Sperm Cryosurvival Research Articles

Cryotolerance of equine spermatozoa correlates with specific fatty acid pattern: A pilot study

Sperm cryopreservation represents a powerful tool for horse breeding. To improve the efficiency of artificial insemination in the horse using cryopreserved spermatozoa, an adequate understanding of the underlying biophysical properties that affect sperm cryosurvival needs to be reached yet. In this pilot study, we described isolation and analysis of the main fatty acids from sperms of stallions classified as good and poor freezers (7 GF and 5 PF, according to sperm motility and viability, before and after cryopreservation). Fatty acid profiles were only assessed in pre-thaw sperms. Eight main fatty acids were identified, using gas chromatography, and their contents were expressed as percentage of the total lipid content. We found that lauric, myristic and oleic acid (C12:0, C14:0 and C18:1n9c) turned out to be about 2-fold more abundant in the sperm cells of the GFs compared with PFs. Moreover, we described for the first time the presence of a very high amount of a trans geometrical isomer of linoleic acid, linolelaidic acid (C18:2n6t), in pre-thaw PF spermatozoa. Notably, we found in fresh sperms of PF stallions a ratio of unsaturated fatty acids to saturated fatty acids which was twice that of those of GF group, suggesting a positive effect of a high saturated-to-unsaturated fatty acid ratio for the “freezability” of equine spermatozoa. Finally, principal component analysis (PCA) confirmed the relationships between specific fatty acids and cryotolerance of equine spermatozoa, also providing a graphical classification and additional information about the dominant variables governing the classification process.

Melatonin added to freezing diluent improves canine (Bulldog) sperm cryosurvival.

SummaryCryopreservation compromises the capacity of sperm fertilizing due to a series of alterations in the structure and physiology of the sperm. The use of antioxidants, such as melatonin, added to freezing media, may help to reduce sperm cryoinjury. To test the effect of melatonin on Bulldog (Canis lupus familiaris) sperm cryosurvival, spermatozoa were diluted in a standard freezing medium and cooled to 5°C. Then, more freezing medium was added to obtain 200 × 106 cells/mL, and 5% glycerol. Diluted spermatozoa were treated with melatonin (0.0, 0.0005, 0.002, and 0.0035 mol/L), and packaged in 0.25 mL straws, which were further cooled to −5°C before freezing in liquid nitrogen. Thawing was carried out at 70°C for 5 s, and the progressive motility, viability, plasma membrane integrity, acrosome integrity, capacitation status, and plasma membrane fluidity of the spermatozoa (at 37°C) were assessed. Data were analyzed using ANOVA to detect differences between the melatonin doses. There were statistical differences (P < 0.05) in the percentage of sperm having hyper-fluid membranes, intact acrosome, capacitated acrosome-intact, and acrosome-reacted. The values for the high melatonin doses (0.002 and 0.0035 mol/L) were better than for the low melatonin doses (0.0 and 0.0005 mol/L). In conclusion, 0.002 and 0.0035 mol/L of melatonin improved the cryosurvival of sperm from male bulldogs.Lay summaryPreservation of sperm by freezing enables breeding of individuals geographically separated; protocols for the dog may be used to preserve the semen from threatened wild canids. To improve fertility of female dogs that become pregnant with frozen and then defrosted sperm, these cells must survive that process which can be damaging whilst keeping their ability to fertilize. Antioxidants are substances capable of retarding or preventing the oxidation of any oxidizing substrate such as lipids, proteins, and DNA, which are structural compounds of the sperm. The use of antioxidants, added to freezing media, may provide the sperm the capacity to neutralize oxidative compounds, such as reactive oxygen species, produced during the freezing and thawing process. In this work we tested different levels of melatonin, a natural antioxidant, on dog (English Bulldog) sperm survival and quality after freezing. We found that adding melatonin to the freezing media improved sperm quality after thawing.

Inhibin immunization enhances the sperm quality, cryosurvival, and kinematics during low breeding season in Beetal bucks

Inhibin immunization enhances the sperm quality, cryosurvival, and kinematics during low breeding season in Beetal bucks

Converting seminal plasma with extender – a simple procedure to improve cryosurvival of spermatozoa derived from low-concentration high progressive motility sperm ejaculate

Converting seminal plasma with extender – a simple procedure to improve cryosurvival of spermatozoa derived from low-concentration high progressive motility sperm ejaculate

Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality

Sperm cryopreservation represents a powerful tool for livestock breeding. Several efforts have been made to improve the efficiency of sperm cryopreservation in different ruminant species. However, a significant amount of sperm still suffers considerable cryodamage, which may affect sperm quality and fertility. Recently, the use of different “omics” technologies in sperm cryobiology, especially proteomics studies, has led to a better understanding of the molecular modifications induced by sperm cryopreservation, facilitating the identification of different freezability biomarkers and certain proteins that can be added before cryopreservation to enhance sperm cryosurvival. This review provides an updated overview of the molecular mechanisms involved in sperm cryodamage, which are in part responsible for the structural, functional and fertility changes observed in frozen–thawed ruminant sperm. Moreover, the molecular basis of those factors that can affect the sperm freezing resilience of different ruminant species is also discussed as well as the molecular aspects of those novel strategies that have been developed to reduce sperm cryodamage, including new cryoprotectants, antioxidants, proteins, nanoparticles and vitrification.

A Simple and Efficient Semen Cryopreservation Method to Increase the Genetic Variability of Endangered Mediterranean Brown Trout Inhabiting Molise Rivers.

Simple SummaryThe sperm cryobank is an effective strategy for protecting the biodiversity of the local brown trout population. Thus, the aim of the present work was to test, for the first time, the effectiveness of a semen cryopreservation protocol developed for cultivated salmonid fish on wild trout inhabiting the Molise rivers. Moreover, the effects of two different thawing rates (40°C/5 s and 10°C/30 s) were evaluated in vitro and in vivo. In addition, different sperm-to-egg ratios (6 × 105:1, 4.5 ×105:1 and 3 × 105:1) were also investigated in vivo in order to find an effective freezing protocol useful for the creation of the first European semen cryobank for the Mediterranean brown trout (S. macrostigma = S. cettii). From results obtained in vitro 40 °C turned out to be the best thawing rate, which in combination with a sperm-to-egg ratio of 4.5 × 105:1 was, in vivo, the highest fertilization rate. The important results obtained in the present work corroborated the remarkable validity and effectiveness of the freezing protocol used in this study on the native trout populations from Molise.The aim of our study was to test the effectiveness of a simple semen cryopreservation procedure, developed for cultivated salmonid, on the wild salmonid of the Mediterranean area and to evaluate the effect of different thawing rates and sperm-to-egg ratios. The semen of five individual males was diluted into a final extender concentration of 0.15 M glucose and 7.5% methanol and loaded into 0.25 mL plastic straws, and a final sperm concentration of 3.0 × 109 sperm/mL was obtained. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above the liquid nitrogen level for 5 min. The semen was thawed at 40 °C/5 s or 10 °C/30 s. The sperm cryosurvival was evaluated by examining in vitro the sperm motility parameters using the CASA system, followed by fertilization trials in vivo, using three different sperm-to-egg ratios 6 × 105, 4.5 × 105 and 3 × 105:1. The applied cryopreservation procedure resulted in remarkably high (85.6%) post-thaw sperm total motility, when the semen was thawed at 40 °C/5 s, whilst the highest fertilization rate (53.1%) was recorded for a sperm-to-egg ratio of 4.5 × 105:1. According to these outcomes, the cryopreservation procedure that was tested turned out to be effective for the wild population of Mediterranean brown trout and practical for the creation of the first European semen cryobank foreseen as part of our “LIFE” Nat.Sal.Mo. project.

Bovine sperm selection procedure prior to cryopreservation for improvement of post-thawed semen quality and fertility

BackgroundThe application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction. However, a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation, rendering the frozen-thawed semen impractical for routine use. Cryopreservation is known to reduce sperm lifespan and fertility. Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination (AI) centers. Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process.ResultsOur strategy of sperm selection based on rheotaxis and thermotaxis (SSRT) on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity, viability and biological function related to fertilization competence. This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders. This technique protected further sublethal damage to bovine spermatozoa (higher % cryosurvival than the control) and resulted in the improvement of DNA integrity. Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization.ConclusionsThese results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival, longevity of post-thawed sperm, and optimization of in vivo fertilization, embryo development and calving as supported by the favorable results of field fertility study.

Effect of butylated hydroxytoluene supplementation on ultrastructure of crossbred bovine spermatozoa

The present study was planned to observe the effect of Butylated hydroxytoluene (BHT) on ultra structure changes in bovine crossbred spermatozoa. The sperm plasma membrane is the primary site of damage induced by cryopreservation. There are many harmful conditions such as cold shock, osmotic stress, ice crystal formation or oxidative damage etc which cause sperm cryo-injury and loss of sperm viability and fertility during cryopreservation. At the time of cryopreservation there were considerable ultra structure changes to the acrosomes and middle piece studied by transmission electron microscopy. Semen from two crossbred bulls were collected and extended with egg yolk tris dilutor along with addition of different concentrations of BHT i.e. 0.0mM (Control), 0.5mM (T 1 ) and 1.0mM (T 2 ), respectively. The effect of BHT on ultrastructural alterations was revealed by Transmission electron microscopy during post dilution (Stage I), post equilibration (Stage II) and post thaw (Stage III), respectively. TEM results of spermatozoa at Stage I, II and III showed that major ultrastrucural alterations occur in plasma membrane and outer acrosomal membrane. Group T 1 and T 2 showed significantly lower number of ultrastructural defects compared to control at Stage I, II and III. The study indicated that 1.0mM BHT increases the cryosurvival of sperm as revealed by post thaw lower ultrastructural defects of bovine bull spermatozoa

Improvement of sperm cryo-survival of cynomolgus macaque (Macaca fascicularis) by commercial egg-yolk–free freezing medium with type III antifreeze protein

When nonhuman primate sperm undergoes cryopreservation in an egg yolk medium there is an increased risk that the egg yolk might adversely affect the sperm due to containing of avian pathogens. Although commercial egg-yolk-free medium for human sperm cryopreservation has been used for macaque sperm, the cryo-survival remains less than optimal. The present study, therefore, was conducted to determine the optimal concentration of antifreeze protein (AFP) III supplemented in a commercial egg-yolk-free medium for cynomolgus macaque (Macaca fascicularis) sperm cryo-survival. The function of frozen-thawed sperm was evaluated by post-thaw sperm motility, acrosome integrity, and mitochondrial function. Results indicate that the sperm motilities were greater when 0.1, 1, and 10 μg/ml of AFP III were supplemented into the sperm freezing medium (P < 0.05). In addition, the mitochondrial membrane potential was greater in the sperm cryopreserved with the medium that was supplemented with 0.1 μg/ml of AFP III (P < 0.05). The addition of AFP III at any of the concentrations, however, did not have any cryoprotection effect on the sperm acrosome, and the greatest concentrations of AFP III at 100 and 200 μg/ml had detrimental effects on acrosomal integrity (P < 0.05). Results of the present study indicated the methods used are effective for the cryopreservation of cynomolgus monkey sperm while reducing associated health risks due to avian pathogens being present in egg yolk-based extenders.

The effect of pre-freeze cooling and melatonin on pig sperm plasma membrane fluidity and cryosurvival

The effect of pre-freeze cooling and melatonin on pig sperm plasma membrane fluidity and cryosurvival

Differential motility parameters and identification of proteomic profiles of human sperm cryopreserved with cryostraw and cryovial

BackgroundAlthough sperm cryopreservation has been widely used in human reproductive medicine as an integral infertility management in infertility clinics and for banking sperm in sperm banks, the freezing/thawing protocols are not optimal. The freezing and thawing processes result in changes at both structural and molecular levels, some even detrimental, in human sperm when compared with fresh sperm. The change of sperm proteins after cryopreservation may play negative roles for fertilization and early embryo development. Conventionally, cryostraws (CS) and cryovials (CV) are the most widely used cryopreservation carriers (CPCs) for human sperm cryopreservation accompanied with the use of egg yolk free commercial media. However, the influence of cryopreservation on the proteomic profile of human sperm preserved with the two CPCs is unknown. Therefore the purpose of the present study was to compare the frozen-thawed motility, investigate the proteomic profile of human sperm cryopreserved with the two types of CPCs, and identify the susceptible proteins that play key roles for sperm function and fertility.MethodsThe present study compared the cryosurvival of human sperm frozen with the two different CPCs and identified the sperm proteomic changes by using the isobaric tags for relative and absolute quantification labeling technique coupled with 2D LC–MS/MS analysis after freezing and thawing.ResultsOur results indicated that sperm cryopreserved with CV showed higher values for percentage of motile sperm and forward activity rate than those with CS. Compared to fresh sperm, 434 and 432 proteins were differentially identified in human sperm cryopreserved with CS and CV, respectively.ConclusionThe proteomic profiles of human sperm are greatly affected by cryopreservation with either type of CPC. GO analysis revealed that most of the differentially identified sperm proteins enriched in the extracellular membrane-bounded organelles, cytoplasm and cytosol. In addition, 106 susceptible proteins having known identities related to sperm functions were identified. In general, cryovial seems to be the preferred CPC for human sperm cryopreservation based on the post-thaw motility parameters and the effect on sperm proteomic profiles. These results are beneficial for the insight into the understanding of the cryoinjury mechanism of sperm and the development of human sperm cryopreservation strategies.

Optimization of Sperm Cryopreservation Protocol for Mediterranean Brown Trout: A Comparative Study of Non-Permeating Cryoprotectants and Thawing Rates In Vitro and In Vivo

Simple SummaryCryobanking is an important tool to preserve the genetic resources of fish species. Semen cryopreservation has been extensively used in conservation programs for endangered species. Here, we aimed to find an effective cryopreservation protocol for the autochthonous Mediterranean brown trout inhabiting the Biferno river (south Italy), in order to create a sperm cryobank. Low-density lipoproteins and sucrose were tested as non-permeating cryoprotectants (NP-CPAs) to replace the egg yolk. Moreover, the thawing rate (10 °C for 30 s vs. 30 °C for 10 s) was also studied. From results obtained in vitro and in vivo, egg yolk emerged as the best NP-CPA and the lower thawing rate recorded better post-thaw semen quality in vitro and higher fertilization and hatching rates in vivo. These findings are important because they will contribute to the creation of a sperm cryobank for Molise’s native trout, which is a milestone of our European project (Life Nat.Sal.Mo).The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C (p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions.

The effect of non-traditional cooling on dog sperm cryosurvival and ability to perform the acrosome reaction

Abstract: The objective of this study was to assess cryosurvival, plasma membrane fluidity, and capability of cryopreserved dog (Canis lupus familiaris) spermatozoa, cooled to -5 °C before freezing, to perform the acrosome reaction under the effect of progesterone and calcium ionophore. In the first experiment, fresh spermatozoa diluted in Tyrode’s medium plus albumin, lactate, and pyruvate (TALP) were incubated at 38 °C in 5% CO2 in air, with progesterone or calcium ionophore added at 2, 4, and 6 h after incubation and sampled 30 min later to assess the acrosome reaction. In the second experiment, diluted sperm were packaged in plastic straws, cooled to either +5 °C or -5 °C and cryopreserved. Progressive motility, plasma membrane integrity and fluidity, capacitation status and acrosome integrity were assessed before and after freeze-thawing. After thawing, sperm were assessed, resuspended in TALP and incubated to assess the acrosome reaction. Parameters for sperm cryosurvival were similar in sperm cooled to either +5 °C or -5 °C, except in the percentage of hyper-fluid membranes which was lower (P<0.05) in sperm cooled to -5 °C. There were no differences in the percentages of frozen-thawed spermatozoa with acrosome reaction, induced by progesterone or calcium ionophore, between cooling treatments. In conclusion, cooling of dog spermatozoa to -5 °C did not improve sperm cryosurvival but had a positive effect on plasma membrane fluidity.

Lecithin nanoparticles enhance the cryosurvival of caprine sperm

This study was designed to compare the effects on goat spermatozoa cryosurvival of nano-lecithin-based (NL), lecithin-based (L) and egg yolk-based (EY) extenders. Ejaculates were collected from four fertile goats using artificial vagina and diluted with nine extenders. NL and L were tested at concentrations 1%, 2%, 3% and 4% (w/v), versus 15% (v/v) egg yolk-based extender. Overall, sperm quality (higher motility, viability and HOST, and lower apoptosis) was higher for NL than for L treatments (P < 0.05 for most cases, except for 1%). NL at 1% and especially at 4% showed lower motility and viability than 2% or 3% NL. NL at 2% achieved a better performance (P < 0.05) than EY for VCL (131.5 ± 1.3 vs. 120.3 ± 1.9 μm/s), VSL (43.9 ± 1.5 vs. 35.8 ± 1.4 μm/s), LIN (35.7 ± 0.6 vs. 29.3 ± 0.8%), WOB (47.0 ± 0.5 vs. 43.9 ± 0.9%) and viability (66.4 ± 1.7 vs. 52.7 ± 1.9%). Late apoptotic spermatozoa were also lower in 2% NL compared to EY (16.0 ± 0.5 vs. 26.3 ± 1.1%, P < 0.001). EY and 2% NL were compared in an IVF trial, with no significant differences in cleavage (68.8 vs. 70.8%) or blastocyst ratios (21.3 vs. 20.8%). In conclusion, using 2% nanolecithin in semen dilution could improve sperm cryosurvival of goat.

The proteome of frozen-thawed pig spermatozoa is dependent on the ejaculate fraction source

The preservation of sperm functional parameters and fertility post-cryopreservation largely varies in the porcine, a species with a fractionated ejaculate. Although intrinsic individual differences have primarily been linked to this variation, differences in protein abundance among frozen-thawed (FT)-spermatozoa are far more relevant. This study, performed in two experiments, looked for proteomic quantitative differences between FT-sperm samples differing in post-thaw viability, motility, apoptosis, membrane lipid peroxidation and nuclear DNA fragmentation. The spermatozoa were either derived from the sperm-rich ejaculate fraction (SRF) or the entire ejaculate (Experiment 1) or from the first 10 mL of the SRF, the remaining SRF and the post-SRF (Experiment 2). Quantitative sperm proteomic differences were analysed using a LC-ESI-MS/MS-based SWATH approach. In Experiment 1, FT-spermatozoa from the SRF showed better preservation parameters than those from the entire ejaculate, with 26 Sus scrofa proteins with functional sperm relevance showing relative quantitative differences (FC ≥ 1.5) between sperm sources. In Experiment 2, FT-spermatozoa from the first 10 mL of the SRF and the remaining SRF were qualitatively better than those from the post-SRF, and 187 proteins showed relative quantitative differences among the three ejaculate sources. The results indicate that quantitative proteome differences are linked to sperm cryosurvival.

Short communication: The percentage of egg yolk in the freezing media affects mouflon (Ovis musimon) epididymal sperm cryosurvival

Sperm cryopreservation protocols are not well defined in many wild species such as the mouflon. The aim was to study the effect of different concentrations of EY on mouflon epididymal sperm cryosurvival. Samples were collected by the flushing method and cryopreserved by the conventional freezing technique in straws using a TEST (TES, Tris, glucose) 5% v/v glycerol medium containing either 6% v/v (n=16) or 12% v/v (n=13) clarified EY. The membrane integrity, acrosome integrity, motility, and morphological abnormalities were assessed in fresh and frozen/thawed samples. Fresh sperm quality parameters did not differ between groups except for the acrosome integrity that was lower in the TEST-6% EY than in the TEST-12% EY group (88.9 ± 2.1% vs 94.7 ± 0.8%). Membrane integrity (31.6 ± 4.6% vs 11.6 ± 4.5%), total motility (32.8 ± 4.6% vs 17.2 ± 5.6%), progressive motility (13.3 ± 2.7% vs 6.1 ± 2.9) were higher in frozen-thawed sperm with TEST-6% EY than with TEST-12% EY (p&lt;0.05). Other motility parameters such as curvilinear velocity, straight-line velocity, average path velocity and amplitude of lateral head displacement were also higher (p&lt;0.05) in frozen-thawed sperm with TEST-6% EY. Frozen-thawed acrosome integrity (85.1 ± 3.3% vs 91.9 ± 2.3) and morphological abnormalities (34.0 ± 3.7% vs 29.1 ± 3.6%) did not differ between extenders. In conclusion, high EY concentration had detrimental effects on post-thaw quality parameters, therefore the use of TEST based extender containing 6% EY is recommended for the cryopreservation of mouflon epididymal sperm.

Semen extender type and cold storage of tissue affect cryosurvival of alpaca epididymal sperm

The objectives of this study were to determine the ability of liposome- and DMFA-containing semen extenders to support cryosurvival of alpaca epididymal sperm, and to evaluate the effect of epididymal cold storage on post-thaw viability. It was hypothesized that both semen extenders provide appropriate cryoprotection, and post-thaw sperm viability does not differ when alpaca epididymal sperm are cryopreserved immediately after castration or after 24 h of cold storage. Sperm were recovered from one epididymis from each male (n = 10) on the day of castration, and from the other one after 24 h of tissue storage at 4C. On each day, each sample was divided into two aliquots, which were cryopreserved in semen extender OptiXcell or BotuCRIO. Pre-freezing sperm parameters did not differ between samples obtained on the day of castration or 24 h later. Motility of sperm cryopreserved in BotuCRIO on the day of castration did not differ from pre-freezing values. Use of OptiXcell or delaying sperm cryopreservation decreased total (p = 0.002) and progressive sperm motility (p = 0.041) compared with pre-freezing values seen in sperm recovered on the day of castration. Membrane integrity was lowest when sperm were cryopreserved in OptiXcell after cold storage. Treatment had no effect on DNA integrity. It was concluded that the DMFA-containing semen extender BotuCRIO was superior to the liposome-containing semen extender OptiXcell at preserving post-thaw viability of alpaca epididymal sperm. Cold storage of epididymides for 24 h negatively affected post-thaw motility and membrane integrity of sperm. Therefore, cryopreservation with BotuCRIO immediately after castration is recommended for best preservation of post-thaw viability of alpaca epididymal sperm.

Effect of lecithin nanomicelles in extender on bull sperm cryosurvival

Effect of lecithin nanomicelles in extender on bull sperm cryosurvival

The effect of melatonin and cooling to -5°C on bulldog sperm cryosurvival

The effect of melatonin and cooling to -5°C on bulldog sperm cryosurvival

Use of dimethylsulfoxide for semen cryopreservation in Indian red jungle fowl (Gallus gallus murghi)

Glycerol is a least toxic and most effective cryoprotectant for cryopreservation of poultry semen, but due to its contraceptive properties removal of glycerol is usually needed prior to artificial insemination. Dimethylsulfoxide (DMSO), a small amphiphilic molecule used as penetrating cryoprotectant for biological cells, has been recognized as an adequate alternative for cryopreservation of sperm from several species. This study was designed to evaluate the efficacy of different concentrations of DMSO as cryoprotectant for Indian red jungle fowl (Gallus gallus murghi) sperm. Semen was collected from Indian red jungle fowl cocks, pooled and divided into five aliquots. Different concentrations of DMSO (0%, 4%, 6%, 8% and 10%) were compared. Diluted semen was cooled from 37 °C to 4 °C (−0.275 °C min−1), 20% glycerol added to control and equilibrated for 10 min. After equilibration, semen was filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Semen samples were thawed at 37 °C for 30 s. Cryo-survival of Indian red jungle fowl sperm was affected by cryopreservation stages and different concentrations of cryoprotectant used. Highest sperm motility (85.0 ± 2.9; 80.0 ± 3.5; 71.3 ± 4.3; 60.0 ± 1.3), plasma membrane integrity (79.5 ± 3.8; 75.3 ± 2.4; 72.8 ± 3.3; 60.3 ± 2.8), viability (80.8 ± 4.6; 75.5 ± 2.9; 71.0 ± 7.6; 58.8 ± 1.3) and acrosomal integrity (76.3 ± 2.4; 72.0 ± 6.0; 62.5 ± 4.3; 55.0 ± 3.2) were recorded in a diluent having 8% DMSO at post-dilution, cooling, equilibration and freeze-thawing. Highest fertility results were obtained after artificial insemination with 8% DMSO compared to 20% glycerol (73.0 ± 4.4 vs 53.1 ± 4.3, P < 0.05). It is concluded that 8% DMSO as a permeable cryoprotectant improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol.