• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.113-120
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• 2022

Sperm cryopreservation is a fundamental process for the long-term conservation of livestock genetic resources. Yet, the packaging method has been shown, among other factors, to affect the frozen-thawed (FT) sperm quality. This study aimed to develop a new mini-straw for sperm cryopreservation. In addition, the kinematic patterns, viability, acrosome integrity, and mitochondrial membrane potential (MMP) of boar spermatozoa frozen in the developed 0.25 mL straw, 0.25 mL (minitube, Germany), or 0.5 mL (IMV technologies, France) straws were assessed. Post-thaw kinematic parameters were not different (experiment 1: total motility (33.89%, 32.42%), progressive motility (19.13%, 19.09%), curvilinear velocity (42.32, 42.86), and average path velocity (33.40, 33.62) for minitube and the developed straws, respectively. Further, the viability (38.56%, 34.03%), acrosome integrity (53.38%, 48.88%), MMP (42.32%, 36.71%) of spermatozoa frozen using both straw were not differ statistically (p > 0.05). In experiment two, the quality parameters for semen frozen in the developed straw were compared with the 0.5 mL IMV straw. The total motility (41.26%, 39.1%), progressive motility (24.62%, 23.25%), curvilinear velocity (46.44, 48.25), and average path velocity (37.98, 39.12), respectively, for IMV and the developed straw, did not differ statistically. Additionally, there was no significant difference in the viability (39.60%, 33.17%), acrosome integrity (46.23%, 43.23%), and MMP (39.66, 32.51) for IMV and the developed straw, respectively. These results validate the safety and efficiency of the developed straw and highlight its great potential for clinical application. Moreover, both 0.25 mL and 0.5 mL straws fit the present protocol for cryopreservation of boar spermatozoa.

• Journal of Sericultural and Entomological Science
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• v.34 no.1
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• pp.1-7
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• 1992

For a long-term preservation of silkworm stocks by frozen gonad storage, fundamental topics such as freezing rate and transplanting stage of the gonad, proper cryoprotectant, and super-cooling temperature and freezing point of the freezing medium were examined and following results were obtained. Proper method to anesthetize the ovary-recipient silkworm was to dip the animal to cold water for 10 minutes, and the ovary taken from the 4th instar larvae was more suitable for freezing-preservation than that from the 5th. Concerning the cryoprotectant, glycerol and DMSO were effective to prevent cryoinjury of the ovary, but sorbitol was not. The supercooling temperature and freezing point of the medium to freeze the ovary and testes were checked, and consulting with the results desirable cooling rate was confirmed. On the desirable conditions of transplanting methods, freezing rate and cryoprotectant concentration ect., the next generation was obtained when the females implanted frozen-thawed ovaries mated with normal males, but none of the normal females mated with the males implanted frozen-thawed testes laid fertilized eggs. Now it is needed to improve the connecting ration of the ducts associated with the transplanted testis to those of the hosts.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.9
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• pp.1247-1255
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• 2016

Heparin binding proteins (HBPs) are produced by accessory glands. These are secreted into the seminal fluid, bind to the spermatozoa at the time of ejaculation, favour capacitation, acrosome reaction, and alter the immune system response toward the sperm. The present study was conducted with an objective to assess the effect of purified seminal plasma-HBPs (SP-HBPs) on cross bred cattle bull sperm attributes during two phases of cryopreservation: Pre freezing and freezing-thawing. SP-HBPs were purified from pooled seminal plasma by heparin affinity chromatography. Three doses of SP-HBPs i.e. 10, 20, $40{\mu}g/mLs$ semen were standardized to find out the optimum dose and $20{\mu}g/mLs$ was found to be an optimum dose. Semen as such and treated with SP-HBPs was diluted with sodium citrate-egg yolk diluter and cryopreserved as per the standard protocol. Sperm parameters i.e. motility, viability, Hypo-osmotic swelling test (HOST), acrosome damage, in vitro capacitation and lipid peroxidation were evaluated in SP-HBP treated and untreated (control) semen at both phases of cryopreservation. A considerable variation in percent sperm motility, viability, membrane integrity (HOST), acrosome damage, acrosome reaction and lipid peroxidation was observed at both phases among the bulls irrespective of the treatment. Incubation of neat semen with $20{\mu}g/mL$ SP-HBP before processing for cryopreservation enhanced the average motility, viability, membrane integrity by 7.2%, 1.5%, 7.9%, and 5.6%, 6.6%, 7.4% in pre-frozen and frozen-thawed semen in comparison to control. There was also an average increase of 4.1%/3.9% in in vitro capacitation and acrosome reaction in SP-HBPs-treated frozen-thawed semen as compared to control. However, binding of SP-HBPs to the sperm declined acrosome damage and lipid peroxidation by 1.3%/4.1% and 22.1/$32.7{\mu}M$/$10^9$ spermatozoa in SP-HBP treated pre-frozen/frozen-thawed semen as compared to control, respectively. Significant (p<0.05) effects were observed only in motility, HOST and in vitro acrosome reaction. It can be concluded that treatment of neat semen with SP-HBPs before cryopreservation minimized the cryoinjury by decreasing the generation of reactive oxygen species.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.129-134
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• 1998

The objective of this study was to test whether the developmental capacity of human multi-pronuclear (PN) zygotes after ultra-rapid freezing using EM grid can be maintained. For this experiment, multi-PN zygotes which produced in human IVF program were used as an alternatives of normal 2PN zygotes, and they were separated into 3PN or $\geq4PN$ zygotes to compare their in vitro development and cryoinjury according to PN number. As freezing solution, EFS30 which consisted of 30% ethylene glycol, 18% bcoll, 0.5 M sucrose and 10% FBS added D-PBS was used. The result obtained in this experiment was summarized as follows; When the multi..PN zygotes were ultrarapidly frozen and thawed, the high mean percentages (85.5%) were survived. Also when the cleavage rates between control and freezing group were compared with PN number, there were not significantly different in each group (3PN; 81.3% & 85.4% and $\geq4PN$; 90.0% & 95.7%). When the in vitro development rates after thawing were examined, freezing 3PN group (22.0%) was not differed to control 3PN group (38.5%), although the development result of freezing $\geq4PN$ group (45%) was significantly lower than that of control $\geq4PN$ group (44.4%) (p<0.05). These results demonstrate that developmental capacity of human zygote can be obtained by ultra-rapid freezing method using EM grid and EFS30.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.1
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• pp.24-30
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• 2011

Objective: The objectives of this study were to analyze efficacy of immature and mature mouse oocytes after vitrification and warming by applying various combinations of cryoprotectants (CPAs) and/or super-rapid cooling using slush nitrogen ($SN_2$). Methods: Four-week old ICR female mice were superovulated for GV- and MII-stage oocytes. Experimental groups were divided into two groups. Ethylene glycol (EG) only group: pre-equilibrated with 1.5 M EG for 2.5 minutes and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 seconds. EG+dimethylsulfoxide (DMSO) group: pre-equilibrated with 1.3 M EG+1.1 M DMSO for 2.5 minutes and equilibrated with 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 20 seconds. The oocytes were loaded onto grids and plunged into $SN_2$or liquid nitrogen ($LN_2$). Stored oocytes were warmed by a five-step method, and then their survival, maturation, cleavage, and developmental rates were observed. Results: The EG only and EG+DMSO groups showed no significant difference in survival of immature oocytes vitrified after warming. However, maturation and cleavage rates after conventional insemination were greater in the EG only group than in the EG+DMSO group. In mature oocytes, survival, cleavage, and blastocyst formation rates after warming showed no significant difference when EG only or EG+DMSO was applied. Furthermore, cleavage and blastocyst formation rates of MII oocytes vitrified using $SN_2$ were increased in both the EG only and EG+DMSO groups. Conclusion: A combination of CPAs in oocyte cryopreservation could be formulated according to the oocyte stage. In addition, $SN_2$ may improve the efficiency of vitrification by reducing cryoinjury.