• Advances in nano research
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• v.11 no.6
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• pp.649-655
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• 2021

Nanotechnology is widely considered a major technology of the twenty-first century. Nanoparticles (NPs) has been shown to pass through reproductively significant biological barriers such as the blood-testicle and placental barriers. Thus, the purpose of this study was to determine the effect of silver Nanoparticles (Ag-NPs) on sperm-egg interaction and spermatozoa quality parameters in quail spermatozoa. Semen was suspended in Ringer solution containing Ag-NPs levels at 5.5 × 106 sperm/ml (0, 0.01, 0.1, 1 and 10 ppm). The results indicated that when sperm were counted at 0.1 ppm, the number of holes formed on the inner perivitelline layer was significantly increased compared to the control. The 10 ppm group had a significant reduction in sperm viability. At 0.1 and 1 ppm, the membrane integrity was significantly decreased (P < 0.05). All treatments (except 0.01 ppm Ag-NPs) had a significant (P < 0.05) effect on the percentage of spermatozoa with an intact acrosome when compared to the control group. At 0.1, 1, and 10 ppm Ag-NPs, morphological defects in the acrosome were observed. As a result, Ag-NPs is likely capable of destroying the acrosome membrane. This research indicates that Ag-NPs may be cytotoxic to spermatozoa by impairing sperm functionality and increasing sperm mortality.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.5
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• pp.646-651
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• 2016

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

• Reproductive and Developmental Biology
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• v.40 no.4
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• pp.33-37
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• 2016

This study was conducted to evaluate effect of ${\alpha}$-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or $20\;{\mu}g/mL$ BSA. Cryo-preserved boar sperms were thawed in $37^{\circ}C$ water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

• CELLMED
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• v.10 no.1
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• pp.7.1-7.6
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• 2020

Plants produce a wide range of active principles, making them a rich source of different types of medicines. Without any knowledge of the phytoconstituents or active principles the medicinal plants itself or in the form of polyherbal formulations, were used by all society of human being from ancient times for prevention and cure of disease, but most of the traditional formulations including the formulation of Ayurveda and Unani have not been scientifically validated in order to establish the pharmacopoeial standards to improve the efficacy. Globally, the people become conscious that uses of synthetic drugs for a long period is not safe; the trend of medical society at large is looking at alternatives from natural, safe sources to combat diseases. Due to this comprehension, it has been increased the demand for plant-derived medicine, and on the other side, it is extremely important to standardize the polyherbal formulations and validate them scientifically to improve their safety and efficacy. The polyherbal Unani formulation Safuf-e-Muallif is widely used and recommended in Unani system of medicine (USM) as a spermatogenic agent, semen thickening agent, etc. to treat sexual disorders viz. premature ejaculation, nocturnal emission, etc. The study mainly deals with phytochemical screening for the detection of nature of phytoconstituents and analytical technique like High-performance thin-layer chromatography (HPTLC) for developing fingerprint of Safuf-e-Muallif revealing specific identities of the drug. The phytochemical screening and HPTLC fingerprint profile for SM reported here may be used as a reference standard for quality control purpose in future.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.1
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• pp.22-29
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• 2015

Objective: Sperm must be properly prepared in in vitro fertilization (IVF)-embryo transfer (ET) programs in order to control the fertilization rate and ensure that embryos are of high quality and have appropriate developmental abilities. The objective of this study was to determine the most optimal sperm preparation method for IVF. Methods: Patients less than 40 years of age who participated in a fresh IVF-ET cycle from November 2012 to March 2013 were included in this study. Poor responders with less than three mature oocytes were excluded. Ham's F-10 medium or sperm-washing medium (SWM) was used in combination with the density-gradient centrifugation/swim-up (DGC-SUP) or SUP methods for sperm preparation. A total of 429 fresh IVF-ET cycles were grouped according to the media and methods used for sperm preparation and retrospectively analyzed (DGC-SUP/Ham's F-10, n=82; DGC-SUP/SWM, n=43; SUP/Ham's F-10, n=181; SUP/SWM, n=123). Results: There were no significant differences among these four groups with respect to the mean age of the female partners, duration of infertility, number of previous IVF cycles, and retrieved oocytes. We determined that both the DGC-SUP and SUP methods for sperm preparation from whole semen, using either Ham's F-10 or SWM media, result in comparable clinical outcomes, including fertilization and pregnancy rates. Conclusion: We suggest that both media and both methods for sperm preparation can be used for selecting high-quality sperm for assistive reproductive technology programs.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.275-279
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• 2009

The beneficial effect of glycerol as a cryoprotectant, especially for sperm cryopreservation, has been shown in many studies. However, glycerol is toxic to living cells, and boar sperm in particular show greater sensitivity to glycerol than sperm from other domestic animals. Amides have been studied as alternative cryoprotectants for freezing stallion sperm. Sperm frozen in methylformamide or dimethylformamide as cryoprotectants show similar motility when thawed compared with sperm frozen in glycerol. We evaluated the cryoprotective effects of dimethylformamide on boar sperm freezing. To test the effect of amides, the concentration of boar semen was adjusted to $10^9sperm/mL$, and seminal plasma was removed using Hulsen solution. After centrifugation, the pellet was diluted in modified-Modena B extender. Lactose-egg yolk (LEY) extender was used as the cooling extender. The freezing extender was madeed aaddition of the optimal amount of glycerol and amides to LEY-Glycerol-Orvus ES Paste extender, and this extender was used for the second dilution. Diluted sperm were frozen in liquid nitrogen using the 0.5 mL straw method. Sperm frozen in extender with glycerol as a cderol were compared with those frozen in extender including the different amides. Sperm were tested for motility, viability, the sperm chromatin structure assay, and normal apical ridge after thawing. The percent of motile sperm diluted in glycerol was as high as that in the stallion study (61%). Dimethylformamide showed positive effects on sperm quality and was better than glycerol. Methylformamide provided similar sperm quality as glycerol. Therefore, dimethylformamide is useful for reducing cryoinjury in boar sperm and is expected to be useful as an alternative cryoprotectant.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.53-60
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• 2006

Objective: We tested the usefulness of swim-down technique using human follicular fluid (hFF) in sperm preparation. Methods: Semen samples were obtained from twelve male partners showing asthenozoospermia (sperm motility < 50%) at the time of routine andrologic evaluation in Seoul National University Bundang Hospital. After dividing into two aliquots, each samples were processed either by swim-down using 100% hFF or density gradient using SpermGrad. Sperm quality was assessed by computer-assisted semen analyzer (CASA). Results: Motility, Rapid motility, VCL (curvilinear velocity), ALH (amplitude of lateral head displacement), and hyperactivated sperms were significantly increased, and LIN (mean linearity) was decreased significantly after sperm preparation in both groups. Motility was significantly higher after swim-down using 100% hFF when compared with density gradient using SpermGrad ($81.2{\pm}4.7$ vs. $67.6{\pm}2.3$, p=0.02) The other parameters assessed by CASA were not different between the two methods. Conclusion: Swim-down method with 100% hFF may be a useful method in preparation of sperm from asthenozoospermia.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.229-235
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• 2010

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

• Animal Bioscience
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• v.35 no.3
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• pp.385-398
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• 2022

Objective: The objective was to evaluate the influence of different dietary crude protein (CP) levels during the growth phase on reproductive characteristics and reproductive efficiency as well as the body development of adult male Japanese quail. Methods: Three hundred one-day-old male quails were distributed into five treatments with diets containing different CP levels (18%, 20%, 22%, 24%, and 26%) in a completely randomized design, with six replicates of ten birds each. The CP diets were applied only during the growth phase (1 to 35 days). At 36 days of age, the birds were transferred to 30 laying cages with three males and nine females each, and all birds received the same diet formulated to meet production-phase requirements until 96 days of age. Results: The growth rate of the birds increased linearly (p<0.01) with increasing dietary CP, but the age of maximum growth decreased (p<0.05). At growth maturity, all birds had the same body weight (p>0.05). At 35 days of age, higher weight gain was obtained (p<0.05) with diets containing 22% CP or higher. No effects on feed conversion were observed in this phase. The increase in dietary CP enhanced (p<0.01) nitrogen intake and nitrogen excretion but did not affect (p>0.05) nitrogen retention. Testis size, seminiferous tubular area, number of spermatogonia, and germinal epithelial height at 35 days of age increased linearly (p<0.05) with dietary CP, while the number of Leydig cells decreased (p<0.01). The Sertoli cell number at 60 days of age increased linearly (p<0.01) with dietary CP. Dietary CP levels did not affect cloacal gland size, foam weight, foam protein concentration, semen volume, or flock fertility at 90 days of age. Conclusion: Dietary CP concentration affected body and testicular development in male Japanese quails but did not affect reproductive efficiency.

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.35-40
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• 2009

The objectives of this study was to evaluate the efficiency of the bacteria eliminated sperm by percoll gradient method on sperm quality and embryo cleavage in vitro in pig. The semen of miniature pig collected by gloved-hand method pre-warmed ($37^{\circ}C$) in thermos bottle, and separated by 65% percoll. Analysis of sperm ability was estimated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) and the abnormality. Also, fertility of sperm was monitored with cleavage rate of embryo after IVF using separated and un-separated sperm by percoll. The result, viability of separated sperm was significantly(p<0.05) higher($83.6{\pm}$2.0 vs $59.0{\pm}4.4%$) than un-separated sperm. The results of CTC analysis showed the percentage of F- and B-patterned separated sperm was higher in separated that than un-separated sperm. On the contrary, the percentage of AR-patterned form unseparaed sperm was significantly(p<0.05) higher($13.6{\pm}0.8$ vs $8.1{\pm}0.6%$) than separated sperm. Also, abnormality of un-separated sperm was significantly(p<0.05) higher($2.2{\pm}0.4$ vs $16.8{\pm}2.8%$) than separated sperm. However, the cleavage rates of embryo using separated sperm by percoll and un-separated sperm had not significantly difference on 2 cell stage(9.25 vs 11.88%), 4 cell stage(26.76 vs 24.51%) and >4 cell stage(63.99 vs 63.61%) at 48h of IVF. Therefore, the sperm separated by percoll method showed improvement in sperm quality than un-separated sperm in miniature pig.