A total of 18 ejaculates were collected, once per week, from six fertile stallions for three consecutive weeks in October and November, to compare motility over time between extenders using four semen processing treat...A total of 18 ejaculates were collected, once per week, from six fertile stallions for three consecutive weeks in October and November, to compare motility over time between extenders using four semen processing treatments. Four total aliquots of semen were used. Two aliquots of each semen collection were extended in either INRA96 or an experimental proprietary milk-based extender Walworth （WW） extender, and each was designed for multi-day storage of fresh chilled semen. Each aliquot was divided again, and either centrifuged at 600 μg for 10 min without cushion, or not centrifuged and extended to a final concentration of 25 × 10^6 spermatozoa/mL. The treatments evaluated were INRA96 without centrifugation （INRA-NC） or with centrifugation （INRA-C）, and Walworth extender without centrifugation （WW-NC） or with centrifugation （WW-C）. Total and progressive motility were measured using Sperm Vision~ CASA at 0, 24, 48 and 72 h post-collection. Samples were stored at 4 ℃. No differences were found between extenders in progressive （P = 0.13） or total motility （P = 0.14） over the four different time points without centrifugation. However, ejaculates processed in INRA-C group had the greater total and progressive motility （P 〈 0.05） over the four time points than ejaculates in the WW-C group. It was found that centrifugation and re-suspension of stallion semen in INRA96 improved the longevity of fresh chilled semen. However, when not using centrifugation, the Walworth extender proved to be as effective for maintaining spermatozoa motility across all time points as 1NRA96 , and may be an alternative for use in the equine breeding industry.展开更多
The present study evaluated the ef fects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm mot...The present study evaluated the ef fects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm motility and the activities of superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GPx), glutathione reductase(Gr), and lipid peroxidation(measured via malondialdehyde(MDA) content) were determined after the milt was stored at 4°C for 12 h, cryopreserved without cryoprotectant in 12% propylene glycol(PG), cryopreserved in 12% PG+0.1 mol/L trehalose, or cryopreserved in 12% PG spermatozoa but centrifuged to decant the supernatant prior to cryopreservation(only sperm cells were cryopreserved). After chilled storage or cryopreservation, the SOD, CAT and GPx activities were reduced in sperm cells and increased in seminal plasma in almost all treatments; sperm motility parameters were also decreased. However, the addition of trehalose into the cryoprotectant could signifi cantly improve the postthaw sperm quality as revealed by the sperm average path velocity. This improvement might be attributed to the function of trehalose in scavenging reactive oxygen species. Chilled storage and cryopreservation had signifi cant eff ects on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod.展开更多
Objective: To evaluate penetration capacity of human sperm preserved in electrolyte-free (EF) solution at 4 ℃.Methods: The motility, acrosomal status penetration rate and fertility index of human sperm were assessed ...Objective: To evaluate penetration capacity of human sperm preserved in electrolyte-free (EF) solution at 4 ℃.Methods: The motility, acrosomal status penetration rate and fertility index of human sperm were assessed before and after cold-preservation in EF solution, respectively.Results: The motility of human sperm cold-preserved in EF solution for 1 week was significantly higher than that of human sperm cold-preserved in modified human tubal fluid (mHTF) (43.4%±7.9% vs 9.5%±2.5%, P<0.01 ).Although acrosomal status of human sperm cold-preserved in the EF solution before reinitiation was not different from those of the fresh sperm (capacitated sperm: 7.6%±1.8% vs 6.4±1.8%; acrosome-reacted sperm: 3.0%±1.7% vs 2.4±1.1%, P>0.05), the percentage of capacitated and acrosome-reacted sperm in the EF solution significantly increased after reinitiation (capacitated sperm: 16.0%±2.3% vs 7.6±1.8%, acrosome-reacted sperm: 9.4%±2.1% vs 3.0%±1.7%, P<0.01).The penetration rate and fertility index of cool-preserved human sperm in the EF solution were comparable with those of fresh sperm (48.1% vs 50.9%; 1.38±0.16 vs 1.29±0.13, respectively, P>0.05).Conclusion: Cold-preservation did not induce capacitation and acrosome reaction of human sperm in the EF solution, but human sperm cold-preserved in the EF solution for 1 week possesses as much penetration capacity as fresh sperm.展开更多
This study aimed to identify and characterize the sperm subpopulations existing in water buffalo semen using a computer assisted sperm analyzer （CASA）, as well as assess the effects of cryopreservation on the sperm ...This study aimed to identify and characterize the sperm subpopulations existing in water buffalo semen using a computer assisted sperm analyzer （CASA）, as well as assess the effects of cryopreservation on the sperm subpopulation structure and evaluate bull variability. The semen of eight Bulgarian Murrah bulls was collected by four times in an interval of one week each. The semen was cryopreserved following a standard protocol and sperm kinematics was assessed. Clustering methods were applied to individual sperms, forming two significantly different （P 〈 0.05） subpopulations （P1 and P2）. Subpopulation P1 represents those spermatozoa that moved most rapidly and progressively （46.29%）, and subpopulation P2 includes spermatozoa with relatively low velocity or poorly motile but with high progressiveness （53.41%）. There was a decline on the population of P1 sperms from fresh （52.52%）, pre-freeze （45.73%） to post-thaw （35.17%） stages and significant difference on the sperm kinematics between P1 and P2. A significant decline in the values of distance, velocity and amplitude of lateral head （ALH） parameters were observed at post-thaw stage, while an increase was observed on trajectory and beat cross frequency （BCF） kinematics. Values of sperm kinematics were also significantly different （P 〈 0.05） among all bulls. The frequency distribution of spermatozoa on both subpopulations P1 and P2 was quite similar for all bulls in pre-freeze and post-thaw stages, but with significant （P 〈 0.05） variability on fresh stage. Bulls with the highest maintained frequency of P1 sperms are denoted as good freezer bulls. In sum, kinematic characterization of water buffalo sperm and clustering into subpopulation enable to identify bulls that are more resistant to cryopreservation and production of quality semen for genetic propagation.展开更多
The experiment was designed to study the preparation, cryopreservation and culture of calf spermatogonial stem cells in order to provide some data for theoretical research and practical use of calf spermatogonial stem...The experiment was designed to study the preparation, cryopreservation and culture of calf spermatogonial stem cells in order to provide some data for theoretical research and practical use of calf spermatogonial stem cells. As for enzymolysis of testis tissue, two digestive method A and B. The cryopreservation cooling procedure was that spermatogonial stem cells was equilibrated at 4℃ for lh, at -20℃ for lh, at -40℃ for 2h, at -80℃ overnight and then stored in liquid nitrogen. The different concentration of three eryoprotectants were compared. The results indicated that both method A and method B achieved the same high motility rates of cells with method B needing time longer, calf spermatogonial stem cells could be well cryopreserved by the stage cooling procedure. Satisfactory cryopreservative results were gotten by adding 10% DMSO or 10% EG to cryopreservative medium, the former was better and adding sucrose could improved cryopreservative effect. The culture behaviors of spermatogonial stem cells kept the same before and after cryopreservation. It was concluded that an effective method was verified for preparation, cryopreservation and culture of new calf spermatogonial stem cells.展开更多
Cryopreservation of spermatozoa is a strategy that has been used to conserve the sperm of animal species and animal strains that are valuable for biomedical research. A simple method for preserving spermatozoa after a...Cryopreservation of spermatozoa is a strategy that has been used to conserve the sperm of animal species and animal strains that are valuable for biomedical research. A simple method for preserving spermatozoa after application of intracytoplasmic sperm injection （ICSI） is much needed. It has been shown previously that spermatozoa frozen at -20~C can activate oocytes and support full-term embryo development. However, epigenetic reprogramming could be affected by the environment and by the in vitro manipulation of gametes. Here, we investigated embryo epigenetic reprogramming including DNA methylation and histone modification, in embryos derived from sperm preserved at -20~C without cryoprotectants. The results showed that alt- hough both fertilization and embryo developmental competence were decreased, the dynamic epigenetic reprogramming of embryos derived from frozen sperm was similar to the reprogramrning of embryos derived from fresh sperm. The results re- ported in this study indicate that sperm frozen without cryoprotectant is epigenetically safe for ICSI.展开更多
文摘A total of 18 ejaculates were collected, once per week, from six fertile stallions for three consecutive weeks in October and November, to compare motility over time between extenders using four semen processing treatments. Four total aliquots of semen were used. Two aliquots of each semen collection were extended in either INRA96 or an experimental proprietary milk-based extender Walworth （WW） extender, and each was designed for multi-day storage of fresh chilled semen. Each aliquot was divided again, and either centrifuged at 600 μg for 10 min without cushion, or not centrifuged and extended to a final concentration of 25 × 10^6 spermatozoa/mL. The treatments evaluated were INRA96 without centrifugation （INRA-NC） or with centrifugation （INRA-C）, and Walworth extender without centrifugation （WW-NC） or with centrifugation （WW-C）. Total and progressive motility were measured using Sperm Vision~ CASA at 0, 24, 48 and 72 h post-collection. Samples were stored at 4 ℃. No differences were found between extenders in progressive （P = 0.13） or total motility （P = 0.14） over the four different time points without centrifugation. However, ejaculates processed in INRA-C group had the greater total and progressive motility （P 〈 0.05） over the four time points than ejaculates in the WW-C group. It was found that centrifugation and re-suspension of stallion semen in INRA96 improved the longevity of fresh chilled semen. However, when not using centrifugation, the Walworth extender proved to be as effective for maintaining spermatozoa motility across all time points as 1NRA96 , and may be an alternative for use in the equine breeding industry.
基金Supported by the National Natural Science Foundation of China(Nos.31072212,41076100)the CAS Scientific and Technological Innovation Program for Cross and Cooperative Team,Marine Economy Innovative Development Project(No.12PYY001SF08)the National Key Basic Program of Science and Technology Platforms of Aquaculture Stock Resources,Shandong Technology Development Project(No.2013GHY11509)
文摘The present study evaluated the ef fects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm motility and the activities of superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GPx), glutathione reductase(Gr), and lipid peroxidation(measured via malondialdehyde(MDA) content) were determined after the milt was stored at 4°C for 12 h, cryopreserved without cryoprotectant in 12% propylene glycol(PG), cryopreserved in 12% PG+0.1 mol/L trehalose, or cryopreserved in 12% PG spermatozoa but centrifuged to decant the supernatant prior to cryopreservation(only sperm cells were cryopreserved). After chilled storage or cryopreservation, the SOD, CAT and GPx activities were reduced in sperm cells and increased in seminal plasma in almost all treatments; sperm motility parameters were also decreased. However, the addition of trehalose into the cryoprotectant could signifi cantly improve the postthaw sperm quality as revealed by the sperm average path velocity. This improvement might be attributed to the function of trehalose in scavenging reactive oxygen species. Chilled storage and cryopreservation had signifi cant eff ects on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod.
文摘Objective: To evaluate penetration capacity of human sperm preserved in electrolyte-free (EF) solution at 4 ℃.Methods: The motility, acrosomal status penetration rate and fertility index of human sperm were assessed before and after cold-preservation in EF solution, respectively.Results: The motility of human sperm cold-preserved in EF solution for 1 week was significantly higher than that of human sperm cold-preserved in modified human tubal fluid (mHTF) (43.4%±7.9% vs 9.5%±2.5%, P<0.01 ).Although acrosomal status of human sperm cold-preserved in the EF solution before reinitiation was not different from those of the fresh sperm (capacitated sperm: 7.6%±1.8% vs 6.4±1.8%; acrosome-reacted sperm: 3.0%±1.7% vs 2.4±1.1%, P>0.05), the percentage of capacitated and acrosome-reacted sperm in the EF solution significantly increased after reinitiation (capacitated sperm: 16.0%±2.3% vs 7.6±1.8%, acrosome-reacted sperm: 9.4%±2.1% vs 3.0%±1.7%, P<0.01).The penetration rate and fertility index of cool-preserved human sperm in the EF solution were comparable with those of fresh sperm (48.1% vs 50.9%; 1.38±0.16 vs 1.29±0.13, respectively, P>0.05).Conclusion: Cold-preservation did not induce capacitation and acrosome reaction of human sperm in the EF solution, but human sperm cold-preserved in the EF solution for 1 week possesses as much penetration capacity as fresh sperm.
文摘This study aimed to identify and characterize the sperm subpopulations existing in water buffalo semen using a computer assisted sperm analyzer （CASA）, as well as assess the effects of cryopreservation on the sperm subpopulation structure and evaluate bull variability. The semen of eight Bulgarian Murrah bulls was collected by four times in an interval of one week each. The semen was cryopreserved following a standard protocol and sperm kinematics was assessed. Clustering methods were applied to individual sperms, forming two significantly different （P 〈 0.05） subpopulations （P1 and P2）. Subpopulation P1 represents those spermatozoa that moved most rapidly and progressively （46.29%）, and subpopulation P2 includes spermatozoa with relatively low velocity or poorly motile but with high progressiveness （53.41%）. There was a decline on the population of P1 sperms from fresh （52.52%）, pre-freeze （45.73%） to post-thaw （35.17%） stages and significant difference on the sperm kinematics between P1 and P2. A significant decline in the values of distance, velocity and amplitude of lateral head （ALH） parameters were observed at post-thaw stage, while an increase was observed on trajectory and beat cross frequency （BCF） kinematics. Values of sperm kinematics were also significantly different （P 〈 0.05） among all bulls. The frequency distribution of spermatozoa on both subpopulations P1 and P2 was quite similar for all bulls in pre-freeze and post-thaw stages, but with significant （P 〈 0.05） variability on fresh stage. Bulls with the highest maintained frequency of P1 sperms are denoted as good freezer bulls. In sum, kinematic characterization of water buffalo sperm and clustering into subpopulation enable to identify bulls that are more resistant to cryopreservation and production of quality semen for genetic propagation.
文摘The experiment was designed to study the preparation, cryopreservation and culture of calf spermatogonial stem cells in order to provide some data for theoretical research and practical use of calf spermatogonial stem cells. As for enzymolysis of testis tissue, two digestive method A and B. The cryopreservation cooling procedure was that spermatogonial stem cells was equilibrated at 4℃ for lh, at -20℃ for lh, at -40℃ for 2h, at -80℃ overnight and then stored in liquid nitrogen. The different concentration of three eryoprotectants were compared. The results indicated that both method A and method B achieved the same high motility rates of cells with method B needing time longer, calf spermatogonial stem cells could be well cryopreserved by the stage cooling procedure. Satisfactory cryopreservative results were gotten by adding 10% DMSO or 10% EG to cryopreservative medium, the former was better and adding sucrose could improved cryopreservative effect. The culture behaviors of spermatogonial stem cells kept the same before and after cryopreservation. It was concluded that an effective method was verified for preparation, cryopreservation and culture of new calf spermatogonial stem cells.
基金supported by the National Basic Research Program of China (Grant No. 2011CB944501)
文摘Cryopreservation of spermatozoa is a strategy that has been used to conserve the sperm of animal species and animal strains that are valuable for biomedical research. A simple method for preserving spermatozoa after application of intracytoplasmic sperm injection （ICSI） is much needed. It has been shown previously that spermatozoa frozen at -20~C can activate oocytes and support full-term embryo development. However, epigenetic reprogramming could be affected by the environment and by the in vitro manipulation of gametes. Here, we investigated embryo epigenetic reprogramming including DNA methylation and histone modification, in embryos derived from sperm preserved at -20~C without cryoprotectants. The results showed that alt- hough both fertilization and embryo developmental competence were decreased, the dynamic epigenetic reprogramming of embryos derived from frozen sperm was similar to the reprogramrning of embryos derived from fresh sperm. The results re- ported in this study indicate that sperm frozen without cryoprotectant is epigenetically safe for ICSI.
