• Journal of Animal Science and Technology
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• 제63권1호
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• pp.58-68
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• 2021

Several herbs including Artemisia are known to possess conceptive property. In the present study, mouse spermatozoa were incubated with ethanol extract of Artemisia vulgaris leaves. The effect of extract on acrosome exocytosis was studied by labeling spermatozoa with fluorescein isothiocyanate (FITC) peanut agglutinin and by staining with Coomassie blue. Viability and membrane integrity were studied by Trypan-blue staining and hypo-osmotic swelling test. Artemisia extract at very low concentration caused precocious acrosome reaction and loss of sperm viability. Acrosome reaction increased remarkably from 22.63% to 88.42% with increasing extract concentration from 0 to 2,000 ㎍/mL. However, the viability loss of spermatozoa was increased from 11.71% in control to 63.73% in samples treated, evaluated by Trypan-blue staining method. Membrane damage caused by the extract, evaluated by hypo-osmotic swelling test was even low, ranging from 2.27% to only 24.23%. These results indicate that Artemisia extract might block fertilization by causing precocious acrosome exocytosis in spermatozoa. A direct contraceptive effect was tested by injecting the plant extract into the vagina of female mice and then allowing them to mate with normal males. The treated female mice delivered significantly fewer litters in comparison to the control.

• 대한수의학회지
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• 제37권4호
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• pp.925-934
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• 1997

This study was directed at inducing the production of antibodies by immunizing heifers with bovine sperm antigen and on measuring the serum antibodies using indirect immunofluorescence assay(IFA) and agglutination test. The effect of antisperm antibodies on fertilizing capacity of bovine spermatozoa was evaluated. 1. Three heifers between 12- and 15- month old were immunized with bovine spermatozoa or phosphate-buffered saline. In heifers immunized with bovine spermatozoa serum IgG level was highest between 3 weeks and 5 weeks postimmunization detected by IFA. The antibody levels persisted through week 7 and slowly declined until week 20 and then antisperm antibodies were localized on spermatozoa. The fluorescent antisperm antibodies were detected at 2~20 weeks and at 6~9 weeks postinoculation on acrosome and tail, respectively. Among 21 sera from repeat breeder cows, only one cow has shown positive antisperm antibody response detected by IFA. 2. In spite of vital rate of bovine sperm after swim-up was not significantly affected by different concentration of antisperm antibodies in sera, the numbers of bovine sperm after swim-up were significantly reduced in proportion to the increased concentration of antibodies. Above 1/512 dilution of antibody neither influence on vital rate and numbers of bovine sperm nor sperm agglutination after swim-up. The study has also shown that the vital rate and number of sperm after swim-up and capacitation were also significantly reduced by the addition of antisperm antibodies. Although antisperm antibodies did not influence on the acrosome reaction rate of sperm during swim-up, did significantly reduce the sperm acrosome reaction rate after capacitation. The studies have resulted that the bovine antisperm antibodies can prevent the sperm motility by agglutination and block the capacitation and acrosome reaction of bovine sperm.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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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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• 제19권1호
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• pp.57-64
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• 1992

The present study was undertaken to clarify the role of TEST-Yolk Buffer(TYB) as a factor for the improvement of human sperm fertility potential. We examined the effects of low temperature capacitation using TYB on sperm motility (%), motility pattern, normal morphology, true acrosome reaction, sperm penetration assay and human in vitro fertilization. Comparing the TYB method and swim-up method, the sperm motility(%) of selected sperm was not significantly different, but statistically significant differences were found in curvilinear velocity, linearity, lateral head displacement, normal morphology(%) and true acrosome reaction(%)(p<0.05). Results obtained from the sperm penetration assay demonstrated that the penetration index and penetration rate were increased significantly(p<0.05) when the spermatozoa were incubated in TYB, as compared with swim-up method. And fertilization of intact human oocytes was more succesful when spermatozoa were pretreated with TYB at $4^{\circ}C$ for 48 hours as compared with swim-up method. Our results show that TYB method have advantages in terms of enhancement of sperm hyperactivation, increased true acrosome reaction, increased ability to penetrate zona-free hamster ova and augmented fertilization of human oocytes, suggesting that TYB is superior in its ability to preserve sperm motility and fertilizing ability.

• Clinical and Experimental Reproductive Medicine
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• 제26권1호
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• pp.89-96
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• 1999

당뇨병은 생식내분비 조직의 구조나 기능 변화를 유발하여 호르몬 합성 및 분비량의 변화를 야기하고, 정자의 운동성 등에 영향을 미치는 것으로 알려져 왔다. 그러나 부정소와 수정관내 정자의 농도 변화나 수정능력 획득 및 침체반응에 미치는 영향은 잘 알려져 있지 않다. 본 실험에서는 Wistar 쥐에 streptozotocin을 투여하여 당뇨병을 유발시킨 후 3일과 14일에 부정소 각 부위와 수정관내 정자 농도 변화를 조사하고 부정소 꼬리와 수정관내 정자의 침체반응 유도 실험 (acrosome reaction to ionophore challenge test, ARIC test)을 이용하여 정자의 수정 능력을 평가하였다. Streptozotocin을 주사한 후 혈액내 인슐린 및 포도당 농도는 당뇨병 경과 기간이 길어짐에 따라 반비례 관계를 보였다. 부정소 머리와 몸통내 정자의 농도는 3일군에서부터 감소하기 시작하나 꼬리에서는 14일군 $(15.2{\pm}2.1)$에서 대조군 $(28.1{\pm}4.0)$이나 3일군에 $(24.8{\pm}2.9)$비해 유의하게 감소하였다. 수정관내 절자 농도는 14일군이 $0.025{\pm}0.013$으로 대조군과 $(0.108{\pm}0.03)$ 3일군에 $(0.067{\pm}0.046)$ 비해 유의하게 감소하였으며, 3일군도 대조군에 비해 유의한 차이를 보였다. 자발적 첨체반응율은 대조군의 부정소 꼬리정자는 $37.1{\pm}2.4$이고 수정관내 정자는 $49.3{\pm}2.4$로 두 부위간 유의한 차이를 보였다. 3일군과 14일군의 부정소 꼬리와 수정관내 정자의 자발적 첨체반응율은 대조군에 비해 유의하게 증가하였다. 한편 14일군의 수정관내 정자의 자발적 첨체반응율은 대조군이나 3일군에 비해 유의하게 증가하였다. ARIC test 결과 대조군과 3일군에서는 20%이상 차이를 보였으나 14일군에서는 약 8.4% 차이를 보였다. 위의 결과가 부정소의 성숙 조절기능 이상 또는 정자형성 이상에 기인한 것인지는 더 연구되어야 하나 당뇨병 병력이 길어짐에 따라 정자의 수적인 감소, 자발적 침체반응의 증가나 침체반응의 약화가 유발되어 생식능력의 감소 원인으로 작용하는 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제22권2호
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• pp.131-141
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• 1995

This study was designed to determine the relationship between sperm acrosome reaction following ionophore challenge(ARIC) and hamster ovum sperm penetration assay(SPA) as assessment of fertilizing capacity of male. ARIC test and SPA were performed in 23 fertile and 19 subfertile men. The results were as follows; Sperm concentration was significantly higher in fertile group compared with subfertile group: $114.6{\pm}64.40$ vs $61.3{\pm}46.50{\times}10^6/ml$. However, there were no significantly differences in seminal volume, motility and motility index, respectively. There was a significantly correlation between spontaneous and induced AR in fertile and subfertile group, respectively. ARIC value was significantly higher in fertile group, compared with subfertile group: $12.0{\pm}5.57%$ vs $2.6{\pm}4.96%$. Both Penetration rate(PR) and Penetration index(PI) were significantly higher in fertile group, compared with subfertile group: $97.4{\pm}7.40%$ vs $64.9{\pm}36$. 20% and $5.4{\pm}2.88$ vs $1.5{\pm}1.47$, respectively. The Positive predictive value(PPV), Negative predictive value(NPV), sensitivity and specificity of ARIC test (cut-off: 8.5) and SPA(PI cut-off : 3.0) in predicting fertility were 95.0%, 81.8%, 82.6%, 94.7% and 95.2%, 85.7%, 87.0% and 94.7%, respectively. There was no significantly difference in predicting fertility between ARIC test and SPA. In conclusion, ARIC test was shown to have a predictive value for fertilizing capacity comparable to that of the hamster ovum sperm penetration assay. Therefore, ARIC test may be a simple and cost-effective addition to existing semenology instead of SPA.

• Asian-Australasian Journal of Animal Sciences
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• 제19권3호
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• pp.335-340
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• 2006

A large number of toxicological substances and pharmacological and physical agents can cause reproductive intervention at the cellular and molecular level. The present study was designed to assess the effect of mercury ($HgCl_2$) at 50 to $550{\mu}M$ concentration ranges, in vitro, on the sperm membrane and DNA integrity, viability, and acrosomal status of normal bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid (PBS, pH 7.2). We recorded a sharp increase in the lipid peroxidation/LPO rate; the highest was at $550{\mu}M$ mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and % viable spermatozoa (R = 0.987, p<0.001). Data obtained from a comet assay technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 92% of DNA breaks were double-stranded. The correlation between LPO rate and % DNA breaks was 0.984. Performing the gelatin test indicates that mercury is able to alter the integrity of acrosomal membranes showing an abnormal acrosome reaction. In this regard, a strong link was found between LPO rate and % halos (R = 0.990, p<0.001). Collectively, mercury proved to be a potent oxidant in the category of environmental factors affecting bull spermatozoa. Hence, considering the wide spread use of mercury and its compounds, these metals should be regarded with more concern.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.41-46
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• 2010

The objective of this study was to evaluated the efficiency of sperm cryosurvival using each extenders Triladyl and LEY containing egg yolk to the cryopreservation of separated sperm by percoll in miniature pig. The ejaculated semen from miniature pig was separated by 65% percoll and un-separated sperm as a control before freezing. The freezing of diluted semen added with Triladyl containing egg yolk and LEY solution (solution I: 11% Lactose or Triladyl + egg yolk; solution II: solution I + glycerol + OEP). Analysis of sperm ability was estimated by viability, capacitation acrosome reaction using chlortetracycline (CIC) the morphologic abnormality and hypoosmotic swelling test(HOST). The groups were designed that as separated sperm by Percoll with Triladyl(ST) or LEY(SL) for cryopreservation. And unseparated sperm with Triladyl(UT) or LEY(UL). As a results, the viability was higher significantly(p<0.05) in ST, SL, UT than UL extender. The morphologic abnormality was measured significantly (p<0.05) lower in ST than other extenders. The AR-patterned in CTC analysis was measured significantly(p<0.05) lower in SL and UL than other extenders. In conclusion, using Triladyl extender resulted in viability and morphology of separated sperm by percoll that were effective than using LEY extender, but it resulted in capacitation acrosome reaction was lower than using LEY extender.

• 한국가축번식학회지
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• 제10권1호
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• pp.91-99
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• 1986

This experiment was undertaken to examine the effects of HIS treatment on the motility and acrosome reaction of frozen bovine spermatozoa and to test their abilities to interact with zona-free hamster eggs in vitro. Also, in vitro results were compared with those of bull's fertility in AI. The frozen semen from four Holstein bulls were exposed to HIS-DM for 5 minutes after thawing and then preincubated for 60 minutes in DM prior to insemination. The hamster eggs were mounted, fixed and stained 6 hours after exposure to boving spermatozoa and examined under a phase-contrast microscope. 1. The sperm motility expressed as a mobility index dro, pp.d significantly from 60-75 to 12-24 after exposure to HIS-DM, but increased in 32 to 41 at insemination. Bull C showed a low motility index than those of the orher bulls. The percentage of acrosome reaction by staining procedure were increased by HIS-DM treatment but did not change during 7 hours incubation period in DM. 2. The overall percentage of hamster eggs interacting with bull spermatozoa was 56.3%, 58.3%, 66.6% and 70.0%, respectively. Although there was no significant difference among bulls in the penetration rate of spermatozoa into hamster eggs, high proportions of eggs interacted with spermatozoa from Bull C and D than those from Bull A and B. 3. The conception rates (60-90 day RP) resulting from AI were 62.5%, 67.5% and 70.9% for Bull A, B and C, respectively. These results were in good agreement with the invitro results that the proportions of bull sperm-egg interction were greater for Bull C than for Bull A and B.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.57-66
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• 2002

Objective : This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. Materials and Methods: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. Results: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. Conclusion: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.