-thawed sperm viability. Therefore, 8% ethylene glycol was more effective sperm cryoprotectant than 8% glycerol in Jeju Horse.