• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.9
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• pp.251-258
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• 2020

This study investigated the effect of Zardaverine supplementation in freezing extender, on kinetic characteristics of post-thawed boar sperm. Cryopreservation of boar sperm is an important technique of assisted reproductive technology and genetic resource banking. Although this technique is particularly useful, freeze-thaw cycles associated with sperm cryopreservation significantly reduce sperm quality. Semen from mature Duroc boars were collected and cryopreserved in freezing extenders (LEY) treated with varying concentrations of Zardaverine (0, 20, 50, 75, 100 𝜇M). The time-dependent kinetic characteristics of post-thawed spermatozoa were determined after thawing by applying computer-assisted sperm analysis (CASA). We observed that the motility immediately after thawing was significantly higher in 20 𝜇M stocks than in control (0 𝜇M) and the other treatments (p<0.05). Curvilinear velocity (VCL) in 0 𝜇M and 20 𝜇M stocks were significantly higher than the other treatment groups, except 75 𝜇M (p<0.05). Higher average path velocity (VAP) was obtained at 20 𝜇M as compared to 100 𝜇M, whereas amplitude of head lateral displacement (ALH) was significantly higher at 20 𝜇M than 50 𝜇M and 100 𝜇M (p<0.05). No differences were obtained for Straight-line velocity (VSL) and Linearity (LIN). In conclusion, our results indicate that Zardaverine improves the motility, VCL, VAP, and ALH of post-thawed boar sperm.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.3
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• pp.199-205
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• 2020

Cryopreservation of semen is useful for animal breeding via artificial insemination (AI). However, the use of frozen-thawed boar semen is limited due to cryodamage. The aim of this study was to investigate the effects of different concentrations of MitoTEMPO (a mitochondria-targeted antioxidant) in lactose-egg yolk (LEY) extenders on kinetic characteristics of frozen-thawed boar sperms. Semen samples were collected from mature Duroc boars (2~3 years old) and cryopreserved in LEY extenders containing 0, 0.5, 5, 50, and 500 μM MitoTEMPO. The kinetic characteristics of frozen-thawed sperms were determined 0 and 30 min after thawing using computer-assisted sperm analysis (CASA). Results indicated that sperm motility immediately after thawing was significantly higher with 5 and 50 μM (50.46±2.71% and 46.96±2.66%, respectively) than with 500 μM MitoTEMPO (35.40±2.95%) (P<0.05). However, there were no significant differences in other kinetic characteristics except motility. In conclusion, the addition of MitoTEMPO to the sperm freezing extender may have a beneficial effect on motility of post-thawed boar semen.

• Reproductive and Developmental Biology
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• v.40 no.3
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• pp.27-31
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• 2016

The aim of this study was to evaluate effect of ${\alpha}$-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at $37.5^{\circ}C$ for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.

• Journal of Animal Science and Technology
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• v.63 no.1
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• pp.36-45
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• 2021

Presently, there is an increased demand for livestock products all over the world which has led to more devotion on improving livestock population. Although goats have been bred for a long time in Korea, but there is not much research conducted on traditional Korean black goat (Capra hircus coreanae) compared to other livestock populations. Mutton consumption has been dramatically changing from medicinal use to edible meat and this trend directs the black goat populations declining and also mutton import quantities are increasing consistently. The present study introduced a new estrus synchronizing technique with subsequent artificial insemination (AI) for Korean black goats to enable crossbreeding with non-native breeds for the small or subsistent farmers. Our data highlighted that, the percentage of motile sperm from the electro-ejaculated samples declined significantly after freezing and melting. In addition, the sperm motility significantly declined with regard to sperm incubation period (0, 5, 60, and 120 min at 37℃) and was negatively correlated (64.2 ± 7.9%, 63.3 ± 5.8%, 49.9 ± 6.3%, and 35.9 ± 7.6%, respectively) in frozen-thawed sperm samples. Moreover, the E2 levels were unchanged even 24 h after controlled internal drug releas (CIDR) withdrawal. But, 48 h and 72 h after CIDR removal, E2 levels increased significantly. These data helps us to consider the two time points for AI; CIDR removal after 24 h, at which E2 decreases, and after 48 h, as the time at which progesterone increases. Additionally, the AI after 48 h of CIDR removal group exhibited significantly higher pregnancy and parturition rates (42.9%) compared to AI after 24 h after CIDR removal 28.6% group. In conclusion, these studies will propose an optimal estrus synchronisation process with subsequent timing of AI and also will promote the Korean black goat breeding industry.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.363-367
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• 2011

The purpose of this study was to improve of frozen-thawed sperm using magnetized water in Korean native cattle. Before cryopreservation, without egg-yolk Triladyl$^{(R)}$ solution was flowed though magnet [0, 2000, 4000 and 6000 Gauss(G)] for S min. The freezing of dilluted semen added with Triladyl containing 20% egg-yolk. Analysis of frozen-thawed sperm was estimated viability with SYBR14/PI double stain, membrane intact with hypoosmotic swelling test (HOST), acrosome reaction with FITC-PNA, mitochondria membrane function with Rhodamin 123 by flow- cytometry. Sperm viability was significantly higher in 4000G group than other groups (p<0.05). However, the Hypoosmotic Swelling Test(HOST) was significantly higher in fresh, 4000 and 6000G than 0 and 2000G (p<0.05). In addition, mitochondria membrane damage and acrosome damage were significantly lower in 6000G group than other groups (p<0.05). in conclusion we suggest that magnetized water could be improve ability of sperm on cryopreservation in Korean native cattle.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.84-84
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• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.301-306
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• 2004

본 연구는 개의 신선 및 동결 정소상체 정액과 정소상체 정액을 saline가 tris-buffer액으로 희석하고 원심분리에 의해 정장성분을 제거한 정액의 성상과 및 동결보존 시의 생존성 및 난포란과 정소상체 정자의 체외수정 후 체외수정율과 분할율에 대해 조사하였다. 개 정소상체 정액을 saline으로 희석한 정액을 20분간 배양했을 때 정자농도는 3.50$\pm$0.80${\times}$l0$^{6}$ cells/$m\ell$, 정자의 활력은 72.45$\pm$4.55%, 기형정자 수는 7.40$\pm$1.20%로 나타났으며, 대조군인 사출정액의 정자농도는 5.45$\pm$0.50${\times}$$10^{6}$cells/$m\ell$, 정자의 활력은 92.55$\pm$4.65%, 기형정자 수는 3.25$\pm$0.45%와 비교할 때 정자농도와 활력은 낮았으며, 기형정자 수는 많았다. 개 정소상체 정액과 tris-buffer로 희석한 정액을 20분간 배양했을 때 정자농도는 3.80$\pm$0.36${\times}$$10^{6}$ cells/$m\ell$, 정자활력은 78.45$\pm$3.50%, 기형정자 수는 5.54$\pm$0.85%였으며, 희석하지 않은 정소상체 정액의 성상에 비해 약간 높게 나타났다. Tris-buffer로 희석한 개 정소상체 정액을 동결 융해했을 때 생존율은 57.50$\pm$4.20%, 활력은 52.70$\pm$5.50%였으며, 희석하지 않은 대조군의 생존을 74.50$\pm$6.25%와 활력 78.50$\pm$5.20%에 비해 현저히 높게 나타났다. 개 난포란과 신선 몇 동결 정소상체 정자와 체외수정시켰을 때 체외수정율은 각각 63.10$\pm$6.45%, 49.50$\pm$4.28% 및 42.84$\pm$5.90%, 22.30$\pm$5.60%로서 신선 정자에 비해 동결 정소상체 정자로 수정시킨 군의 분할율이 유의하게 낮았다.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.307-313
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• 2004

본 연구는 고양이의 신선 및 동결 정소상체 정액과 정소상체 정액 성상과 및 동결보존시의 생존성 및 난포란과 정소상체 정자의 체외수정 후 체외수정율과 분할율에 대해 조사하였다. 고양이 정소상체 정액의 정자농도는 3.25$\pm$0.75${\times}$$10^{6}$ cells/$m\ell$, 정자의 활력은 70.85$\pm$4.20%, 기형정자 수는 8.55$\pm$1.85%로서 대조군인 사출정액의 정자농도는 5.05$\pm$0.40${\times}$$10^{6}$ cells/$m\ell$, 정자의 활력은 90.24$\pm$455%, 기형정자 수는 4.20$\pm$0.50%와 비교할 때 정자농도와 활력은 낮았으며, 기형정자 수는 많았다. 고양이 정액과 tris-buffer로 희석한 정액을 20분간 배양했을 때 정자농도는 3.50$\pm$0.40${\times}$$10^{6}$ cells/$m\ell$, 정자활력 은 75.50$\pm$2.55%, 기 형정자 수는 6.75$\pm$0.58%로서 희석하지 않은 정소상체 정액의 성상에 비해 약간 높게 나타났다. Tris-buffer로 희석한 고양이 정소상체 정액을 동결 융해했을 때 생존율은 54.50$\pm$4.45, 활력은 47.50$\pm$6.40%로서 희석하지 .않은 대조군의 생존을 74.50$\pm$6.25%와 활력 78.50$\pm$5.20%에 비해 현저히 높게 나타났다. 고양이의 난포란과 신선 및 동결 정소상체 정자를 수정시켰을 때 체외수정율과 분할율은 68.30$\pm$5.35%, 57.25$\pm$4.35% 및 48.65$\pm$4.95%, 35.65 $\pm$4.75%로서 신선 정자에 비해 동결 정소상체 정자로 수정시킨 군의 분할율이 유의하게 낮았다.

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.151-158
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• 1996

This study was carried out to investigate the effect of equilibration time, sucrose concentration and age of embryo on survival and developmental rates of bovine IVF expanding blastocysts frozen-thawed by direct transfer method. The bovine oocytes were collected from 2~5mm follicles, matured for 20~24hrs in 5% $CO_2$incubator and then fertilized with frozen-thawed semen. Expanding blastocysts at day 7, 8, 9, 10 and 11 after IVF were frozen in 1.8M ethylene glycol(EG). Survival and hatching rates of frozen-thawed IVF embryos were examined. The results were as follow ; Survival and hatching rate of TVF expanding blastocysts after 10, 20, 3Omin exposure in 1.8M EG were 100,0,90.9, 47.1, 85.0, 75.0 and 62.5% respectively. Survival rates of IVF expanding blastocysts frozen with 1.8M EG and various concentration(0, 0.25, 0.5, 1M) of sucrose were 73.3, 25. 0, 16.7, 9.1% respectively. Survival and hatching rates of IVF expanding blastocysts frozen-thawed according to age of embryo(Day 7, 8, 9,10, 11) were 86.1, 84.8, 79.3, 61.4, 51.3, 74.2, 76.9, 71.7, 63.0 and 65.0% respectively. In conclusion, the age of the embryo(Day 7, 8) is very important for the successful freezing of IVF bovine embryos and 1.8M ethylene glycol not containing sucrose may be effective cryoprotectant for direct transfer method.

• 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.