69 AN EASY-TO-PERFORM METHOD TO ASSESS VIABILITY OF FELINE FREEZE-DRIED SPERM

2013 ◽  
Vol 25 (1) ◽  
pp. 182 ◽  
Author(s):  
L. C. O. Magalhães ◽  
C. M. Melo-Oña ◽  
M. J. Sudano ◽  
D. M. Paschoal ◽  
L. F. Crocomo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Acridine Orange ◽  
Freeze Drying ◽  
Membrane Damage ◽  
Daily Basis ◽  
Control Group ◽  
Dna Integrity ◽  
Sperm Cells ◽  
Significance Level ◽  
Freeze Dried

Freeze-drying sperm seems to be the ultimate alternative to preserve sperm cells from endangered species because of its easiness in transportation and storage of samples. This technique has already been used for equine, bovine, murine, rabbit, canine, and feline sperm cells. However, it is still important to verify the DNA integrity of such samples to produce viable offspring. The objective of this study was to verify the reliability of acridine orange (AO) staining to assess DNA damage of feline freeze-dried sperm samples. Three normospermic cats were used as sperm donors, and sperm samples were collected with the use of an artificial vagina without the presence of an oestrous queen. The control group (G1) used only fresh semen. To increase the range of sperm variability before freeze-drying, the sperm samples were supplemented with 2 extenders, namely SOF (G2) and human tubal fluid (G3). These extenders were selected because of their lack of cryoprotectant agents with a view to producing membrane damage and thus putting DNA integrity at risk. Samples were taken to a freeze-dryer (Edwards do Brasil, Brazil) to produce stable free-dried sperm. Over 50 samples were assessed for DNA damage by using AO and alkaline comet assay (CA). Acridine orange taints red/orange the sperm cells that present sperm damage, and CA shows long comet tails for cells with DNA damage. The CA was selected to countercheck the AO results owing to its higher accuracy. Nevertheless, both AO and CA count 100 cells per analysis, and a total of 3 repetitions per sample were sufficient. For the statistical analysis, SAS PROC GLM was used, and the significance level was set at 0.05. The AO staining showed that the G1 samples had a greater DNA damage (27.1%) if compared with G2 (15.2%) and G3 (23.1%; P < 0.0001). Nevertheless, G2 and G3 displayed sufficient DNA integrity throughout the whole lyophilization process. The CA, which is a more precise evaluation technique, proved that the groups had distinguished results but still were ordered in the same way: G1 was the one with more DNA damage (17.7%), G3 was second (14.4%), and G2 was third (3.2%) in DNA alterations. Just like AO, the CA showed difference among treatments (P < 0.0001). Since AO is a technique that requires very little sophistication and presented about the same results as the very elaborate CA method, it might be used on a daily basis to assess feline sperm DNA that underwent the freeze-drying process.

scholarly journals Selenium supplementation prevents DNA damage in ram spermatozoa

2021 ◽  
Vol 51 (1) ◽  
Author(s):  
Carla Fredrichsen Moya ◽  
Marcelo Piagentini ◽  
Danilo da Cunha Silva ◽  
Fábio Henrique Fernandes ◽  
Daisy Maria Fávero Salvadori ◽  
...  
Keyword(s):  
Dna Damage ◽  
Latin Square ◽  
Negative Control ◽  
Mineral Salt ◽  
Control Group ◽  
Dna Integrity ◽  
Protective Effects ◽  
Mineral Supplement ◽  
Significance Level ◽  
Mean Differences

ABSTRACT: In the present study, we aimed to evaluate the effects of different concentrations of selenium (Se) ovine nutritional supplementation on spermatozoa DNA integrity. Thirty male ovines (age: 10 months) were used. They were fed with hay and ram food in an intensive system, which was divided into stalls (5 m long and 3 m wide) with feeding troughs, and had ad libitum access to food and water. Ovines in group 1 (G1, the negative control) received mineral salt supplementation without Se; ovines in G2 received the same mineral salt mixed with 5 mg Se (as sodium selenite)/kg mineral supplement;ovines in G3 received 10 mg Se/kg mineral supplement; ovines in G4 received 15 mg Se/kg mineral supplement; and ovines in G5 received 20 mg Se/kg mineral supplement. Ovines in all groups remained untreated for 14 days, followed by a treatment period of 56 days. Semen samples were obtained by electroejaculation. The DNA damage in semen samples was evaluated using the comet assay. The experimental design was implemented using a 5 × 5 Latin Square, i.e., five treatments and five experimental periods. The mean differences were compared using Tukey’s test at a significance level of 5%. The control group (G1) showed a high percentage of DNA damage compared to the Se-treated groups (G2-G5). Therefore, Se supplementation could decrease the basal level of DNA damage in sperm cells, suggesting that Se might exert protective effects on sperm DNA.

Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 15-24 ◽  
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
A. Takizawa ◽  
N. Maedomari ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Freeze Drying ◽  
Chelating Agents ◽  
Sperm Head ◽  
Control Group ◽  
Sperm Dna Fragmentation ◽  
Freeze Dried ◽  
Boar Sperm

SUMMARYSuccessful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1–4) did not increase, even at 180 min (0.7–4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120–180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0–60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39.The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

scholarly journals Prolonged use of finasteride-induced gonadal sex steroids alterations, DNA damage and menstrual bleeding in women

2020 ◽  
Vol 40 (2) ◽  
Author(s):  
Gadah Albasher ◽  
May Bin-Jumah ◽  
Saleh Alfarraj ◽  
Fatimah Al-Otibi ◽  
Nouf K. Al-Sultan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Sex Steroids ◽  
Serum Levels ◽  
Treated Group ◽  
Heavy Menstrual Bleeding ◽  
Menstrual Bleeding ◽  
Control Group ◽  
Dna Integrity ◽  
Adverse Health Effects

Abstract The aim of the present study was to examine the effect of prolonged use of finasteride on serum levels of dihydrotestosterone (DHT), estradiol (E2), progesterone, testosterone and androstenedione in women during the menstrual period. Further, to screen and compare the 5α-reductase activities through the expression of SRD5A1, SRD5A2 and AR gene and to determine the level of VEGF, VKOR and SAA gene expression and DNA damage. A total of 30 Saudi women aged between 25 and 35 years were enrolled in the study. The selected women were divided into two groups. The first group (n = 15) received 5 mg finasteride/day for prolonged period of one year and second group (n = 15) was taken as a healthy control. ELISA technique was used for measuring the serum levels of the targeted hormones, and Comet assay was used for checking the DNA integrity. Our findings revealed significant decrement of DHT, E2, progesterone and androstenedione levels and elevated levels of testosterone in group treated with daily oral doses of 5 mg finasteride/day compared with the control subjects. mRNA expression suggested that finasteride has concrete effects on the gene expression of the selected genes from the treated group in comparison with the control group. In addition, finasteride induced DNA damage, and heavy menstrual bleeding was noted in women treated with finasteride. In conclusion, the present findings revealed that finasteride has adverse health effects in women associated with gonadal sex steroids alterations, DNA damage and heavy menstrual bleeding with no consensus in the treatment of androgenetic alopecia in women.

scholarly journals Effect of addition an amino acid or its combination with EDTA on DNA integrity and morphometry sperm heads of freeze-dried bovine spermatozoa

2020 ◽  
Vol 45 (3) ◽  
pp. 189-196
Author(s):  
S. Said ◽  
T. Maulana ◽  
S. Setiorini ◽  
G.E. Ibrahim ◽  
M.N. Ramadhan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Freeze Drying ◽  
Sperm Head ◽  
Test Method ◽  
Dna Integrity ◽  
Drying Process ◽  
Freeze Dried ◽  
Bovine Spermatozoa ◽  
Amino Acid Glycine

The objective of the current study was to investigate the effect of addition an amino acid or its combination with EDTA on DNA integrity and morphometry sperm heads of freeze-dried bovine spermatozoa. Spermatozoa were freeze-dried in medium with the addition of an amino acid glycine, cysteine, glutamine, or its combination with EDTA. Sperm head morphometry was identified at 400X magnification using Axio Vision(Zeiss Company, Germany), while for membrane plasma integrity (MPI) determined by calculation of the percentage of spermatozoa having intact plasma membrane by osmotic resistance test method and DNA integrity analysis using acridine orange staining. Sperm head had declined in size after the freeze-drying process, MPI of FD spermatozoa gradually increased significantly when FD solution was added with an amino acid solution (glycine, cysteine) and its combination with EDTA. DNA integrity of all freeze-dried spermatozoa treatments was remaining intact, no significantly different (P>0.01) among treatments. The present study concluded that the addition of an amino acid (glycine, cysteine) or its combination with EDTA could be reduced morphometric sperm heads and plasma membrane damage of freeze-dried bovine spermatozoa, however, DNA integrity of bovine sperm nucleus remaining intact after the freeze-drying process without addition both amino acids and EDTA. 

scholarly journals Differences in donor compatibility for fresh and freeze-dried homologous platelet rich plasma studied using crossmatch test

2020 ◽  
Vol 28 ◽  
pp. 03003
Author(s):  
Nandini Anindita Sumitro ◽  
Wiwin Winda Kusumadewi ◽  
Fitri Yuniawati ◽  
Naila Amalia ◽  
Hendrawati Hendrawati ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Blood Donors ◽  
Platelet Rich Plasma ◽  
Test Method ◽  
Control Group ◽  
Test Results ◽  
Chi Square ◽  
Freeze Dried ◽  
Periodontal Tissue Regeneration ◽  
Chi Square Test

Platelet-rich plasma (PRP) rich in growth factors has evolved as an important therapy for periodontal tissue regeneration. A healthy blood donors obtained for homologous PRP (h-PRP). The Freeze-drying h-PRP sample provides an effective method to ensure a longer shelf-life. The h-PRP samples are subjected to crossmatch testing in clinics to prevent any immune response in recipients. The present study aimed to evaluate the differences in donor compatibility on crossmatch test results towards fresh and freeze-dried h-PRP (FD h-PRP). This was a laboratory experiment, h-PRP prepared according to the protocol of blood bank, and 40 recipients blood samples divided into two groups, fresh h-PRP (control group) and FD h-PRP. The crossmatch test was performed to evaluate h-PRP compatibilities by using the gel-test method. The data were analyzed using the chi-square test. The results of the study showed that the crossmatch test on FD h-PRP samples was 100 % compatible and could increase the compatibility results of the donor. the FD h-PRP was safe to become donors and clinical applications.

scholarly journals Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Luca Palazzese ◽  
Debora Agata Anzalone ◽  
Federica Turri ◽  
Marco Faieta ◽  
Anna Donnadio ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Structure ◽  
Mechanical Damage ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Residual Water ◽  
Gravimetric Analysis ◽  
Technological Advancement ◽  
Developmental Potential ◽  
Freeze Dried

Abstract Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. In order to minimize the physical/mechanical damage resulting from lyophilization, here we focused on the freezing phase, comparing two cooling protocols: (i) rapid-freezing, where ram sperm sample is directly plunged into liquid nitrogen (LN-group), as currently done; (ii) slow-freezing, where the sample is progressively cooled to − 50 °C (SF-group). The spermatozoa dried in both conditions were analysed to assess residual water content by Thermal Gravimetric Analysis (TGA) and DNA integrity using Sperm Chromatin Structure Assay (SCSA). TGA revealed more than 90% of water subtraction in both groups. A minor DNA damage, Double-Strand Break (DSB) in particular, characterized by a lower degree of abnormal chromatin structure (Alpha-T), was detected in the SF-group, comparing to the LN-one. In accordance with the structural and DNA integrity data, spermatozoa from SF-group had the best embryonic development rates, comparing to LN-group: cleaved embryos [42/100 (42%) versus 19/75 (25.3%), P < 0.05, SL and LN respectively] and blastocyst formation [7/100 (7%) versus 2/75 (2.7%), P < 0.05, SF and LN respectively]. This data represents a significant technological advancement for the development of lyophilization as a valuable and cheaper alternative to deep-freezing in LN for ram semen.

scholarly journals Assessment of three generations of mice derived by ICSI using freeze-dried sperm

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 239-251 ◽  
Author(s):  
Ming-Wen Li ◽  
Brandon J. Willis ◽  
Stephen M. Griffey ◽  
Jimmy L. Spearow ◽  
K. C. Kent Lloyd
Keyword(s):  
Genomic Instability ◽  
Freeze Drying ◽  
Mouse Strains ◽  
Control Group ◽  
Three Generations ◽  
Phenotypic Abnormality ◽  
Freeze Dried ◽  
Two Generations

SummaryAlthough the derivation of mice by intracytoplasmic sperm injection (ICSI) using freeze-dried sperm has been demonstrated previously, a comprehensive analysis of their viability, health, and fertility has not. The purpose of the present study was to determine the extent to which ICSI using freeze-dried sperm stored at 4 °C for 1–2 months from mice on either an inbred (C57BL/6J) or hybrid (B6D2F1/J) genetic background results in genomic instability and/or phenotypic abnormality in mice and two generations of their progeny. Fertilization rates (number of 2-cells per injected oocytes) using ICSI of fresh and freeze-dried sperm were similar within and between mouse strains, although fewer freeze-dried sperm-derived embryos than fresh sperm-derived embryos developed to blastocystsin vitro(C57BL/6J and B6D2F1/J) and liveborn pupsin vivo(B6D2F1/J only). Nevertheless, once born, mice derived by ICSI using freeze-dried sperm in both mouse strains were healthy and reproductively sound. No major differences in litter size, weaning rate, and sex ratio were noted in the two generations of progeny (F2 and F3) of ICSI-derived offspring using freeze-dried sperm compared with that in the natural mating (control) group. Further, there was no evidence that either ICSI or freeze drying induced genomic instability, as determined by microsatellite analysis of the derived mice and subsequent generations when compared with both parental genotypes, nor were there differences in the number or types of pathological changes in any of the three generations of progeny. We conclude that viable, healthy and genomically stable mice can be derived by ICSI using freeze-dried mouse sperm stored in the refrigerator for at least 2 months. Further, because freeze drying is a simpler and more economical technique compared with embryo and sperm cryopreservation, the results of this study justify additional research to continue to develop and enhance the technique for the preservation, storage, and sharing of genetically altered mice.

scholarly journals Collapsed mitochondrial cristae in goat spermatozoa due to mercury result in lethality and compromised motility along with altered kinematic patterns

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bhawna Kushawaha ◽  
Rajkumar Singh Yadav ◽  
Dilip Kumar Swain ◽  
Priyambada Kumari ◽  
Akhilesh Kumar ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mercuric Chloride ◽  
Control Group ◽  
Sperm Cells ◽  
Functional Dynamics ◽  
Transmission Electron ◽  
Middle Piece ◽  
Acrosomal Membrane ◽  
Bax Gene

AbstractEarlier we have reported mercury-induced alterations in functional dynamics of buck spermatozoa through free radicals-mediated oxidative stress and spontaneous acrosome reaction. Based on our earlier findings, we aimed to investigate the effect of mercury exposure on motility, kinematic patterns, DNA damage, apoptosis and ultra-structural alterations in goat spermatozoa following in vitro exposure to different concentrations (0.031–1.25 µg/ml) of mercuric chloride for 15 min and 3 h. Following exposure of sperm cells to 0.031 µg/ml of mercuric chloride for 3 h, livability and motility of sperms was significantly reduced along with altered kinematic patterns, significant increase in per cent necrotic sperm cells and number of cells showing DNA damage; and this effect was dose- and time-dependent. Contrary to up-regulation of Bax gene after 3 h in control group, there was significant increase in expression of Bcl-2 in mercury-treated groups. Transmission electron microscopy studies revealed rifts and nicks in plasma and acrosomal membrane, mitochondrial sheath, and collapsed mitochondria with loss of helical organization of mitochondria in the middle piece of spermatozoa. Our findings evidently suggest that mercury induces necrosis instead of apoptosis and targets the membrane, acrosome, mid piece of sperms; and the damage to mitochondria seems to be responsible for alterations in functional and kinematic attributes of spermatozoa.

scholarly journals THE EFFECT OF ALLOGENIC FREEZE DRIED PLATELET-RICH PLASMA IN RESPONSES INFLAMMATION REACTION OF RABBIT

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Trio Rachmawati
Keyword(s):  
Inflammatory Response ◽  
Freeze Drying ◽  
Transforming Growth Factor ◽  
Platelet Rich Plasma ◽  
T Test ◽  
Control Group ◽  
Freeze Dried ◽  
Before And After ◽  
Inflammation Reaction ◽  
Tgf Β1

This study aims to analyze the effects of allogenic freeze dried platelet-rich plasma in responses inflammation reaction of rabbit. The designs of this study are one group pretest posttest conducted to determine the effect of freeze drying on levels of TGF-β1 PRP and post test only control group design conducted to determine the effect of allogenic freeze dried PRP. Nine samples of PRP which examined levels of TGF-β1 before and after freeze drying were obtained from blood centrifugation of three rabbits. These nine samples were used as allogenic donor which injected intramuscularly in nine rabbits for the treatment groups. The control group used nine rabbits which was injected intramuscularly using autologous PRP. Both groups were observed inflammatory response. Measurement of TGF-β1 levels before and after freeze drying were tested statistically using T- test dependent. Data inflammatory response were tested statistically using T- test independent. The results showed that no effect of freeze drying process on levels of TGF-β1. Allogenic freeze dried PRP did not cause an iflammatory response.Keywords : autologous, allogenic, freeze dried platelet rich plasma, transforming growth factor- β1.

scholarly journals Efficacy of Melatonin against Oxidative Stress, DNA Damage and Histopathological Changes Induced by Nicotine in Liver and Kidneys of Male Rats

2021 ◽  
Vol 10 (1) ◽  
pp. 31-36
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Histopathological Examination ◽  
Harmful Effect ◽  
Male Rats ◽  
10Mg Dose ◽  
Control Group ◽  
Dna Integrity ◽  
Histopathological Changes ◽  
Antioxidant Parameters

The present study was carried out to investigate and compare the effect of nicotine alone and in combination with melatonin on some oxidants and antioxidant parameters, histopathological changes and DNA integrity in the liver and kidneys of male rats. For this purpose 75 mature male rats weighing 120-140g were randomly divided into five groups; control group (1% ethanol in saline), nicotine group (rats administrated nicotine at a dose of 0.6mg/kg body weight; BW) and nicotine and melatonin groups (rats administrated the same dose of nicotine plus 1, 5 or 10mg/kg BW melatonin, respectively). Nicotine and ‏ melatonin were injected intraperitoneally daily for 21days. Fasting blood samples were collected from each rat one day after the end of last injection (at 22nd day) and sera were collected for determination of total antioxidant capacity (TAC). Five rats were sacrificed from each group; Liver and kidneys were collected for estimation of oxidative stress parameters (MDA, SOD and GSH), histopathological examination and for estimation of DNA damage. The results revealed that nicotine increased MDA, decreased TAC, SOD and GSH, induced histopathological changes and increased the percentage of DNA damage in the liver and kidneys Melatonin administration with nicotine counteracted the effect of nicotine on previous parameters. The effect of melatonin was dose dependent and the 10mg dose produced the highest protective effect. It is concluded that melatonin can ameliorate the harmful effect of nicotine on the liver and kidneys of male rats.

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