Dimethyl acetamide and dimethyl sulfoxide associated at glucose and egg yolk for cryopreservation of Pseudoplatystoma corruscans semen

This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility > 80%. After thawing, all treatments provided semen with total sperm motility > 40%, with no significant difference (P < 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P. corruscans semen samples.(AU)

Este estudo teve como objetivo desenvolver um protocolo para a criopreservação do sêmen de Pseudoplatystoma corruscans. Para tal, machos maduros foram induzidos hormonalmente com uma dose única de extrato de hipófise de carpa (5 mg/kg de peso vivo). O sêmen foi coletado e avaliado. Sendo testados para compor os diluentes, dois crioprotetores: dimetil acetamida (DMA) e dimetil sulfóxido (Me2SO), em duas concentrações (8% e 10%), + 5,0% glicose + 10% gema de ovo. O sêmen foi diluído na proporção 1: 4 (sêmen: extensor), embalado em palhetas de 0,5 mL e congelado em container dryshipper em vapores de nitrogênio líquido. Após o descongelamento, foram avaliados os aspectos cinéticos espermáticos, a morfologia espermática e a integridade do DNA dos espermatozoides criopreservados. Os machos de P. corruscans produziram sêmen com motilidade espermática > 80%. Todos os tratamentos proporcionaram após o descongelamento sêmen com motilidade espermática total > 40%, sem diferença significativa (P < 0,05) entre eles, como também entre os demais parâmetros cinéticos espermáticos avaliados. Os tratamentos com DMA proporcionaram uma menor fragmentação do DNA dos gametas. Malformações espermáticas foram identificadas, tanto no sêmen fresco, como no criopreservado, sendo identificado um aumento discreto dessas malformações nos espermatozoides das amostras de sêmen descongeladas de P. corruscans.(AU)

Seminal characteristics and sensitivity of Astyanax lacustris (Characiformes: Characidae) sperm to cryoprotective solutions based on dimethylsufoxide and methylglicol

This study aimed to determine the semen characteristics of Astyanax lacustris after hormonal induction and to evaluate the sensitivity of the species sperm to cryoprotective solutions based on the cryoprotectants dimethyl sulfoxide and methyl glycol. Volume, color, sperm concentration, total motility and aspects of sperm movement were analyzed using "Integrated Semen Analysis System". Three different extenders were tested: A) glucose 5%+egg yolk 10%, B) BTS®5% and C) glucose 5% and two permeable cryoprotectants: dimethyl sulfoxide (Me2SO) and methyl glycol (MTG). Fresh A. lacustris semen presented total motility of 76.6±11.2%, motility duration of 33.0±2.2s, sperm concentration of 7.22±3.2×109sptz/mL and seminal osmolality of 219±0.03mOsm/kg-1. The toxicity test showed the highest total motility values at the MTG15%+A, Me2SO15%+B and Me2SO10%+C dilutions, and the Me2SO10%+C and Me2SO15%+C dilutions presented the highest values for curvilinear velocity, linear velocity and average velocity. The tested protocol was not effective at maintaining the viability of A. lacustris semen after freezing because no motility was observed in any of the dilutions. However, the Comet Assay demonstrated that cryoprotectant solutions were effective in protecting the genetic material of cells, as DNA damage levels were low, with no difference between control and Me2SO10% + A, dilutions MTG10%+C, Me2SO10%+B and Me2SO15%+B.(AU)

O objetivo deste estudo foi determinar as características do sêmen de Astyanax lacustris após indução hormonal e avaliar a sensibilidade dos espermatozoides da espécie a soluções crioprotetoras baseadas nos crioprotetores dimetilsulfóxido e metilglicol. Volume, cor, concentração espermática, motilidade total e aspectos do movimento espermático foram analisados usando o "Sistema Integrado de Análise de Sêmen (ISAS®CASA)". Três extensores diferentes foram testados: A) glicose 5%+gema de ovo 10%, B) BTS® 5% e C) glicose 5% e dois crioprotetores permeáveis: dimetilsulfóxido (Me2SO) e metilglicol (MTG). O sêmen fresco de A. lacustris apresentou motilidade total 76,6±11,2%, duração da motilidade 33,0±2,2s, concentração de espermatozoides 7,22±3,2×109sptz/mL e osmolalidade seminal 219±0,03mOsm/kg-1. O teste de toxicidade apresentou maiores valores de motilidade total nas diluições MTG15%+A, Me2SO15%+B e Me2SO10%+C, e as diluições Me2SO10%+C e Me2SO15%+C apresentaram maiores valores de velocidade curvilínea, velocidade linear e velocidade média. O protocolo testado não foi eficaz em manter a viabilidade do sêmen de A. lacustris pós-congelamento, pois não foi observada motilidade em nenhuma das diluições. No entanto, o Ensaio Cometa demonstrou que as soluções crioprotetoras eram eficazes na proteção do material genético das células, pois os níveis de dano ao DNA eram baixos, sem diferença entre controle e Me2SO10%+A, MTG10%+C, Me2SO10%+B e Me2SO15%+B.(AU)

Chromosome polymorphisms and their influence on semen quality and sperm DNA integrity in males undergoing IVF/ICSI / 中华男科学杂志

Objective@#To investigate the incidence of chromosome polymorphisms and their influence on semen quality and sperm DNA integrity in male patients receiving in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI).@*METHODS@#We retrospectively analyzed the chromosomal karyotypes and the types and incidence rate of chromosome polymorphisms in 2 370 male patients undergoing IVF/ICSI between June 2016 and June 2018. We classified the patients into groups A (with variation in the secondary constriction region in the autosomal long arm), B (with variation in the short arm of the D/G group chromosomes), C (with interbrachial inversion of chromosome 9) and D (with Y chromosome polymorphisms), and compared the semen parameters and sperm DNA fragmentation indexes (DFI) between the patients with chromosome polymorphisms and those with normal chromosomes.@*RESULTS@#Totally, 154 (6.50%) of the patients undergoing IVF/ICSI were found with chromosome polymorphisms, including 34 cases of secondary constriction variation in the long arm of the autosome (1.43% ［34/2 370］, 22.08% ［34/154］), 82 cases of short arm polymorphisms of the D/G group chromosomes (3.46% ［82/2 370］, 53.25% ［82/154］), 26 cases of interbrachial inversion of chromosome 9 (1.10% ［26/2 370］, 16.88% ［26/154］), 10 cases of Y chromosome polymorphisms (0.42% ［10/2 370］, 6.50% ［10/154］), and 2 cases of mixed chromosome polymorphisms (0.08% ［2/2 370］, 1.42% ［2/154］). The total sperm count was lower in group D than in the other polymorphism groups and the normal chromosome group, but with no statistically significant difference among the five groups (P > 0.05). The sperm progressive motility was also lower in group D than in the other five groups, with statistically significant difference from group B (27.5 ± 13.5 vs. 41.5 ± 21.1, P = 0.027), but not from the other groups (P > 0.05). No statistically significant difference was observed in the sperm DFI between the polymorphism groups and the normal chromosome group (P > 0.05), or among the polymorphism groups (P > 0.05). The proportion of normal semen was lower in group D than in the other four groups, but with no statistically significant difference among the five groups (P > 0.05). The incidence rate of asthenospermia was higher in group D than in the other four groups, but with no statistically significant difference among the five groups (P > 0.05), and so was that of oligoasthenospermia, with statistically significant difference from the normal chromosome group (30.0% vs 8.0%, P = 0.041), but not from the other polymorphism groups (P > 0.05).@*CONCLUSIONS@#Short arm polymorphisms of the D/G group chromosomes are the most common type of chromosome polymorphisms in male patients undergoing IVF/ICSI. Polymorphisms of the Y chromosome have a negative effect on semen quality, while those of the other chromosomes do not significantly affect semen quality and sperm DNA integrity.

Value of the concentration and integrity of serum cell-free DNA for the clinical diagnosis of esophageal carcinoma / 中华肿瘤杂志

Objective@#To explore the diagnostic value of serum cell-free DNA (cfDNA) concentration and integrity for esophageal carcinoma.@*Methods@#Venous blood samples from 68 patients with esophageal cancer, 36 patients with benign esophageal lesions and 45 healthy subjects were collected. Circulating cfDNA was verified through quantitative real-time PCR (Alu-qPCR) using Alu-115 and Alu-247 primers. DNA integrity index was calculated as the ratio of Alu-qPCR results (Alu247/115). Concentrations of carcino-embryonic antigen (CEA) and squamous cell carcinoma associated antigen (SCC) were detected by chemiluminescence analyzer assay. Statistical analysis was performed using Mann-Whitney U test, Kruskal-Wallis H test and Spearman correlation test. The Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficiency of each index to esophageal carcinoma.@*Results@#The median absolute serum Alu115 and the Alu247/115 index (1 162.0 ng/ml, 0.57) in esophageal cancer group were significantly higher than those in benign esophageal disease group (496.7 ng/ml, 0.43) and in healthy control group (432.3 ng/ml, 0.42) (all P<0.01, respectively). The Alu115 and Alu247/115 index of serum DNA in benign esophageal disease group were no statistically different from those in the healthy control group (all P>0.05, respectively). The levels of cfDNA and its integrity were not significantly correlated with age, gender, tumor differentiation, or disease stage according to American Joint Committee on Cancer (AJCC) staging system in the esophageal cancer group (all P>0.05). The serum Alu247/115 index of Stage Ⅲ patients was higher than that of Stage Ⅰ~Ⅱ patients(P<0.05). The serum Alu247/115 index of Stage Ⅳ was higher than that of Stage Ⅲ(P<0.05). In the esophageal cancer group, both of serum Alu115 and Alu247/115 index had no correlation with CEA or SCC (all P>0.05). The area under the ROC curve (AUC) of Alu115 and Alu247/115 index were 0.867 and 0.854, respectively, which were both higher than that of CEA (0.622) and SCC (0.753). The addition of Alu115 or Alu247/115 index to CEA and SCC detection increased the sensitivity of the diagnosis of esophageal cancer by 95.6% and 94.1%, respectively.@*Conclusions@#The detection of serum cfDNA concentration and integrity is helpful to the early diagnosis and monitoring of esophageal cancer. Their diagnostic value of esophageal cancer is better than that of the traditional tumor markers CEA and SCC.

Yam polysaccharide improves sperm viability and protects sperm DNA integrity in vitro / 中华男科学杂志

Objective@#To clarify the roles of yam polysaccharide (YPS) in improving sperm viability and protecting sperm DNA integrity in vitro and provide a new approach to the treatment of oligoasthenozoospermia.@*METHODS@#We collected samples by masturbation from 36 normal fertile males aged 27－39 years. Each sample was divided into six groups: blank control or treated with normal saline, vitamin C solution, and YPS solution at low (0.25 mg/ml), medium (1.0 mg/ml) or high concentration (5.0 mg/ml). Using eosin-Y staining, sperm hypotonic swelling (HOS) and sperm chromatin diffusion (SCD) test, we observed the effects of different concentrations of YPS on sperm viability, membrane integrity and nuclear DNA.@*RESULTS@#After 24 and 48 hours of treatment, sperm viability was markedly reduced in the vitamin C (［28.5 ± 3.1］ and ［6.5 ± 1.2］%), low-YPS (［31.3 ± 3.5］ and ［6.5 ± 2.2］%), medium-YPS (［37.1 ± 3.5］ and ［9.5 ± 2.8］%) and high-YPS groups (［38.3 ± 3.3］ and ［9.0 ± 3.2］%) as compared with the blank control (［17.3 ± 2.1］ and ［3.2 ± 1.3］%) (P 0.05).@*CONCLUSIONS@#Yam polysaccharide can improve sperm viability and protect sperm DNA integrity in vitro.

Correlation of oxidative stress with sperm DNA integrity and semen parameters in infertile men with varicocele / 中华男科学杂志

<p><b>Objective</b>To investigate the relationship of oxidative stress with DNA integrity and semen parameters in infertile men with varicocele (VC).</p><p><b>METHODS</b>This prospective study included 98 infertile males with VC. According to the levels of reactive oxygen species (ROS) in the semen, we divided the patients into a high ROS group (n=44) and a low ROS group (n=54), determined the sperm DNA fragmentation index (DFI), motility and morphology, and analyzed their correlation with ROS in the two groups of patients.</p><p><b>RESULTS</b>Compared with the patients of the low ROS group, those of the high ROS group showed a significantly higher DFI (27.38±8.10 vs 34.49±6.05, P=0.039) and a higher concentration of seminal leukocytes (［0.65±0.15］×10⁶/ml vs ［0.86±0.41］×10⁶/ml, P=0.022), but lower sperm motility (［36.16±22.83］% vs ［18.22±25.21］%, P=0.017), percentage of progressively motile sperm (［23.34±11.53］% vs ［16.34±9.22］%, P=0.041), sperm curvilinear velocity (［27.03±6.21］ vs ［20.62±4.38］ μm/s, P=0.013), and sperm linearity (［29.75±8.24］% vs ［18.30±7.93］%, P=0.024). Spearman correlation analysis indicated that the ROS level was correlated positively with the concentration of seminal leukocytes (r=0.41, P<0.01) and DFI (r=0.21, P=0.006), but negatively with sperm curvilinear velocity (r=－0.24, P=0.017), linearity (r=－0.24, P=0.021), motility (r=－0.31, P=0.002), and the percentage of progressively motile sperm (r=－0.41, P=0.012). Additionally, the sperm DFI manifested a significant negative correlation with sperm motility (r=－0.29, P<0.01) and the percentage of progressively motile sperm (r=－0.34, P<0.01).</p><p><b>CONCLUSIONS</b>The level of seminal ROS is positively correlated with the sperm DFI in infertile men with varicocele, and both the ROS level and DNA integrity are associated with semen parameters.</p>

Association of sperm DNA integrity with seminal plasma oxidative stress and its influence on in vitro fertilization in infertile males / 中华男科学杂志

<p><b>Objective</b>To investigate the association of sperm DNA integrity with semen routine parameters and seminal plasma oxidative stress and its influence on in vitro fertilization (IVF) in males with infertility.</p><p><b>METHODS</b>Using sperm chromatin dispersion (SCD), we detected sperm DNA damage in 433 infertile men undergoing IVF. Based on the sperm DNA fragmentation index (DFI), we divided the patients into a low DFI (lt;30%) and a high DFI ( ≥30%) group and then compared sperm concentration, the percentage of progressively motile sperm (PMS), the contents of malondialdehyde (MDA) and total antioxidant capacity (TAC) in the seminal plasma, and the rates of fertilization, cleavage and high-quality embryos between the two groups of patients.</p><p><b>RESULTS</b>Compared with the low DFI group, the high DFI group showed significantly decreased rates of PMS (［48.6±16.7］ vs ［29.2±16.8］%, P<0.01) and fast PMS ［19.0±9.1］ vs ［9.4±6.6］%, P<0.01), but no statistically significant difference in sperm concentration (［51.4±30.9］ vs ［52.3±32.4］ ×106/ml, P>0.05). The content of MDA in the seminal plasma was markedly higher in the high DFI than in the low DFI group (［2.28±0.26］ vs ［0.95±0.18］ nmol/L, P<0.01) but that of TAC remarkably lower in the former than in the latter (［10.2±3.5］ vs ［33.2±7.9］ U/L, P<0.01). The rate of fertilization was significantly lower in the high DFI than in the low DFI group (［58.9±30.0］ vs ［77.2±25.0］%, P<0.01), but there were no significant differences between the two groups in the rates of cleavage (［70.7±35.6］ vs ［80.4±15.6］%P>0.05) and high-quality embryos (［40.4±31.3］ vs ［41.7±29.4］%,P>0.05).</p><p><b>CONCLUSIONS</b>Sperm DNA damage is associated with seminal oxidative stress and may affect the outcomes of IVF by reducing the rate of fertilization.</p>

Testicular and epididymal sperm comparison of DNA integrity rate and ICSI outcome / 重庆医学

Objective To comparative analysis the intracytoplasmic sperm injection (ICSI)result and rate of sperm DNA in-tegrity (DNA fragmentation index,DFI)about testicular and epididymis sperm.Methods Totally 183 obstructed azoospermia pa-tients were choosed to use ICSI.80 cycles by PESA and 103 cycles by TESA,compared two groups of sperm DNA integrity rate and ICSI outcome.Results Sperm DNA integrity rate,fertilization rate,cleavage rate,good-qualityembryo rate and pregnancy rate com-pared with no difference by ICSI(P >0.05).Conclusion DNA integrity rate and ICSI outcomes of the testis and epididymis sperm have no significant differences,clinicians can be based on personal experiences or patients,wills to select sperm for ICSI.

Cell-free DNA concentration and integrity as a screening tool for cancer.

AIM OF THE STUDY: This study aims to evaluate cell-free DNA (CFDNA) concentration and integrity in patients with malignant and nonmalignant diseases and in controls to investigate their value as a screening test for cancer, and to correlate them with clinicopathological parameters of cancer patients. MATERIALS AND METHODS: The study included three groups; group I: 120 cancer patients, group II: 120 patients with benign diseases and group III: 120 normal healthy volunteers as control. One plasma sample was collected from each subject. CFDNA was purified from the plasma then its concentration was measured and integrity was assessed by PCR amplification of 100, 200, 400, and 800 bp bands. RESULTS: There was a highly significant difference in CFDNA levels between cancer group and each of benign and control groups. AUC of ROC curve for cancer group versus normal and benign groups were 0.962 and 0.895, which indicated the efficiency of CFDNA as a marker of cancer. As for integrity, normal and benign subjects showed only two bands at 100 and 200 bp, while all cancer patients demonstrated the 400 bp band and 78% of them had the 800 bp whose presence correlated with vascular invasion. CONCLUSION: The combined use of CFDNA concentration and integrity is a candidate for a universal screening test of cancer. Upon setting suitable boundaries for the test it might be applied to identify cancer patients, particularly among subjects with predisposing factors. Being less expensive, CFDNA concentration could be applied for mass screening and for patients with values overlapping those of normal and benign subjects, the use of the more expensive, yet more specific, integrity test is suggested.

Novel insights into the genetic and epigenetic paternal contribution to the human embryo

The integrity of the sperm genome and epigenome are critical for normal embryonic development. The advent of assisted reproductive technology has led to an increased understanding of the role of sperm in fertilization and embryogenesis. During fertilization, the sperm transmits not only nuclear DNA to the oocyte but also activation factor, centrosomes, and a host of messenger RNA and microRNAs. This complex complement of microRNAs and other non-coding RNAs is believed to modify important post-fertilization events. Thus, the health of the sperm genome and epigenome is critical for improving assisted conception rates and the birth of healthy offspring.

Comet assay: A prognostic tool for DNA integrity assessment in infertile men opting for assisted reproduction.

Background & objectives: The growing concern on transmission of genetic diseases in assisted reproduction technique (ART) and the lacunae in the conventional semen analysis to accurately predict the semen quality has led to the need for new techniques to identify the best quality sperm that can be used in assisted procreation techniques. This study analyzes the sperm parameters in the context of DNA damage in cytogenetically normal, AZF non deleted infertile men for DNA damage by comet assay. Methods: Seventy infertile men and 40 fertile controls were evaluated for the semen quality by conventional semen parameters and the sperms were also analyzed for DNA integrity by comet assay. The patients were classified into oligozoospermic (O), asthenozoospermic (A), teratozoospermic (T), oligoasthenoteratozoospermic (OAT) categories and infertile men with normal semen profile. The extent of DNA damage was assessed by visual scoring method of comets. Results: Idiopathic infertile men with normal semen profile (n=18) according to conventional method and patients with history of spontaneous abortions and normal semen profile (n=10) had high degree of DNA damage (29 and 47% respectively) as compared to fertile controls (7%). The O, A, T and OAT categories of patients had a variably higher DNA damage load as compared to fertile controls. Interpretation & conclusions: The normal range and threshold for DNA damage as a predictor of male fertility potential and technique which could assess the sperm DNA damage are necessary to lower the trauma of couples experiencing recurrent spontaneous abortion or failure in ART.

Evaluación de la calidad del espermatozoide humano: comparación entre la integridad del ADN espermático y variables del semen / Evaluation of the quality of the human spermatozoon: comparison between spermatic DNA integrity and semen variables

El análisis del semen no tiene valor predictivo absoluto de fertilidad, pero informa sobre el potencial de fertilidad del varón, el cual está relacionado con la calidad de sus espermatozoides y de otras variables del semen. Se ha comprobado que los valores del semen pueden mostrar gran variabilidad en un mismo individuo. Esto explica por que un hombre cuyas variables no son absolutamente normales, puede lograr un embarazo en su pareja. Dentro de los parámetros tradicionalmente utilizados en la evaluación clínica de la fertilidad masculina se encuentran: la concentración, la movilidad y la morfología espermática; además de medir estas variables, nuevos procedimientos han sido incorporados para evaluar la capacidad funcional de los espermatozoides, uno de los que ha alcanzado particular importancia en la última década es la medida de la integridad del ADN nuclear. La fragmentación del ADN consiste en interrupciones en las cadenas simples o dobles del ADN que ocurre frecuentemente en la muestra de pacientes no fértiles. Se ha llevado a cabo un estudio clínico, en muestras de semen provenientes de pacientes que acudieron al laboratorio de Andrología de la Universidad de los Andes, colectadas entre marzo del 2007 y marzo del 2009, a fin de establecer comparaciones entre los parámetros convencionales y la medición de la integridad de la cromatina espermática, mediante citometría de flujo. Los resultados obtenidos mostraron correlaciones evidentes entre los parámetros convencionales y la integridad del ADN espermático y aportan datos de gran utilidad en el estudio clínico integral de la infertilidad masculina.

Semen analysis does not have an absolute predictive value on fertility, however it is a reflection of male fertility potential, which is related to its spermatozoa quality and other semen variables. Great variability in human semen parameters has been demonstrated within a single individual, an observation that could explain why a male with low semen quality can successfully fertilize an egg. Although conventional semen analysis, such as sperm concentration, motility and morphology, provide important information about the clinical status of male fertility, new procedures to predict the sperm functional capability have been developed in the last decade, such as analysis of nuclear DNA integrity, which have improved considerably the clinical diagnosis of male infertility, and increased the knowledge about spermatozoa function. DNA fragmentation consist in interruptions, both in single and double DNA strains, that frequently occur in sperm samples from infertile patients. We have conducted a clinical study in semen samples from patients who have attended the Andrology laboratory of the University of Los Andes, between March 2007 and March 2009. The aim of this study was to compare sperm DNA integrity, analyzed by flow cytometry, with traditional semen parameters. Our results show remarkable correlations between conventional human semen variables and sperm chromatin integrity, contributing to asses an integral evaluation of sperm quality allowing the analysis of its fertilizing potential in clinical studies.

Freezing effect on post-thawed sperm characteristics especially sperm DNA integrity comparing between liquid nitrogen vapour and computerized program freezer.

The aim of the study was to evaluate the cryodamage effects on human sperm characteristics, especially on sperm DNA integrity, after 6 months of freezing comparing between liquid nitrogen vapour (LNV) and computerized program freezer (CPF). Forty normal semen samples were collected for semen analysis. Each sample was mixed with cryoprotective media and devided into 2 straws. The first straw was frozen with LNV and the second one with CPF. After 6 months of cryostorage, semen samples were thawed, and sperm chromatin integrity as well as sperm motility, morphology, vitality and cryosurvival rate were determined. Percentages of DNA damage were higher (p<0.01) following freezing with LNV than with CPF. Sperm vitality was greater (p<0.05) after CPF than after LNV, as well as cryosurvival rate (p<0.001). Post-thawed sperm motility was greater after CPF than after LNV, either in grade A (p<0.001) or in grade B (p<0.05). No significant difference was observed in the percentage of normal sperm morphology comparing the two freezing methods. The current study demonstrated post-thawed decrease in sperm DNA integrity as well as other sperm characteristics after freezing in both methods. The CPF significantly provided superior results in post-thawed sperm DNA integrity, sperm motility and vitality than LNV did. In case of 6 months of cryostorage, therefore, we recommend the computerized program freezer as a preference for sperm cryopreservation.