• Asian-Australasian Journal of Animal Sciences
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• 제22권12호
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• pp.1614-1619
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• 2009

The present study was conducted to observe the effect of increased osmolality of basic tris extender supplemented with trehalose and sucrose on post-thawing quality (motility, progressive motility, viability, the rate of acrosome abnormality, total abnormality and membrane integrity) of Markhoz goat spermatozoa. Fresh semen samples were evaluated for motility and sperm concentration. Only semen samples with motility more than 70% and sperm concentration higher than $3{\times}10^{9}$ sperm/ml were used for cryopreservation. In Exp. 1, trehalose (50, 75 or 100 mM) and sucrose (40, 60 or 80 mM) were added to a basic tris diluent. Based on the results of experiment 1, the goal of Exp. 2 was to investigate the combinational effects of the highest and lowest concentrations ($T_{100}+S_{80}$ or $T_{50}+S_{40}$) of trehalose and sucrose. As the control, semen was diluted and frozen in the tris diluent without trehalose or sucrose. The results in Exp. 1 showed that all evaluated spermatozoa characteristics improved significantly after freezing and thawing (p<0.05) and at the same time the increase of trehalose and sucrose concentrations in basic extenders was seen, with the best results obtained for extenders containing 70 and 100 mM trehalose and 80 mM sucrose. Comparing these results with those of control diluents, the effects of supplementation were significantly (p<0.05) better. In Exp. 2, the results showed no significant differences (p>0.05) between $T_{100}+S_{80}$ and $T_{50}+S_{40}$ extenders, but the results of $T_{50}+S_{40}$were slightly better than obtained with $T_{100}+S_{80}$ diluents. Furthermore, the results of this experiment indicated that the sperm characteristics in the isotonic control extender were significantly (p<0.05) lower than examined extenders. In conclusion, the results of this study indicated that goat sperm can tolerate hypertonic trehalose and sucrose solutions better than isotonic control diluents in the freezing period. In particular, these positive effects have been shown for acrosome integrity, which is very important for the fertilization capacity of sperm. The data indicated that addition of trehalose plus sucrose to the freezing extender can be recommended for cryopreservation of goat spermatozoa, but more data is needed on pregnancy rate, acrosome reaction and IVF to ascertain the real effect.

• Reproductive and Developmental Biology
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• 제38권4호
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• pp.159-163
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• 2014

The objective of this study was to determine the effects of E. coli on boar sperm quality and reproductive performance in sows after artificial insemination. Three different levels of E. coli were artificially inoculated to semen with following concentrations; Control, 500, 5,000 and 50,000 colony forming unit (cfu)/ml. Semen samples were preserved at $17^{\circ}C$ for 5 days. Sperm motility was significantly decreased (p<0.05) on day 3 in the group inoculated with 5,000 cfu/ml compared to control groups. In all treatment groups, sperm motility was gradually decreased as storage time increased, but the decline pattern was more drastic in the groups inoculated with 5,000 and 50,000 cfu/ml groups from day 3 (p<0.05) compared to control group. After 3 day of storage at $17^{\circ}C$, sperm viability in sample inoculated with the highest concentration (50,000 cfu/ml) of bacteria was less (p<0.05) than that of control group. The pH of semen sample pH was maintained 7.2~7.5 in all groups during the experimental period. No differences (p>0.05) were found for both storage time and bacterial concentration. The pregnancy rate and live born piglets tend to decrease by increasing the concentration of E. coli in semen. In particular, the rate of pregnancy was lower in the group inoculated with 50,000 cfu/ml (58.3%) compare to the other groups (81.8, 75.0, 76.5%). These results suggest that the contamination of E. coli in boar semen negatively affects fertilizing ability of boar sperm and the reproductive performance obtained from sows after artificial insemination.

• Journal of Animal Science and Technology
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• 제48권6호
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• pp.785-796
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• 2006

본 연구는 인공수정 센터에서 이용되고 있는 종모돈의 인공수정 분만율의 차이를 기준으로 하여 정액의 보존 일에 따른 정자의 생존율, CASA, HOST, IVP 및 SCSA를 조사하여 이들 정자의 분석법들과 인공수정 분만율과의 상관관계를 조사하여 종모돈의 수정 능력을 예측할 수 있는 정액 평가법을 개발하고자 실시하였다. 종모돈의 액상정액을 대상으로 CASA, HOST, IVP 및 SCSA 기법을 적용한 정액의 평가 결과는 다음과 같았다. 정액의 보존 일별 HOST 결과는 보존 일이 경과함에 따라 유의적으로 감소하였고(P<0.05), IVP 결과 난자 당 침투 정자 수는 보존 3일째에 유의적으로 감소하였다(P<0.05). 보존 0일과 6일에 HOST는 분만율이 80% 이상인 종모돈 군이 70% 미만인 종모돈 군 보다 유의적으로 높았고, 3일에서는 80% 이상인 종모돈 군이 다른 종모돈 군에 비하여 높았다(P<0.05). IVP는 보존 0일에 분만율이 80% 이상인 종모돈 군이 70% 미만인 종모돈 군보다 난자당 침투 정자 수가 유의적으로 많았다(P<0.05). %Red는 보존 0일과 3일에는 분만율이 80% 이상과 70% 미만인 종모돈 군 간에 유의적인 차이를 보였으나 보존 6일에서는 모든 종모돈 군간에 유의적인 차이가 있었다(P<0.05). %Red(r=0.79, P<0.01 ; r=0.86, P<0.01 ; r=0.88, P<0.01)는 정액의 보존 일이 증가함에 따라 분만율과의 부의 상관 계수가 증가하였다. 이상의 결과를 종합해 보면 SCSA는 정액의 수정 능력을 평가하는데 있어서 매우 유용한 방법이며, 정액의 보존 일은 여러 가지 정자 기능과 SCSA 결과에 크게 영향을 주는 것으로 나타났다. 정액의 수정 능력을 평가함에 있어서 정액의 보존 일령을 고려하고 정자 기능 검사와 SCSA를 병행하여 실시할 경우 종모돈의 수정 능력 평가 시 정확성을 더욱 높일 수 있는 유용한 방법이 될 수 있을 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제21권12호
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• pp.1721-1727
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• 2008

Four experiments were conducted to study the following: i) the influence of different concentrations of sucrose (0.15, 0.3 and 0.5 M with osmolality of 308, 500 and 760 mOsm/kg, respectively) in diluents and control diluent (370 mOsm/kg) on intensity of motility and progressive motility of goat sperm without rehydration and freezing step in four incubation periods (0, 0.5, 2 and 4 h after dilution); ii) the influence of gradual dilution (in 3 steps) on improvements in ascertained results of the first experiment; iii) cryoprotective effects of different concentrations of sucrose (0.15, 0.22, 0.29 and 0.37 M with osmolality of 450, 560, 740 and 920 mOsm/kg, respectively) plus 7% glycerol and 20% egg yolk in basic diluent (Tris-Citric acid-Fructose) and iv) the effect of two concentrations of sucrose (0.15 and 0.22 M) with and without glycerol (7%). In experiment 1, we obtained better results for control diluent, 0.15 and 0.3 M sucrose supplemented diluents with 0 and 0.5 h incubation periods. In experiment 2, apart from a slight improvement, similar tendencies to experiment 1 were observed. In experiment 3, we obtained the best result for diluent with 0.22 M sucrose with regard to intensity of motility, progressive motility, live sperm and normal acrosomes ($40{\pm}4%$, $3.1{\pm}0.2$, $37{\pm}4%$ and $37{\pm}4%$, repectively). In experiment 4, we obtained the best result for diluent with 0.22 M sucrose plus 7% glycerol in regard to intensity of motility, progressive motility and live sperm ($39{\pm}3%$, $3.6{\pm}0.4$ and $41{\pm}4%$, respectively). The characteristic normal acrosomes in diluents without glycerol, i.e. diluents with 0.15 and 0.22 M sucrose showed better results ($39{\pm}8$ and $42{\pm}6%$ respectively). With regard to the release of hyaluronidase enzyme there were no significant differences between diluents (p>0.05). The results of the diluents with 0.15 and 0.22 M sucrose without glycerol were slightly lower than those with glycerol ($69{\pm}11$ and $70{\pm}11$ vs. $72{\pm}11$ and $70{\pm}11{\times}120{\times}10^6$ units $ml^{-1}$, respectively). In conclusion, the use of concentrated sucrose solutions showed that goat sperm can tolerate osmolality up to 560 mOsm (0.22 M) in the freezing period. In addition, glycerol proved to be a necessary cryoprotective agent in the cryopreservation of goat sperm, particularly for intensity of motility, progressive motility and live sperm.

• Reproductive and Developmental Biology
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• 제40권3호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제37권3호
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• pp.245-251
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• 2010

목 적: 인간 정자 핵 내의 DNA integrity는 배아의 발달 및 임신 유지에 중요한 역할을 하여 DNA integrity가 손상된 경우 불임과 유산의 원인이 된다고 하며, 정계정맥류는 DNA 손상을 일으키는 대표적인 원인 중 하나이다. 본 연구에서는 미세술기를 이용한 정계정맥류절제술로 정계정맥류를 교정을 하였을 때 정자 핵 내 DNA integrity가 어떠한 영향을 받는지에 대하여 알아보았다. 연구방법: 2006년 4월부터 2007년 4월까지 불임을 주소로 미세술기를 이용한 정계정맥류절제술을 받았던 18명의 환자에서 수술 전 후에 정액검사의 다른 지표들과 함께 정자 핵 내 DNA integrity가 어떻게 변화하였는지 조사하였다. 정자 핵 내 DNA integrity를 측정하는 방법으로 comet assay를 시행하였고, comet assay를 통한 DNA 손상 정도는 DNA fragmentation index (DFI)로 나타내었다. 결 과: 수술 후 4개월에 모든 환자에서 재발의 소견은 보이지 않았으며, DNA 손상 정도를 나타내는 평균 DFI는 수술 전에 19.3%, 수술 후에 13.7%로 유의한 변화를 보였다. 수술 전 DFI가 10 이상으로 비정상인 14명의 환자들 중 12명 (85%)에서 개선 소견을 보였으나, 수술 전 DFI가 10 미만인 정상 환자 4명에서는 1명 (25%)만이 개선 소견을 보였다. 수술 후 정자의 밀도, 운동성, 생존성에서 호전 양상을 보였으나 유의한 차이는 없었다. 결 론: 미세술기를 이용한 정계정맥류절제술을 통한 수술적 교정은 정액검사상의 다른 지표의 개선 뿐 아니라, 정자 핵 내 DNA 손상을 감소시킬 수 있다. 이상에서 정계정맥류의 수술적 교정으로 정자 핵 내 DNA integrity의 개선을 기대할 수 있으며, 이는 보다 양호한 정자를 많이 얻을 수 있어 자연임신이나 보조 생식술의 성공 가능성을 높일 수 있다는 점을 제시한다.

• 한국임상수의학회지
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• 제30권6호
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• pp.442-448
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• 2013

개 정액 동결을 위한 glucose가 첨가된 glycerol-free TRIS 희석액을 개발하기 위해 glycerol-free TRIS내 알맞은 glucose의 양과 0.3 M glucose가 첨가된 glycerol-free TRIS내 정자의 적정 노출시간을 조사하였다. 여섯 마리의 수캐의 사출액을 0.04 M glucose가 첨가된 glycerol-free TRIS내에서 $4^{\circ}C$까지 100분 동안 냉각한 후 서로 다른 glucose농도 (0 M, 0,04 M, 0.1 M, 0.2 M, 0.3 M)의 glycerol-free TRIS에서 30분 동안 냉각하여 동결하였다. $37^{\circ}C$에서 25 초 동안 융해한 후 정자의 막 고유성과 첨단체 고유성을 검사하였다. 부가적으로 0.3 M glucose가 첨가된 glycerol-free TRIS내 정자의 적정 노출시간에 따른 정자의 동결 후 운동성, 생존성, DNA 고유성을 확인하였다. 막 고유성과 첨단체 고유성은 각각 6-carboxyfluoresceindiacetate(6-CFDA)/propidium iodide(PI) fluorescent staining와 Pisum sativum agglutinin conjugated-fluorescein isothiocyanate 방법에 의해 검사하였다. DNA 고유성은 terminal deoxynucleotidyl transferase dUTP nick end labeling로 염색하여 flow cytometry로 검사하였다. 0.2 M 또는 0.3 M glucose가 첨가된 glycerol-free TRIS에서 동결된 정자가 낮은 농도의 glucose가 첨가된 희석액에서 동결된 정자보다 막 고유성이 높게 나타났으며(p<0.05), 첨단체 고유성은 0.3 M 군에서 높게 나타났다(p<0.05). 운동성은 50 분 군에서 높게 나타났으나(p<0.05), DNA fragmentation index는 노출시간에 따라 차이가 없었다. 본 연구 결과 개정자가 0.3 M glucose가 첨가된 glycerol-free TRIS에서 $4^{\circ}C$, 50 분간 냉각 후 동결과 융해 후 더 높은 생존성을 나타냈다.

• 한국가축번식학회지
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• 제23권2호
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• pp.127-132
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• 1999

본 연구는 소형견 정액의 원정액과 정장제거 정액의 일반성상, 채취분획별 및 단기보존시 정자의 생존성과 아울러 동결보존시의 정자의 생존성에 대해 조사하고자 수행하였다. 1. 원정액과 정장제거 정액에 saline 및 Tris-buffer를 희석 후 각각 일반성상 검사를 실시했을 때 원정액의 경우 정자농도는 5.07$\pm$2.32$\times$$10^{6}$cells/$m\ell$, 정자의 운동성은 95.42$\pm$2.65%, 기행정자수는 4.42$\pm$0.15%로 나타났으며, RSP(saline 및 Tris-buffer)군의 경우 정자농도는 4.69$\pm$3.27~4.25$\pm$3.65$\times$$10^{6}$cells/$m\ell$, 정자의 운동성은 91.17$\pm$3.85~88.52$\pm$3.85%, 기형정자수는 6.57$\pm$0.43~5.54$\pm$0.52%로 나타났다. 2. 분획별 채취 전정액중 제 1 분획에서는 정액량이 0.92$\pm$0.7$m\ell$, 정자농도는 4.57$\pm$0.78$\times$$10^{6}$cells/$m\ell$, 정자활력은 10.72$\pm$3,21%, 기형정자수는 5.50$\pm$0.70%로 나타났으며, 제 2분획에서는 정액량이 2.14$\pm$0.19$m\ell$, 정자농도는 2.01$\pm$0.12$\times$$10^{6}$cells/$m\ell$, 정자활력은 95.44$\pm$4.21%, 기형정자수는 4.31$\pm$0.53%로 나타났다. 또한, 제3 분획에서는 정액량이 2.66$\pm$0.23$m\ell$, 정자농도는 2.35$\pm$0.21$\times$$10^{6}$cells/$m\ell$, 정자활력은 90.71$\pm$2.63%, 기형정자수는 6.33$\pm$0.91%로 나타났다. 3. 원정액과 정장제거 정액을 4$^{\circ}C$ 와 2$0^{\circ}C$ 및 37$^{\circ}C$ 에서 각각 보존했을 때 보존시간별 정자의 활력은 2$0^{\circ}C$ 의 경우 1, 6, 13, 24, 30 및 40 시간에서 각각 98.51%와 98.32%, 86.32%와 92.15%, 83.71% 와 89.20%, 74.29% 와 82.08%, 52.98% 와 72.07%, 15.45% 와 60.02%, 2.41% 와 37.19% 의 정자활력을 나타내어 4$^{\circ}C$와 37$^{\circ}C$에 비해 높은 정자운동성을 나타냈다. 4. 제 2 분획 정액과 정장제거 정액을 각각 제 1차 및 제 2차 희석액으로 희석 평형 시킨 후 동결 융해했을 때 정자의 생존율은 각각 33.3$\pm$8.7, 54.7$\pm$9.5%로서 대조군의 15.4$\pm$5.2% 에 비해 높게 나타났다.

• 한국수정란이식학회지
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• 제26권3호
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• pp.187-193
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• 2011

This study was carried out to establish most suitable freezing condition, to evaluate the different glycerol concentration of freezing and thawing rates on motility, viability, membrane integrity and acrosome intecrity of frozen Korean Jeju Black Bull spermatozoa, Semen was collected from a Korean Jeju Black Bull using an artificial vagina and transported to the laboratory. The semen was extended gradually 1:5 then cooled slowly for 2 hrs to 4$^{\circ}C$. The semen was diluted 1:1 with cryoprotectant extenders (3%, 5% and 7% glycerol) and equilibrated for 2 hrs at cold chamber and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 min and above 8 cm for 10 min. And then the frozen straw was plunged into LN$_2$. The presented straws were examined the viability and motility after thawed at 37$^{\circ}C$ water bath. The viability and membrane integrity immediately post-thawing were significantly higher in samples frozen in 7% glycerol than 3% and 5% glycerol (p<0.05). After CTC staining to assess acrosome integrity, F pattern was significantly increased, but B pattern was significantly decreased in 7% glycerol (p<0.05). Freezing distance of 5 cm from liquid nitrogen and pre-cooling for 10 min yield better survival and membrane integrity, but not significant difference. However, AR pattern according to CTC staining was significantly decreased in 3 cm for 5 min.

• Asian-Australasian Journal of Animal Sciences
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• 제21권1호
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• pp.29-34
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• 2008

The aim of the study was to investigate the effect of dietary fish oil supplementation on the semen characteristics of the Markhoz buck. Sixteen bucks were randomly allocated into 4 groups and received four different diets: unsupplemented control diet, supplemented with fish oil at 2.50% dry matter (DM), supplemented with fish oil (2.50% DM) and vitamin E (0.30 g/kg DM), and supplemented with vitamin E (0.30 g/kg DM). All experimental diets were formulated according to AFRC (1998). Semen was collected at 14 d intervals from June 17, 2006 to September 2, 2006. Semen characteristics were evaluated. Significant effects (p<0.05) of the week (sampling time) were observed for all parameters except semen volume. Also a significant effect (p<0.05) of dietary treatment was observed for all parameters except for percent sperm with normal morphologies and semen volume. Fish oil supplementation with excess vitamin E had a significant effect (p<0.05) on total number and sperm density, motility and progressive motility, percentage viability and dead sperm. The interaction between fish oil feeding and sampling time was significant (p<0.05) for all of the parameters. The bucks that received fish oil in association with vitamin E, effect fish oil showed the greatest improvement in semen characteristics compared with the other groups (p<0.05). This study showed that fish oil supplementation with vitamin E may have a beneficial effect on the semen quality and fertility of Markhoz bucks.