Multiple ejaculates were collected from four male mongrel dogs. The second fraction and the small volume of third fraction from the ejaculates were divided and treated as follows : control; addition of the egg-yolk Tris extender to the semen at $37^{\circ}C$. group I; Removal of seminal plasma, group II; addition of the glycerolated extender at $4^{\circ}C$, group III Removal of seminal plasma and addition of glycerolated extender at $4^{\circ}C$. The semen cooled to $4^{\circ}C$ was equlibrated for 2hrs and preserved in refrigerator at $4^{\circ}C$. The preserved semen was evaluated for kinetics, morphology, motility and thermoresistance daily for 3 days. 1. The kinectics after preserved days 2 and 3 of group I was significantly higher than that of control(p<0.05). 2. There were no significant difference in abnormal morphology of each group between the periods of storage. 3. The motility after preserved day 1 and days 3 of group I was significantly higher than that of others(p<0.05), and the molity after preserved days 2 of group I and III was signficantly higher than that of others(p<0.05). 4. When the molity of preserved semen was measured during incubation at $37^{\circ}C$, the motility of four groups was declined at similar rates. There was no effect of removal of seminal plasma and glycerol addition on thermoresistance.
Highly significant correlation (p < 0.001) was found between body weight and chest circumference of the local Katjang and cross-bred (local Katjang ♀ ${\times}$ German Fawn ♂ bucks under study. Increase in body length, chest circumference, depth of chest and height at withers (p < 0.001) reflected significantly the increase in body weight of the bucks. At the same age the cross-breds were bigger than the indigenous breed. No significant correlation was detected between body weight and scrotal circumference, or the latter with sperm counts in both buck types under study. However, the fluid portion of the semen increased in acidity and volume, the latter being significant (P < 0.01) in the local Katjangs, with increase in scrotal circumference. Although the effects of body condition on buck libido of both groups were not significant, the reaction time taken to mount the teaser females were significantly diminished (p < 0.001) with better body condition, at least in the local Katjangs. The reaction time gad an inverse, though not necessarily significant, relationship with semen characteristics such as volume, pH (in local Katjangs, p < 0.05), concentration, color (in cross-breds, p < 0.05), agglutination and mass movements in both phenotypes.
Eight 2-yr old bulls from Artificial Breeding Center, NLCF were used to determine the effectof collection frequency on semen characteristics and sexual activity. Two successive ejaculates per day were collected by artificial vagina for 4 weeks on weekly or twice a week. Total ejaculate volume included 2nd ejaculates for one time and two time bulls was 6.8ml and 6.0ml, but there was no significant difference between collection intervals. Sperm concentration of one time and two time bulls averaged 0.79$\times$109/ml and 0.89$\times$109/ml, respectively. Total sperm per ejaculate was 5.14$\times$109 for one time bulls and 5.45$\times$10 for two time bulls. Two time bulls had slight more sperm per ml and ejaculate than one time bulls, but there were no significant differences between two group bulls. Sperm motility and semen pH of two time bulls was slightly better than that of one time bulls. In changes of bulk minerals in semen, solium concentration of two time bulls was similar to that of one time bulls. Potassium and calcium was more concentrated in one time bulls than in two time bulls, but these concentrations did not differ significantly. Libido score for two time bulls was higher than that for one time bulls. However, there was no difference between two groups and these scores did not change for 4 weeks in two goups. Total time to 2nd ejaculation was 16.3 sec for one time bulls and 20.5 sec for two time bulls.
In order to protect the spermatozoa against cold shock, hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders for domestic animals. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). The effects of LDL on sperm quality of bull and northern pike (Esox lucius) after freezing-thawing have been reported, but no study has been made to evaluate the effect of LDL on boar sperm motility and other characteristics. The experiment was carried out to investigate the effect of LDL on the freezing of boar sperm in 0.25 ml straws. The aim was to evaluate the quality of boar spermatozoa cryopreserved in the presence of LDL. Motility of semen cryopreserved in LDL was analyzed and compared to semen cryopreserved with Tris-citric acid-glucose (TCG) and Tris-citric acid-fructose (TCF), two basic freezing extenders containing egg yolk. Similarly, acrosome and plasma membrane integrity were also evaluated and compared to semen cryopreserved with TCG and TCF. Analysis of sperm quality after freeze-thaw showed that the motility, acrosome and plasma membrane integrity were improved with LDL in the extender, as compared to the TCG and TCF. The highest post-thaw integrity of acrosome and plasma membrane and motility were obtained with 9% LDL (w/v). Consequently, the optimum LDL concentration in the extender was 9%. It is also suggested that the concentration of LDL addition is important for the effect on boar sperm protection during freezing and thawing. The percentage of motile spermatozoa was significantly higher after freezing in 9% LDL than in TCG and TCF 54.4% versus 30.4% and 30.1% (p<0.05), respectively. The integrity of acrosome and plasma membrane were also significantly higher at 70.3% and 50.5% respectively with semen frozen in 9% LDL extender compared to TCG at 37.8% and 30.3% and TCF at 36.4% and 29.9%, respectively (p<0.05),. In conclusion, we propose that extender containing LDL extracted from hen egg yolk could be used as a cryoprotective media with a better efficiency than TCG and TCF. LDL improved boar semen quality, allowing better spermatozoa motility, acrosome and plasma membrane integrity after the freeze-thaw process. Furthermore, we found out that the extender with 9% LDL concentration significantly enhanced motility, acrosome and plasma membrane integrity of boar sperm after freezing and thawing.
This study was carried out to investigate the general characteristics, such as volume, pH, sperm motility and sperm concentration of the semen collected from Beagle dogs (age $24{\sim}48$ months, weight $10{\sim}15\;kg$) by using the method of digital manipulation of the penis, and the effect of preservation temperature and time on motility of fresh semen. Multiple ejaculates were collected from 4 male Beagles. The average volume, pH, motility and sperm concentration of the second fraction (contained with small volume of the third fraction) per ejaculation were $2.94{\pm}0.24(SD)\;ml$, $6.43{\pm}0.42(SD)$, $97.04{\pm}3.50(SD)%$ and $1.67{\pm}0.23(SD){\times}10^8\;cells/ml$, respectively. Average semen volume per ejaculate, semen pH, sperm motility and sperm concentration of the first fraction from the ejaculate were $1.24{\pm}0.20(SD)\;ml$, $6.03{\pm}0.26(SD)$, $1l.30{\pm}4.02(SD)%$ and $7.25{\pm}1.02(SD){\times}10^5\;cells/ml$. Those of second fraction were $2.52{\pm}0.32(SD)\;ml$, $6.32{\pm}0.31(SD)$, $96.25{\pm}3.52(SD)%$ and $2.35{\pm}0.35(SD){\times}10^8\;cells/ml$. Those of third fraction were $2.71{\pm}0.27\;(SD)\;ml$, $6.52{\pm}0.20(SD)$, $95.65{\pm}2.78(SD)%$ and $5.72{\pm}0.29(SD){\times}10^7\;cells/ml$. Motility of semen was higher at $17^{\circ}C$ preservation temperature than $5^{\circ}C$ or $36^{\circ}C$ during preservation period. When preservation temperature was $17^{\circ}C$, motility was $96.54{\pm}2.05(SD)%$ at 1 h, $90.20{\pm}3.90(SD)%$ at 6 h, $89.05{\pm}2.01(SD)%$ at 12 h, $78.21{\pm}3.50(SD)%$ at 18 h, $45.24{\pm}6.25\;(SD)%$ at 24 h and $30.75{\pm}17.24(SD)%$ at 30 h, respectively.
This study was performed to investigate the freezomg condition especially focused on extender composition to achieve good post-thaw viability and motility of canine sperm. Semen were collected from 6 male dogs which had been proved to be fertile in the past and were treated for freezing. Equex-STM paste was contained in both the 1st(3%) and the 2nd(7%) diluent and the 2nd diluent was added to the 1st diluent following glycerol equilibration for an hour and a half. To investigate the effect of Equex-STM paste in the extender on post-thaw canine sperm characteristics, the post-thaw viability, motility, and HOS(Hypoosmotic swelling) values were evaluated according to the different composition of extender with or without Equex-STM paste, thawing conditions, and different thawing media added to thawed semen. 1. Canine sperm removed from seminal plasma and frozen )n Sweden extender containing Equex showed higher post-thaw viability, motility, and HOS values than those frozen in the extender containing Equex-STM paste with seminal plasma and those frozen in the extender without Equex and seminal plasma. 2. Canine sperm frozen in Sweden extender containing Equex-STM paste with 5% glycerol showed higher post-thaw viability, motility, and HOS values than those frozen with 3%, 8% glycerol or 5% DMSO. 3. The canine semen frozen in Sweden extender with 5% glycerol and Equex-STM paste showed higher viability, motility, and HOS values when thawed at $70^{\circ}C$ for 8 seconds than when thawed at $37.5^{\circ}C$ for 1 min and at $18-20^{\circ}C$ for 5 min. 4. TFC (tris -fructose-citrate) and PB S (phosphate buffered saline) medium added immediately to thawed canine semen brought better viability, motility, and HOS values for the sperm than those semen added with TGC(tris-glucose-citrate) and no medium. These results indicated that Equex-STM paste in Sweden extender for freezing the canine sperm which were removed from seminal plasma brought good post-thaw viability and motility of canine sperm. Also of the freezing conditions of canine sperm with the same extender containing Equex, the concentration of 5% glycerol, the thawing condition at $70^{\circ}C$ for 8 sec, and TFC and PBS medium added to the thawed semen brought better post-thaw viability and motility of canine sperm than the other conditions used in this study.
Curcumin is a major active component of the food flovour tumeric. It has been used for the treatment of many diseases such as inflammatory and infectious diseases, cancer and other disease due to its antioxidant properties. Curcumin is a powerful scavenger of many free radicals such as superoxide anion, hydroxyl radical and nitric oxide. The objective of this study was to investigate the antioxidative effects of curcumin against hydrogen peroxide on semen quality during in vitro storage of boar semen. The sperm treated with different concentration of curcumin (1, 5 and 10 ${\mu}M$) in the presence or absence of hydrogen peroxide (250 ${\mu}M\;H_2O_2$) were incubated for 3, 6 and 9 hr at $37^{\circ}C$ and analyzed sperm characteristics such as motility, membrane integrity (MI), lipid peroxidation (LPO), reactive oxygen species (ROS) and DNA fragmentation (DF). The sperm motility and MI in $H_2O_2$ treated group ($47.8%{\pm}6.8$ and $24.8%{\pm}2.2$) were significantly decreased when compare to curcumin treated group ($79.8%{\pm}2.7$ and $34.6%{\pm}1.0$, respectively) irrespective of incubation periods(p<0.05). The LPO of spermatozoal plasma membrane was measured by thiobarbituric acid (TBA) reactions for malondialdehyde (MDA), MDA level in control ($11.6{\pm}0.6\;nmol/L{\times}10^6$) and curcumin groups ($10.7{\pm}0.3\;nmol/L{\times}10^6$) were lower than those of curcumin plus $H_2O_2$ ($17.1{\pm}0.8\;nmol/L{\times}10^6$) or $H_2O_2$ group ($22.5{\pm}1.9\;nmol/L{\times}10^6$) from 3 to 9 hr incubation periods. The DF by sperm chromatin dispersion (SCD) test and ROS production measured by 2',7'-dichlorofluorescein (DCF) fluorescence intensity were no significantly difference through all experimental groups (p>0.05). Correlation among evaluation methods for sperm quality, motility vs MI and DF vs ROS was positively correlated while motility vs DF and ROS vs LPO were negatively correlated in all treatment groups. These results demonstrate that curcumin can effectively improve the sperm quality during in vitro storage of boar semen through its hydrogen peroxide scavenging mechanism as an antioxidant.
Garole is a prolific, rare, less known and small size Indian sheep breed found in low and humid Sunderban region of West Bengal. Although information on stored Garole ram liquid semen upto 24 h is available, but there is a need to further investigate the short-term and long-term preservability of Garole ram semen for extensive utilization of this valuable germplasm by artificial insemination. The aim of the present study was to apply computer-assisted sperm analysis technique for assessing the motion characteristics of Garole ram semen stored (i) in liquid state at refrigeration temperature for short-term preservation upto 48 h and (ii) in frozen state at $-196^{\circ}C$ for long-term preservation after packaging in mini straws. Short-term preservation had a significant effect on motility (p<0.01) as the motility progressively decreased from 90.1% at 0 h to 85.5% and 73.2% after 24 and 48 h of storage, respectively. Although the decline in rapid moving sperms was also significant (p<0.01) on storage but the decrease was more pronounced at 48 h as compared to 24 h of storage period. Storage of chilled semen had also a significant effect on % linearity (p<0.05), % straightness (p<0.01), sperm velocities (p<0.01), amplitude of lateral head displacement (p<0.01) and beat frequency (pO.Ol) of spermatozoa. The replication had a significant effect for all the variables except average path and straight line velocity. However, the interactions of short-term storage and replication were non-significant for most of the variables except % of medium moving sperms, sperm velocities and beat frequency. On long-term preservation of Garole ram spermatozoa under controlled conditions the mean post-thaw recovery of 70.4 and 71.4% motile spermatozoa was achieved having 48.8 and 48.9% of rapidly motile spermatozoa, respectively in both the replicates. The effect of replication on cryopreservation was significant (p<0.05) on amplitude of lateral head displacement and beat frequency, but there was no significant effect on motility, rapidly motile spermatozoa, linearity, straightness and sperm velocities of frozen-thawed spermatozoa. It can be concluded from these results that an average 70% motility can be achieved on storage of Garole ram semen in chilled liquid state upto 48 h or in liquid nitrogen after freezing under controlled conditions in straws. However, further studies are required to evaluate the fertility of short-term and long-term preserved Garole ram semen for extensive use of this prolific sheep breed.
The purpose of this study was to examine the effects of taurine and vitamin E on sperm characteristics damaged by bromopropane (BP) in pig. We evaluated toxicity of BP on viability, membrane integrity and mitochondrial activity of spermatozoa. 1-BP (0, 2.5, 5.0, 10, and $50{\mu}M$), 2-BP (0, 2.5, 5.0, 10, and $50{\mu}M$), taurine (0, 5.0, 10, and $25{\mu}M$) and vitamin E (0, 50, 100, and $200{\mu}M$) were treated in fresh boar semen for 6 h. 10 and $50{\mu}M$ of 1-BP and 2-BP inhibited sperm viability, membrane integrity and mitochondrial activity in fresh boar semen (P<0.05). $25{\mu}M$ of taurine increased sperm viability and membrane integrity (P<0.05), $100{\mu}M$ of vitamin E enhanced viability and mitochondrial activity of sperm (P<0.05). Finally, $10{\mu}M$ of 1-BP and 2-BP was co-treated with taurine ($25{\mu}M$) and vitamin E ($100{\mu}M$) in the fresh boar semen. The co-treated samples did affected viability, membrane integrity and mitochondrial activity of sperm. In conclusion, taurine and vitamin E can improve and maintain sperm quality in fresh boar semen.
Objectives : This study is to propose the identification keys based on stereoscopic examination of 8 seed herbs in 2 categories (Phyllolobobii Semen (PS) with 4 Astragali Semen (AS), and 2 Lepidii seu Descurainiae Semen (LDS) with 1 Drabae Semen (DS)) which have difficulties in discrimination with visual observation. Methods : We reviewed the description of original plants and their medicinal parts from the literature. The original plants were collected, identified, confirmed as specimens, and compared to the samples distributed in the market. The first identification was made by visual observation, and insufficient points were supplemented by stereoscopic observation. Identification criteria were set by considering morphological characteristics of authentic herbs, percentage of adulterants, and distinction between authentic herbs and adulterants. Results : The original plants of PS and AS could be distinguished by upright or lying form of stem, color of flowers, number of leaflets, and presence of hair of fruits. LDS and DS could be distinguished by leaf arrangement on stem: radical or cauline, whole plants size, leaf division, color of flowers, and shape of fruits. The herbal medicines of PS and AS could be distinguished by seed surface pattern, size, and hardness. LDS and DS could be distinguished by size, shape, viscosity when chewed, and degree of mucous layer formation when soaked in water. Conclusions : This study suggests the identification keys of original plants and herbal medicines. Especially, since fine seed herbs are difficult to distinguish by visual observation, the stereoscope should be applied to the discrimination.
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