• Korean Journal of Animal Reproduction
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• v.16 no.1
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• pp.47-53
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• 1992

This study was carried out to investage the effect of cryoprotectant concentration and equilibration time on volume change and in vitro development of intact and bisected mouse embryos by rapid freezing. When compacted morulae were rapidly frozen in 3.0 to 4.0 glycerol or DMSO with 0.25M sucrose solution, the superior(P<0.05) post-thaw survival rate was obtained at the glycerol concentration of 4.0M(89.4%) than 3.0M(71.4%) or 5.0M(42.4%), but at the DMSO concentration of 3.0M(84.5%) than 4.0M(51.1%) or 5.0M(0.0%). The optimal equilibraton time for rapid freezing of ZP-free or bisected morulae in 4.0M glycerol with 0.25M sucrose was found tobe 3 minutes. The minimal volume of compacted morulaewhich corresponded with 61 to 62% of pre-equilibrated embryo volume was obtained from equilibration for 3 minutes in both 3.0 and 4.0M glycerol solutions with 0.25M sucrose.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.151-159
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• 1997

In vitro fertilization(IVF) derived morula and blastocyst embryos were bisected by a simple method and cultured in vitro without zona pellucida And also bisected embryos were frozen-thawed and cultured in vitro) to evaluate the survival rate. The results obtained were as follows : The average number of grade I or II immature follicular oocytes recovered by slicing method per ovary was 11.9 from 142 ovaries. Following in vitro fertilization, the rates of cleavage and in vitro development to morula and blatocyst were 61.7 and 32.2% respectively. The successful bisection rate of IVE embryos was 67.51%, and the embryos of blastocyst stage were bisected successfully at significantly(P

• Korean Journal of Animal Reproduction
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• v.13 no.1
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• pp.18-25
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• 1989

This study was carried out to investigate the general semen characteristics of the Korean native goat and the effect of temperature, incubation time, dilution rate, freezing rate and glycerol concentration on motility and NAR (normal apical ridge) acrosome of fresh and frozen Korean native goat spermatozoa. 1. Average semen volume per ejaculate, motility, concentration and pH of fresh Korean native goat spermatozoa were 0.19${\pm}$0.09 ml, 94.5${\pm}$0.47%, 26.17${\times}$108${\pm}$1.68/ml and 6.63${\pm}$0.18, respectively. 2. Motility and NAR acrosome of fresh spermatozoa during incubation were higher at 22$^{\circ}C$ than at 5$^{\circ}C$ or 37$^{\circ}C$(P<.01). 3. Motility and NAR acrosome of spermatozoa diluted 1:4 during incubation were higher at 22$^{\circ}C$ than at 5$^{\circ}C$ or 37$^{\circ}C$(P<.01). 4. Motility and NAR acrosome of spermatozoa during incubation were higher for samples diluted 1:1, 1:2, or 1:4 than for samples diluted 1:6(P<.01). 5. Motility and NAR acrosome of post-thaw spermatozoa were higher at freezing rate of 12$^{\circ}C$/min than at freezing rate of 1$^{\circ}C$/min or 24$^{\circ}C$/min when glycerol concentration was 9%(P<.01).

• Journal of Veterinary Clinics
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• v.4 no.2
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• pp.449-455
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• 1987

Quick freezing of bovine embryos was attempted after they were predehydrated at room temperature. Combined solutions of 2M glycerol or 2M ethylene glycol in the presence of either 0.5 or 1.0M sucrose in phosphated buffered saline+20% calf serum were compared. The quick freezing method in which embryos were directly transferred in liquid nitrogen vapor for 2 minutes at - l70$^{\circ}C$ before being plunged into liquid nitrogen was used. Post-thaw survival rates in 2M glycerol and 2M ethylene glycol were high with 0.5 M (55.6% and 53.3%) versus 1.0M(38.1% and 31.6%) sucrose(P < 0.05). But survival rates with 2M glycerol and 2M ethylene glycol were not significantly different. Transfer thawed embryos frozen with 2M glycerol and 2M ethylene glycol by 0.5M sucrose resulted in birthrates of 40.9% and 40.0%, respectively compared to 26.3% and 27.2%, respectively, for 1.0 M sucrose(p<0.05). This was 56.0% for fresh control.

• Development and Reproduction
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• v.5 no.1
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• pp.35-38
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• 2001

This study was peformed to find out the optimum larval stage among trochophore, early Dshaped larva, late D-shaped larva, early umbo and late umbo stages for cryopreservation of surf clam, Spisula sachalinensis larvae. Dimethyl sulfoxide (DMSO)and ethylene glycol were used as cryoprotectant, The larvae were immersed to cryoprotectants for 10 minutes and thereafter, cryopreserved in liquid nitrogen. Survival rates of trochophores frozen-thawed in 2.0 M dimethyl sulfoxide and 2.0 M ethylene glycol were the highest as 97.4% and 85.0%, respectively and post-thaw survival rates were decreased with the larval growth.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.217-224
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• 1992

These studies were carried out ot investigate on the transferred embryo development following ultrarapid frozen for 8-cell and morula of in vitro fertilization mouse embryos. The post-thaw embryo survival was evaluated and compared by cell stage of embryos and by equilibration time before ultrarapid freezing. The results obatined were summerized as follows: 1. The effects of equilibration time of 3 vs. 6 minutes before ultrarapid freezing and after thawing on the morphological survival and the viability of 8-cell and morulas embryos were not significant. 2. When the ultrarapid frozen-thawed 32 eight-cell and 33 morula embryos, and 30 fresh blastocysts were transferred to pseudopregnant recipient mice, the number of normal offsprings produced were 9(28.1%), 14(42.4%) and 18(60.0%), respectively. From the above resutls, it was concluded that the optimal conditions of pH osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOsm and 120 min., respectively,a nd somewhat high conception rate might be resulted from transfer of frozen embryos of morula stage and fresh embryos of blastocyst stage.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.2
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• pp.87-92
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• 2022

Objective: The aim of this study was to determine the effects of melatonin and selenium in freezing extenders on frozen-thawed rat sperm. Methods: Semen samples were collected from 20 adult male Wistar albino rats. Following dilution, the samples were divided into six groups: four cryopreserved groups with 1 mM and 0.5 mM melatonin and selenium supplements, and two fresh and cryopreserved control groups. The rapid freezing technique was used to freeze the samples. Flow cytometry was used to assess plasma membrane integrity, mitochondrial membrane potential, and DNA damage, while computer-assisted sperm analysis was used to assess motility. Results: Total motility was higher in the 1 mM melatonin supplementation group than in the cryopreserved control group (mean±standard error of the mean, 69.89±3.05 vs. 59.21±1.31; p≤0.05). The group with 1 mM selenium had the highest plasma membrane integrity (42.35%±1.01%). The cryopreserved group with 0.5 mM selenium had the highest mitochondrial membrane potential, whereas the cryopreserved control group had the lowest (45.92%±4.53% and 39.45%±3.52%, respectively). Conclusion: Cryopreservation of rat semen supplemented with 1 mM melatonin increased sperm motility after freeze-thawing, while supplementation with 0.5 mM selenium increased mitochondrial activity.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.2
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• pp.110-116
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• 2022

Objective: Sperm vitrification leads to the production of reactive oxygen species (ROS) that can damage the functional parameters of sperm. The present study aimed to investigate the antioxidant effect of Nigella sativa extract on motility, plasma membrane function, mitochondrial membrane potential (MMP), DNA damage, and intracellular ROS production. Methods: A total of 20 sperm samples were used. Samples were divided into six experimental groups, including groups with aqueous extract from N. sativa seeds at concentrations of 1% to 6%, a cryopreserved control group, and a fresh control group. Results: Statistical analysis showed significantly higher total sperm motility at concentrations of 3% to 6% than in the vitrified semen control group. Additionally, progressive motility and all motion characteristics at all concentrations were significantly higher than in the vitrified semen control group. The presence of N. sativa seed extract also improved the quality of the sperm parameters assayed in all experimental groups (1%-6%; intracellular ROS production, DNA damage, MMP, and sperm membrane function) compared to the control group. Conclusion: Higher concentrations of N. sativa led to improvements in all sperm parameters and sperm quality. These findings indicate that N. sativa seed extract is effective for improving the quality of sperm after vitrification.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.7
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• pp.1071-1086
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• 2003

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.157-162
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• 2005

To find out the possible inefficiencies of artificial inseminators at rectovaginal insemination in cows, inseminators' skill were evaluated by controlling the semen thawing procedure adopted and by using the technique of dye deposition in the genital tract of slaughtered cows. This was followed by refreshment training for the inseminators. Thirty seven artificial insemination technicians regularly working in the government, cooperative and NGO (Non Government Organization) artificial insemination programmes at different places of Bangladesh were included in the study. Individual technicians were asked to thaw a semen straw and deposit dye in the genital tract of slaughtered cows following the procedures they would have adopted in their actual practices of insemination. The time and water temperature adopted by technicians were recorded and genital tract after sham artificial insemination was dissected to determine the site of dye deposition. Then, the inseminators took part in a three days intensive training program. The training program was ended up with the same tests for thawing frozen semen straw and dye deposition in the genital tract of slaughtered cows. At pre training evaluation, only $25\%\;and\;72\%\;(n=36)$ inseminators adopted co..ect thawing time and temperature, respectively. At post training evaluation, all inseminators thawed semen straws for proper time and temperature. At pretraining evaluation, $21(57\%),\;11 (30\%)\;and\;3(8\%)$ inseminators deposited dye at the body of uterus, in the vagina or in cervix, and into the horn of uterus, respectively. In $2(5\%)$ cases dye did not pass into the genital tract, instead back flowed through the space between the barrel of insemination gun and sheath. At post training evaluation, all inseminators successfully deposited dye in the body of uterus. Frequent evaluation of inseminators' skill and subsequent training would help improvement of the artificial insemination technicians' skill.