Goat Spermatozoa Research Articles

Effect of trolox added to freezing extenders over goat and ram spermatozoa

Semen cryopreservation is responsible for decrease the gamete fertility, due to structural and functional damages. Among the various causes, oxidative stress, resulting from the higher generation of reactive oxygen species (ROS), has been attributed to affect semen quality. Thus, it was objectified evaluate the effect of Trolox on ram and goat sperm, subjected to freezing. Semen pools of goat (n=5) and ram (n=6) were diluted in skimmed milk (7% glycerol) or Tris-egg yolk (5% glycerol) extender, respectively, added or not of Trolox (0, 20 or 40 μM/ml) and frozen. After thawing (37 °C/30 s), aliquots of semen were evaluated for lipid peroxidation by high performance liquid chromatography, coupled with a photodiode array detector (HPLC-DAD), and flow cytometry (C11-BODIPY581/591), besides of plasma membrane and acrosome integrity by fluorescence microscopy, and sperm kinetics by computerized sperm analysis (CASA). The antioxidant treatment with Trolox did not determine significant effects (p>0.05) on lipid peroxidation, plasma membrane integrity, acrosomal integrity and on the kinetic parameters evaluated. Thus, it is concluded that Trolox (20 or 40 μM) did not have a protective or deleterious effect on goats and ram sperm, submitted to freezing.

Supplementary role of vitamin E and amino acids added to diluent on goat sperm freezability

Supplementary role of vitamin E and amino acids added to diluent on goat sperm freezability

The Effects of Antifreeze Protein III Supplementation on the Cryosurvival of Goat Spermatozoa During Cryopreservation.

Antifreeze protein (AFP) has been shown to have beneficial effects on frozen mammalian spermatozoa. However, rare reports have been published regarding the use of AFPs in storage of goat spermatozoa. The aim of this study was to investigate the effects of AFPIII on the quality of goat semen during cryopreservation. Ejaculates were collected from six Yunshang black goats through an artificial vagina. The collected semen was pooled, divided into five aliquots, and diluted with the commercial bull semen extender containing: no AFPIII (AFP-0, control), 1 μg/mL AFPIII (AFP-1), 10 μg/mL AFPIII (AFP-10), 50 μg/mL AFPIII (AFP-50), and 100 μg/mL AFPIII (AFP-100), respectively. Spermatozoa motility, membrane integrity, acrosome integrity, mitochondrial function, distribution of phosphatidylserine, and formation of reactive oxygen species (ROS) were measured after the freezing and thawing process. The results showed that the spermatozoa motility, membrane integrity, acrosome integrity, and mitochondrial function were significantly higher in frozen spermatozoa using the extender containing 1 μg/mL AFPIII as compared with the other groups (p < 0.05). Furthermore, the extender supplemented with 1 μg/mL of AFPIII resulted in higher viable and lower nonviable spermatozoa compared with the other treated groups (p < 0.05), after staining using Annexin V-fluoresceine isothiocyanate (Annexin V-FITC) and Propidium Iodide. No significant differences were found between these groups in relation to viable cells with lower ROS production. In conclusion, the addition of AFPIII to the freezing extender improved the post-thaw quality of goat semen. The optimal concentration used in this study was 1 μg/mL. However, excessively high concentrations of AFPIII were unable to exhibit their cryoprotective effects on goat spermatozoa. However, the presence of AFPIII cannot mitigate oxidative stress caused by the freezing and thawing process. In addition, in vitro fertilization or artificial insemination can further evaluate the effects of AFPIII on frozen-thawed goat spermatozoa.

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

Earlier 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.

Quantitative sperm characteristics of Tankwa goats with special reference to hyperactivated motility

Computer-assisted semen analysis (CASA) is an automated and objective method of evaluating structural (e.g. morphology) and functional sperm parameters (e.g. motility and hyperactivation). Sperm hyperactivation is essential for successful fertilization and is thus an important aspect in determining the fertility potential of a male. In the current study, CASA was used for standard semen analysis and for comparison of the ability of phosphate buffered saline (PBS), BO sperm wash (10 mM caffeine), 4% lignocaine, and 5 mM procaine hydrochloride to induce hyperactivation in Tankwa goat spermatozoa. Twenty-nine ejaculates were collected from randomly selected male goats by electroejaculation. Although none of the four media affected percentage total sperm motility, lignocaine caused a significant decrease (P &gt;0.05) in percentage progressive motility. Exposure to procaine resulted in an increase in swimming speed (P ≤0.05) and star-spin motility tracks, which are typical of sperm hyperactivation. Using PBS and procaine motility data from individually selected spermatozoa, receiver operator characteristic curves were constructed to distinguish the kinematic parameters employed as cut-off values for sperm hyperactivation. PBS and BO sperm wash did not induce hyperactivation (0.1 + 0.2% and 0.04  0.2% respectively), while lignocaine induced little hyperactivation (3.4 + 3.0%) and procaine hydrochloride had the highest percentage hyperactivation (25.3 + 13.6%). The large variation in hyperactivation (0–54.5%) may reflect inter-individual differences in sperm quality among these males. This study indicated procaine hydrochloride was the most promising hyperactivation-inducing medium for Tankwa goat spermatozoa and should be considered for similar assessments in other animal species&#x0D; Keywords: computer-aided sperm analysis, procaine hydrochloride, sperm kinematics, sperm morphometry, sperm motility

MORPHOLOGICAL CHANGES IN EPIDIDYMAL SPERMATOZOA OF RED SOKOTO (MARADI) BUCKS

Morphological changes in the caput, corpus and cauda epididymides were studied in this experiment. The objective was to study morphological changes in goat spermatozoa using the Red Sokoto goat. Progressive motile spermatozoa were first observed in the caput epididymis (5-7%) and was highest at the cauda epididymis (60%). Progressive motility for the corpus epididymis ranged between 20-30%. Cytoplasmic droplets were lost as the spermatozoa moved up the epididymis. Proximal cytoplasmic droplet was significantly (P&lt;0.05) lowest at the cauda epididymis. Other morphological changes did not follow a particular trend.

Senduro Goat Semen Characteristics as A Candidate for Low Temperature Storage

The goal of this study was to investigate the reproductive profile of male Senduro Goats as superior male candidates based on data of semen quality. The experiment was carried out on ten male Senduro Goats kept at the Singosari Animal Husbandry Service Unit in East Java, Indonesia. An artificial vagina was used to collect sperm. The macroscopic and microscopic qualities of fresh Senduro Goat sperm have been observed. Color, volume, smell, pH, and viscosity were examples of macroscopic observations. Concentration, motility, viability, and membrane integrity were examples of microscopic observations. The results of macroscopic observations showed that the average of Senduro Goat semen was milky white; with volume 1,24 ± 0,22; 6.77 ± 0.05; thick semen. The results of microscopic observations showed that there was concentration 2,5 x 109 ± 0.1; mass motility +++; individual motility 77.5% ± 2.63; viability 88.98% ± 2.07; membrane integrity 89.71 ± 1.69. Based on the results of these studies, it can be concluded that the semen of Senduro Goat has good quality so it can be processed further, namely storage at low temperatures and applied to Artificial Insemination technology.

The effect of adding pyridoxine to soybean lecithin-based extender on goat semen quality parameters after the freeze-thawing process.

Supplementing the semen extender with various antioxidants can increase the quality of semen. The aim of the present study was to investigate the addition of pyridoxine (vitamin B6), a phenolic compound with antioxidant properties, to soybean lecithin extender on motility and quality indices of goat sperm after freezing-thawing process. Semen was collected at weekly intervals from four Mahabadi goats, mixed, and was divided into 7 subsamples. They were then diluted with the basal extender supplemented with 0, 2, 4, 6, 8, 10, and 12 mM pyridoxine. Following freeze-thawing process, quality parameters such as sperm motility characteristics, viability, plasma membrane integrity, and malondialdehyde content were determined. The results showed that pyridoxine at the level of 6 mM caused the highest total motility rate (P<0.05). Progressive sperm motility was highest at the 4, 6, and 8 mM pyridoxine (P<0.05). Although the control group showed the least progressive motility, it was not statistically significantly different from the 12 mM level. Among the pyridoxine levels, the 6 mM level recorded the best performance in term of plasma membrane integrity, sperm viability, and decreasing malondialdehyde concentration compared to the control group (P<0.05). The findings indicated that soybean lecithin extender supplemented with 6 mM pyridoxine can improve motility and quality parameters such as viability, plasma membrane integrity, and reduce oxidative stress of goat sperm after thawing.

Developments in Goat Semen Cryopreservation

Sperm cryopreservation simplifies its storage for longer time for use in artificial insemination and assisted reproductive technologies. This technique is also important for breed conservation process and has paved the way for other reproductive biotechnologies. Despite the significant progress in the field, frozen-thawed sperm have enormous inconsistency in fertility rates. It is well known fact that semen cryopreservation exhibited detrimental effects on post-thaw semen motility, plasma membrane, acrosomal status and DNA integrity which ultimately effect the fertility outcome. In addition, several attributes are responsible for low quality of goat cryopreserved semen such as breeds, seasons, management practices and cryopreservation protocols. The aim of the review article is to give an insight of distant features of goat semen cryopreservation as well as recent development in goat semen cryopreservation. It also provides concise information on progress made in the advancement in the semen extender development and cryopreservation of goat sperm.

Influence of Different Freezing Curves on the Acrosome Integrity of Male Goat Sperm Cells

The aim of the present study was to evaluate the effect of three different freezing curves on male goat’s sperm viability and acrosome integrity. Semen was collected on three adult male goats in February (Azores, Portugal, latitude 38°39′N) using an artificial vagina and a female with induced heat. Three collection sessions were spaced of approximately 5 days. The study followed a factorial design of repeated measurements. After volume, sperm concentration and mass motility evaluation, live and dead spermatozoa (spz) were counted and acrosome integrity was evaluated by Flow Cytometry (FCM). In the meantime semen was frozen using three curves differing in the second cooling ramp rate (curve A = 25°C/min, curve B = 35°C/min and curve C = 45°C/min). After thawing, the percentage of live/dead sperm cells and the acrosome integrity were determined by FCM, using respectively Propidium Iodide (PI) and Pisum Sativum Agglutinin conjugated to Fluorescein Isothiocinate (FITC-PSA). By combining these two fluorochromes, the thawed semen was divided into: Live sperm with intact acrosome, unstained PI and unstained FITC-PSA (LL); live sperm with damaged acrosome, unstained PI and stained FITC-PSA (LR); dead sperm with intact acrosome, stained PI and unstained FITC-PSA (UL); dead sperm with damaged acrosome, stained PI and stained FITC-PSA (UR). Although different freezing curves did not significantly influence live sperm rate with intact (19.14±0.86% spz curve A, 17.72±0.86% spz curve B and 18.18±0.86% spz curve C) or damaged (0.44±0.04 % spz curve A, 0.38±0.04% spz curve B and 0.49±0.04% spz curve C) acrosome, a significant (P<0.05) effect of animals was observed in the four parameters studied. Regarding the percentage of LL spermatozoa, buck 1 presented values lower (P<0.05) by about 50% than bucks 2 and 3. Data also were affected significantly by the day of collection. These results strongly suggest that although goat sperm cells are not influenced by the pattern of cooling curves investigated, a considerable variability in the response of individual animals as well as the collection date may be expected.

Effect of the Refrigeration System on In Vitro Quality and In Vivo Fertility of Goat Buck Sperm.

Simple SummaryArtificial insemination of goats is mostly performed with sperm that has been chilled to 4 °C and stored for up to 12 h. Because the chilling of samples to 4 °C must be done slowly, artificial inseminations have to be performed late in the day, especially in herds located far from artificial insemination centers, which impedes the extensive use of this reproductive technology in goats. In this study, we explored the possibility of chilling sperm doses at a controlled rate (close to −0.18 °C/min) during transportation and compared the quality and fertility of these samples with those chilled in the lab. We observed that the in vitro quality of doses chilled by the prototype procedure developed for transportation was higher than that of samples chilled in the lab, and the fertility of the sperm was similar. Therefore, it is possible to chill doses during transportation without affecting their quality or fertility.Cooling goat sperm insemination doses to 4 °C causes a delay in their delivery. However, chilling these doses during the transportation period could expedite their delivery and the insemination process. In this study, an economical and simple apparatus for chilling goat semen doses in itinere was developed, and the in vitro quality and in vivo fertility of these doses were compared with those chilled by means of a programmable water bath in the laboratory at a rate of −0.18 °C/min. Of the tested prototypes, the one that provided an optimal combination of the chilling rate (average of −0.09 °C/min) and time required to reach 4 °C (3 h 45 min) was selected for further testing. Immediately after chilling and 24 h later, the doses chilled in the prototype were determined to be of higher quality than the samples chilled in the programmable water bath. Finally, the kidding rate was similar between the doses chilled in the programmable water bath (61.7% ± 7.1%) and in the prototype (56.1% ± 5.9%). In conclusion, successful chilling of goat sperm doses during transport is possible, thereby accelerating the delivery of insemination doses.

Comparison of spermatozoal RNA extraction methods in goats

RNA sequencing (RNAseq) has divulged newer role of spermatozoal RNA in male fertility. This study aimed to evaluate different sperm purification and RNA extraction methods for long-read RNA sequencing of poly(A) transcriptome in goat spermatozoa. Sperm samples were purified by swim-up separation using different purification medium. Spermatozoal RNA was extracted by seven different methods with additional supplementation of reducing agents in lysis buffer. poly(A) selected RNA was used for cDNA library preparation and long-read RNAseq in Nanopore sequencer. Sperm purification by 1 h swim-up resulted in higher recovery (89.20 ± 1.15%), motility (82.33 ± 1.53%), viability (88.10 ± 5.03%) and plasma membrane integrity (71.33 ± 4.51%) in sperm TALP (sp-TL) medium. A monophasic solution of GITC with phenol and DTT resulted in the highest yield of large sized RNAs (3.89 ± 0.46 ng/million cells) suitable for long-read RNAseq of poly(A) transcripts. RNAseq resulted in reads of length, ranging from 500bp to 2 Kb. A total of 123 transcripts were identified in goat spermatozoa by de novo assembly and included sperm-specific transcripts such as CATSPERG, PRM2, CYLC2, SPATA6, PLCZ1 etc. This study is the first report of long-read RNAseq of poly(A) transcriptome in goat spermatozoa.

Вплив кріопротекторних середовищ на основі сахарози та гліцерину на кінетичні характеристики сперматозоїдів самців кіз зааненської породи

Вплив кріопротекторних середовищ на основі сахарози та гліцерину на кінетичні характеристики сперматозоїдів самців кіз зааненської породи

Effect of Sperm Concentration and Storage Temperature on Goat Spermatozoa during Liquid Storage.

Simple SummaryOne of the problems with the refrigerated storage of spermatozoa in goats is the short shelf life. The study of the effect of the different storage conditions on the different sperm parameters could help to increase sperm survival. In this work, we studied the effect of temperature and sperm concentration during refrigerated storage on sperm motility, mitochondrial membrane potential and DNA fragmentation. We observed that refrigerated storage of semen doses reduced the majority of sperm quality parameters, however, sperm DNA fragmentation was not affected. Storage at 5 °C preserved higher sperm motility than at 17 °C. Moreover, the reduction of sperm concentration below 500 × 106 sperm/mL did not seem to improve the quality of spermatozoa.The use of cooled semen is relatively common in goats. There are a number of advantages of cooled semen doses, including easier handling of artificial insemination (AI) doses, transport, more AI doses per ejaculate, and higher fertility rates in comparison with frozen AI doses. However, cooled semen has a short shelf life. The objective of this study was to examine the effect of temperature and sperm concentration on the in vitro sperm quality during liquid storage for 48 h, including sperm motility and kinetics, response to oxidation, mitochondrial membrane potential (MMP) and DNA fragmentation in goats. Three experiments were performed. In the first, the effects of liquid preservation of semen at different temperatures (5 °C or 17 °C), durations (0, 24 and 48 h) and sperm concentrations (250 × 106 sperm/mL (1:2 dilution rate), 166.7 × 106 sperm/mL (1:3 dilution rate) or 50 × 106 sperm/mL (1:10 dilution rate)) on sperm motility and kinetics were studied. In the second experiment, the effect of temperature, sperm washing and concentration on sperm motility and DNA fragmentation was studied. Finally, the effect of sperm concentration and duration of storage at 5 °C on sperm motility, response to oxidative stress and MMP was examined. We found that refrigerated liquid storage of goat sperm impaired sperm quality, such as motility, MMP and response to oxidation, as storage time increased; however, sperm DNA fragmentation index was not significantly affected. Liquid storage at 5 °C preserved higher total motility than at 17 °C. Moreover, we observed that the reduction of sperm concentration below 500 × 106 sperm/mL did not seem to improve the quality of spermatozoa conserved in milk-based extender in the conditions tested.

Goat sperm associated antigen 17 protein gene (SPAG17): Small and large fragment genetic variation detection, association analysis, and mRNA expression in gonads

Transcriptome sequencing analyses have suggested that sperm associated antigen 17 protein gene (SPAG17) may play important regulating roles in litter size. In this study, the expression profiles and genetic variations of the SPAG17 were studied in Shaanbei White Cashmere (SBWC) goats (n=1567). SPAG17 was highly expressed in testis and ovary of SBWC goats. At different developmental stages, it also continued to be highly expressed in testis. In addition, two variations of SPAG17, one indel locus and one copy number variation locus, were significantly associated with first-born litter size. Joint analysis results suggested that two polymorphic loci of the SPAG17 gene may regulate host gene expression in goat ovary and testis. Overall, the results indicated the important role of SPAG17 in the reproductive process of goats.

Effects of mint, thyme, and curcumin extract nanoformulations on the sperm quality, apoptosis, chromatin decondensation, enzyme activity, and oxidative status of cryopreserved goat semen

Goat semen cryopreservation is a challenging process as it results in reduced motility, vitality, and fertility of spermatozoa after freezing. In this study, we evaluated the effects of different herbal extract nanoformulations (NFs) [mint (MENFs), thyme (TENFs), and curcumin (CENFs)], supplemented at either 50 or 100 μg into Tris-extender on the cryopreserved goat semen quality. The hydrothermal squeezing method was used for the preparation of the NFs extracts. The morphological evaluation of the NFs extracts was conducted by transmission electron microscopy. All NFs supplements improved (p < 0.05) the progressive motility, vitality, and plasma membrane integrity of sperm compared with the control extender after equilibration (5 °C for 2 h) and thawing (37 °C for 30 s), but had no effect on sperm abnormality and acrosome integrity. All NFs supplements decreased (p < 0.05) the apoptosis, malondialdehyde level, and chromatin decondensation of sperm cells, while increased (p < 0.05) the total antioxidant capacity and catalase activity in the frozen/thawed extender. Particularly, CENFs at a level of 100 μg showed improvement of sperm parameters and antioxidant status during cryopreservation of goat semen more than TENFs and MENFs. The CENFs improved the quality of goat spermatozoa in post-thawed semen in terms of preventing cryodamage and promoting the cryotolerance of spermatozoa when compared with TENFs and MENFs. Therefore, supplementation of Tris-extender with CENFs could enhance goat semen processing during cryopreservation.

Effects of pineapple and apple juices for preservation on refrigerated Kalahari Red goat spermatozoa

Effects of pineapple and apple juices for preservation on refrigerated Kalahari Red goat spermatozoa

Evaluation of pentoxifylline and Basal Medium Eagle supplemented to diluent on cryopreserved goat spermatozoa.

This study investigated the effect of pentoxifylline (PTX) and Basal Medium Eagle (BME) on frozen-thawed goat spermatozoa. Immediately after initial examination of ejaculated semen, samples were pooled and reexamined for quality. Then, samples were divided into eight equal aliquots and diluted with a basic tris-extender containing PTX (3, 6, 9mM) and BME (5mM) to reach a final concentration of 25×109 and frozen. After 24hr, the samples were individually thawed at 37°C for 30s and evaluated for different characteristics. Obtained post-thaw results from Computer-Assisted Sperm Analysis indicate using of 3 and 6mM PTX led significantly to an improvement in total motility, progressive motility and velocity characteristics of spermatozoa, except the beat/cross frequency (BCF) which indicated statistically no differences (p>.05) among control and treatments. Diluents prepared with BME (5mM) and PTX alone (3 and 6mM) improved significantly the membrane integrity-functionality, acrosome integrity and also hyaluronidase activity. Regarding recovery rate, the results showed significantly (p<.05) higher values for diluents containing 3 and 6mM PTX compared to other groups. Malondialdehyde concentration exhibited also a significant difference (p<.05) in diluents supplemented with 5mM BME, 3, 6 and 9mM PTX, and mixture of 3mM PTX and 5mM BME which illustrate a similarity for active mitochondria, apoptotic-like and dead spermatozoa. Finally, the ratio of sperm chromatin dispersion stained spermatozoa presented significant differences (p<.05) among treatments in which the diluents added PTX alone demonstrated significantly lower values than control and extenders containing the mixtures of BME and PTX. In conclusion, the observation in this study indicates using of 3 and 6mM PTX and BME alone may improve significantly (p<.05) the quality of cryopreserved goat spermatozoa.

Effect of Addition of Different Egg Yolks in Basic Tris-Soya Diluent on Quality, Membrane Integrity of Senduro Goat Sperm, and Free Radicals during Storage at Temperature of 4-5° C

Semen diluents need macromolecules to protect sperm from the impact of temperature during storage and the presence of free radicals. The purpose of this study was to examine the effect of adding yolk from different types of eggs to the quality and integrity of the sperm membrane. Fresh semen was diluted with basic tris-soya diluent wich was added with different types of chicken egg yolk, such as local chicken eggs and leghorn chicken eggs. As a control group, fresh semen was diluted with basic tris-soya diluent without the addition of egg yolk. The addition of egg yolk was 20% of the diluent volume. Quality observations include motility and viability of sperm. Sperm motility was observed using a microscope with 200 X magnification. The sperm viability was observed using the eosin and negrosin staining method, then it observed under a microscope with 200 X magnification. The integrity of sperm membrane was observed using the HOST method, and then observed under a microscope with 200 X magnification. Free radicals then observed with MDA test. The results showed that the motility, viability and membrane integrity of sperm only lasted until the 12 hour of storage, with the greatest motility, viability and membrane integrity obtained in diluents with the addition of leghorn chicken egg yolk. The smallest MDA test was 2.72, which obtained from diluents with the addition of leghorn chicken egg yolk. Based on the results of the study it can be concluded that the leghorn chicken egg yolk has the best ability to maintain the quality of Senduro Goat sperm during 4-5°C storage temperature.

Evaluation of used Purslane extracts in Tris extenders on cryopreserved goat sperm

This study aimed to evaluate the comparative effects of Purslane aqueous extract (PAE), Purslane methanolic extract (PME) and Purslane ethanolic extract (PEE on the quality of frozen-thawed goat spermatozoa. Collected semen with motility >75% and sperm concentration >1.0 × 109 sperm/ml was pooled and divided into 10 equal aliquots and supplemented by basic extender containing 25, 50 or 100 μg/ml of Purslane aqueous extract (PAE25μg/ml, PAE50μg/ml, PAE100μg/ml, respectively), basic extender containing 25, 50 or 100 μg/ml of Purslane methanolic extract (PME25μg/ml, PME50μg/ml, PME100μg/ml, respectively), basic extender containing 25, 50 or 100 μg/ml of Purslane ethanolic extract (PEE25μg/ml, PEE50μg/ml, PEE100μg/ml, respectively). Control diluent contained no additives. For the determination of sperm quality, frozen straws were thawed and then the sperm characteristics were assessed. Results indicated that higher (P < 0.05) percentages of total motility, viability, mitochondrial activity and lower percentages of malondialdehyde (MDA) for PAE50μg/ml, PME50μg/ml and PEE50μg/ml than those of the control. In addition, PME50μg/ml resulted in the highest) P < 0.05) total motility and the lowest (P < 0.05) MDA levels compared to other treatments. Compared to the control group, PME50μg/ml resulted in higher integrity (P < 0.05) of plasma membranes and in lower amounts of apoptotic and dead spermatozoa. PME50μg/ml and PAE50μg/ml showed higher (P < 0.05) percentages of progressive motility, DNA integrity and live post-thawed spermatozoa than those of the control. No significant differences in the motility, viability, mitochondrial activity and number of live sperms were observed between PME50μg/ml and PAE50μg/ml treatments. In conclusion, the results of this study indicated that 50 μg/ml purslane extracts could be used for the cryopreservation. However, the results of methanolic extract was more beneficial compared to other extracts.