• Asian-Australasian Journal of Animal Sciences
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• 제15권5호
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• pp.633-636
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• 2002

Six adult Garole rams maintained under a semi-intensive system were used as semen donors for this study. Semen was collectied daily during the monsoon season with the help of an artificial vagina and examined for its quality characteristics. Ejaculates of thick consistency, rapid wave motion, ${\geq}80%$ motility and intense movement of motile spermatozoa were diluted at the rate of 1:1 with egg yolk McIllvaine glucose diluent at $30^{\circ}C$ in water bath. Estrus in ewes was detected by parading aproned rams of proven vigour at 12 h intervals. The ewes (54 Malpura and 23 Avikalin) in estrus were artificially inseminated with fresh diluted ram semen. The overall conception rate was 94.8%, (range 91.7 to 100%). The overall lambing percent was 80.5 with a range of 75.0 to 84.6%. There was no significant (p>0.05) difference in lambing and conception rate because of individual rams. Fertility was significantly lower (p<0.05) in ewes of less than two years and more than six years of age. Breed (Malpura and Avikalin) effect was not observed in conception and lambing rate (p>0.05). No significant difference (p>0.05) in birth weight and 12 month weight was observed between Garole${\times}$Avikalin and Garole${\times}$Malpura crossbred lambs but there was significant (p<0.05) difference at three month and six months body weight of both the crossbred lambs.

• Animal Bioscience
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• 제37권2호
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• pp.203-209
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• 2024

Objective: This study aimed to assess the impact of the dilution ratio of Tris diluent, storage at 0℃, and long-distance transportation on the spermatozoa of Simmental cattle. It also validated the feasibility of the regional distribution of fresh semen. Methods: In experiment 1, semen was diluted at four dilution ratios (1:6, 1:9, 1:12, and 1:15) to determine the optimal dilution ratio of Tris diluent. In experiment 2, we assessed sperm viability, progressive motility (objectively assessed by computer-assisted sperm analyzer), and acrosome intactness in Tris dilutions kept at constant 0℃ for 1, 3, 6, 9, and 12 days. We compared them to Tianshan livestock dilutions (Commercial diluent). In experiment 3, semen was diluted using Tris diluent, and sperm quality was measured before and after long-distance transport. Artificial insemination of 177 Simmental heifers compared to 156 using Tianshan Livestock dilution. Results: The outcomes demonstrated that 1:9 was the ideal Tris diluent dilution ratio. The sperm viability, Progressive Motility, and acrosome integrity of both Tris and Tianshan dilutions preserved at 0℃ gradually decreased over time. sperm viability was above 50% for both dilutions on d 9, with a flat rate of decline. The decrease in acrosome integrity rate was faster for Tianshan livestock dilutions than for Tris dilutions when stored at 0℃ for 1 to 6 days. There was no significant difference (p>0.05) in sperm viability between semen preserved in Tris diluent after long-distance transportation and semen preserved in resting condition. The conception rates for Tris dilution and Tianshan livestock dilution were 49.15% and 46.15% respectively, with no significant difference (p>0.05). Conclusion: This shows that Tris diluent is a good long-term protectant. It has been observed that fresh semen can be successfully preserved for long-distance transport when stored under 0℃ conditions. Additionally, it is feasible to distribute semen regionally.

• 한국가금학회지
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• 제42권2호
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• pp.109-116
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• 2015

본 연구에서는 육종이 필요한 종계장에서 슬라이드 위에 도말된 닭 정액시료를 간편하게 염색하여 정자의 염색질 이상성과 운동성을 유추할 수 있는 기법을 확립하고자 실시하였다. 오계 정자 도말 슬라이드를 Diff-Quik 시약으로 염색하여 관찰하면 신선정액에서는 정자 생존성이 93.53%, 82.42% 및 90.63%일 때, Diff-Quik 염색질의 정상도는 87.96%, 74.25% 및 85.10%로 관찰되었다. 동일한 시료를 동결하고 융해후 정액의 생존성은 조사하면 69.58%, 61.98% 및 72.20%로 관찰되었으며, 염색질의 정상도는 58.91%, 48.49% 및 63.34%로 관찰되었다. 융해된 동결정액에서 활력이 우수한 정자를 쉽게 관찰하기 위하여 정자를 HS-1 희석액으로 재 희석하고, $37^{\circ}C$에서 가온하여 도말하면 염색된 정자의 두부에서 활력이 우수한 정자의 비율을 간단히 유추할 수 있음을 보여주었다. 특히, 신선 정자에서 정상 염색질을 가진 정자의 비율과 생존성이 상관관계는 매우 높은 것으로 판단되며, 동결정자에서도 상관관계가 높다고 추정되었다. 이러한 결과는 Diff-Quik 염색방법으로 닭 정액의 품질을 정자의 염색질의 이상성 유무로 판단할 수 있음을 보여주고 있다. 특히 본 연구에서 제시된 방법은 닭 정자의 우수성을 판단하는데 유용할 것으로 판단되며, 닭 정액 시료를 준비할 때 준비한 도말슬라이드를 현미경적 관찰에 의하여 활성화된 정자의 비율과 정상 염색질을 가진 정자의 비율을 추정하는 방법을 제시하였으며, 이는 인공 수정에 필요한 현장에서 수컷 개체의 종축 이용성을 쉽게 판단할 수 있는 근거를 마련할 수 있음을 의미한다.

• Clinical and Experimental Reproductive Medicine
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• 제17권2호
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• pp.123-128
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• 1990

Capacitation of mouse spermatozoa in vitro is brought about by epididymal secretions released into the m-Tgrode medium at the time of sperm collection. Epididymal mouse sperm suspension achived by centrifugation were preincubated for a total of 120min with aliquants being removed at 5, 30 and 120min. By gently centrifugation and resuspending in fresh medium, the fertilizing rate of unwashed 5-and 30-min suspensions was increased such that 30-min washed samples did not differ significiantly from full capacitated, highly fertile 120-min unwashed samples. When epididymal suspension was added fertilization of cumulus intact oocyte was markedly inhibited, although fertilization of zona free oocytes was unaffected. Washing sperm suspensions preincubated in the absence of $Ca^{2+}$ with the subsequent introduction of exogenous $Ca^{2+}$ resulted in a significant increase in fertilization rates over equivalent unwashed samples.

• Clinical and Experimental Reproductive Medicine
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• 제49권2호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제28권2호
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• pp.155-160
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• 2001

Objective: ICSI with testicular sperm could achieve optimal fertilization and pregnancy. This study was performed to observe the influence on fertilization and pregnancy of motility of fresh testicular sperm and sperm extracted from frozen-thawed seminiferous tubules in obstructive azoospermia. Materials and Methods: We analysed clinical outcome of ICSI using fresh testicular sperm and sperm extracted from thawed seminiferous tubules. The presence of motility were compared to determine the factor for optimal fertilization and pregnancy rates. Results: In 316 cases of TESE-ICSI in obstructive azoospermia, ICSI with fresh testicular sperm (fresh sperm group) were 163 cases and ICSI with sperm testicular sperm extracted from frozen-thawed seminiferous tubule (thawed sperm group) were 153 cases. The fertilization rates were 71.3% and pregnancy rates were 32.5% in fresh sperm group, in thawed sperm group, 65.1% and 33.3% respectively. The fertilization and pregnancy rates of motile and non-motile testicular sperm were 72.9% and 33.6%, 50.0% and 18.2%, respectively (p<0.05). The fertilization and pregnancy rates of motile and non-motile sperm extracted from the thawed seminiferous tubule were 67.8% and 34.7%, 55.1% and 28.1%, respectively (p<0.05). The comparative of the results of ICSI using motile fresh testicular sperm and motile sperm extracted from thawed seminiferous tubule, fertilization and pregnancy rates were not significantly different (72.9% and 33.6%, 67.8% and 34.7%, respectively). Conclusion: These results suggest that successful pregnancy in TESE-ICSI treatment is influenced by the motility of fresh testicular sperm and sperm extracted from thawed seminiferous tubule in obstructive azoospennic patients.

• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.153-162
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• 1992

To solve the logistical problems of the sperm penetration assay (SPA) to provide just a sufficient number of hamster ova exactly when they are needed, a new method to cryopreserve the ova has been devised (1-step dehydration and 2-step thawing). After freezing & thawing of zona-intact (ZI) and zona-free (ZF) hamster ova according to this new method, the frozen-thawed ova were compared with fresh, control ova (FO) in terms of the degree of sperm penetration in SPA using semen samples from fertile donors, subfertile, and infertile male. Each sperm sample was capacitated for 42 hours inTEST-Yolk Buffer before insemination in SPA. In fertile doner, both the penetration rate and penetration index were lower in SPA using frozen ova (ZI; 92.4%, 6.2, ZF; 63.7%, 3.9) than those of SPA using fresh ova (99.3%, 8.4). There was a significant correlation between the penetration index of SPA using FO and ZI (p<0.001), and between those of SPA using FO and ZF and ova (p<0.001). In subfertile patient, both the penetration rate and penetration index were lowered in frozen ova (ZI; 62.3%, 1.3, ZF; 21.8%, 0.4) than those of fresh ova (74.8%, 1.8). There were significant correlation between the penetration rate and penetration index in ZI ova (p<0.05 and p<0.001, respectively). In infertile patient, both the penetration rate and penetration index were ZI; 3.1%, 0.0, ZF;0.0%, 0.0, respectively. There were significant correlation between the penetration rate and penetration index in ZI ova (p<0.05).

• Clinical and Experimental Reproductive Medicine
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• 제50권1호
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• pp.44-52
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• 2023

Objective: The DNA integrity of spermatozoa that attach to fallopian tube (FT) cells is higher than spermatozoa that do not attach. FT epithelial cells can distinguish normal and abnormal sperm chromatin. This study investigated the effects of sperm with a high-DNA fragmentation index (DFI) from men with unexplained repeated implantation failure (RIF) on the Toll-like receptor (TLR) signaling pathway in human FT cells in vitro. Methods: Ten men with a RIF history and high-DFI and 10 healthy donors with low-DFI comprised the high-DFI (>30%) and control (<30%) groups, respectively. After fresh semen preparation, sperm were co-cultured with a human FT epithelial cell line (OE-E6/E7) for 24 hours. RNA was extracted from the cell line and the human innate and adaptive immune responses were tested using an RT2 profiler polymerase chain reaction (PCR) array. Results: The PCR array data showed significantly higher TLR-1, TLR-2, TLR-3, TLR-6, interleukin 1α (IL-1α), IL-1β, IL-6, IL-12, interferon α (IFN-α), IFN-β, tumor necrosis factor α (TNF-α), CXCL8, GM-CSF, G-CSF, CD14, ELK1, IRAK1, IRAK2, IRAK4, IRF1, IRF3, LY96, MAP2K3, MAP2K4, MAP3K7, MAP4K4, MAPK8, MAPK8IP3, MYD88, NFKB1, NFKB2, REL, TIRAP, and TRAF6 expression in the high-DFI group than in the control group. These factors are all involved in the TLR-MyD88 signaling pathway. Conclusion: The MyD88-dependent pathway through TLR-1, TLR-2, and TLR-6 activation may be one of the main inflammatory pathways activated by high-DFI sperm from men with RIF. Following activation of this pathway, epithelial cells produce inflammatory cytokines, resulting in neutrophil infiltration, activation, phagocytosis, neutrophil extracellular trap formation, and apoptosis.

• Clinical and Experimental Reproductive Medicine
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• 제44권4호
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• pp.224-231
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• 2017

Objective: We studied the association between sperm DNA fragmentation (SDF) and several clinical in vitro fertilization outcomes. Methods: We retrospectively analyzed 169 consecutive fresh IVF cycles. Semen was collected on the day of oocyte retrieval, and we assessed standard semen parameters and the SDF level (by terminal deoxynucleotidyl transferase dUTP nick-end labeling). Poor ovarian response (POR) was defined as the collection of three or fewer mature oocytes. Oocytes were inseminated by the conventional method or intracytoplasmic sperm injection. Results: SDF did not affect the fertilization or pregnancy rate, but did have a significant effect on the miscarriage rate. In the miscarriage group (n = 10), the SDF level was significantly higher (23.9% vs. 14.1%) and number of mature oocytes was significantly lower (4.3 vs. 7.6) than in the live birth group (n = 45). Multiple regression analysis showed that SDF was an independent predictor of miscarriage (odds ratio, 1.051; 95% confidence interval, 1.001-1.104). The cutoffs for the SDF level and number of mature oocytes that could predict miscarriage were > 13% and ${\leq}3$, respectively. In the low-SDF group (${\leq}13%$), the miscarriage rate was similar in POR patients and those with a normal ovarian response (NOR; 14.2% vs. 4.3%). In the high-SDF group ( > 13%), the miscarriage rate was significantly higher in the POR group than in the NOR group (60.0% vs. 13.3%, p= 0.045). Conclusion: Our study demonstrated that a high SDF level ( > 13%) was associated with a high miscarriage rate, and that it mainly contributed to miscarriage in the POR group. The results suggest that SDF measurements should be considered in couples with POR in order to predict the prognosis of the pregnancy.

• 한국수정란이식학회지
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• 제28권1호
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• pp.63-71
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• 2013

In this study, we used flow a cytometric assay to evaluate plasma membrane integrity and mitochondrial activity in post-thawed sperm that was supplemented with ginsenoside-$Rg_1$. Varying concentrations of ginsenoside-$Rg_1$ (0, 25, 50 and $100{\mu}M/ml$) were used in the extender during cryopreservation to protect the DNA of thawed sperm, thereby increasing the viability and motility rate as evaluated using a computer-assisted sperm analysis (CASA) method. The results derived from CASA were used to compare the fresh, control, and ginsenoside-$Rg_1$ groups. Sperm motility and the number of progressively motile sperm were significantly (p<0.05) higher in the $50{\mu}M/ml$ ginsenoside-Rg1 group ($61.0{\pm}4.65%$) than in the control ($46.6{\pm}7.02%$), $25{\mu}M/ml$ ($46.2{\pm}4.76%$), and $100{\mu}M/ml$ ginsenoside-$Rg_1$ ($52.0{\pm}1.90%$) groups. However, the velocity distribution of post-thawed sperm did not differ significantly. Membrane integrity and MMP staining as revealed using flow cytometry were significantly (p<0.05) higher ($91.6{\pm}0.82%$) in the $50{\mu}M/ml$ ginsenoside-$Rg_1$ group than in the other groups. Here, we report that ginsenoside-$Rg_1$ affects the motility and viability of boar spermatozoa. Moreover, ginsenoside-$Rg_1$ can be used as a protective additive for the suppression of intracellular mitochondrial oxidative stress caused by cryopreservation.