Computer-aided Sperm Analysis Research Articles

Cryopreservation of epididymal and ejaculated bull spermatozoa

The present study was undertaken to assess viability of frozen–thawed bull semen collected from the bull’s ejaculate and cauda epididymis. A total of 30 ejaculates were collected from three bulls twice per week for 5 weeks (Control). Caudal epididymis were collected from slaughtered beef cattle of unknown origin from the local abattoir. Caudal epididymal sperm was recovered immediately after slaughtering (EP-0 h) and after cooling at 5°C for 24 h (EP-24). The epididymal and ejaculated samples were each pooled together before being extended with Triladyl. Diluted samples per treatment were loaded into a 0.25-mL French straw and cooled to 5°C in 4 h. Cooled straws were placed 4 cm above liquid nitrogen to freeze for 10 min. Frozen straws were immersed into LN2 and kept for 7 days at −196°C. Samples were analysed immediately after dilution and post-thawing using the computer aided sperm analysis for sperm motility rate, viability and acrosome defects. The highest sperm motility rates were observed with EJ-0 h before and after cryopreservation. However, the difference in sperm motility parameters between EP-0 h and EP-24 h evaluated before and after freezing was not significant (P > 0.05). Furthermore, no significant difference in live cells mean values was observed between the three samples on freezing (P > 0.05). In relation to spermatozoa acrosome defects, there was no significant difference observed among the three samples before and on freezing (P > 0.05). In conclusion, the results from the present study revealed that cooling of epididymides at 5°C for 24 h before the recovery of sperm cells was efficient in preserving epididymal sperm viability. However, ejaculated bull spermatozoa had higher sperm motility and viability rate than epididymal sperm.

Variation in post-thaw sperm quality of white-tailed deer bucks (Odocoileus virginianus) during rut

White-tailed deer farming is a growing industry in the United States, with breeding operations contributing significantly to the industry’s economic impact. Artificial insemination with frozen semen allows for selection and dissemination of valuable genetics, yet surprisingly little is known regarding the best time throughout rut to perform semen cryopreservation. The objective of this study was to compare semen quality following cryopreservation of white-tailed deer bucks collected early in the breeding season (September, n = 6), at peak rut (December, n = 8), and late season (March, n = 7). We hypothesized that post-thaw semen quality would be enhanced at peak rut. Mature bucks were anesthetized with tiletamine-zolazepam and xylazine administered intramuscularly via projector. Semen was collected by electroejaculation and cryopreserved using Optixcell extender. Overall and progressive sperm motilities were assessed for each sample before and after cryopreservation using a computer-aided sperm analyzer. Flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and DNA stability (acridine orange). Analysis of variance was applied to normalized data using a general linear mixed model with buck ID as a random variable, and a Tukey HSD test was performed as needed for post-hoc analysis. Pre-freeze overall and progressive sperm motilities were lowest in March, intermediate in September, and highest in December (p ≤ 0.04). Post-thaw overall and progressive motilities were lowest in September (p ≤ 0.02), but did not differ between December and March (p ≥ 0.12). The DNA Fragmentation Index was lowest in December, intermediate in September, and highest in March (p ≤ 0.05). The percentage of spermatozoa with intact plasma membrane was higher in December than September (p < 0.01), but the percentage of intact acrosomes per sperm with intact plasma membrane was highest in September (p = 0.03). This study confirms that post-thaw semen quality appears to be superior during peak rut (December) in bucks. Though semen collected early or late in rut may present acceptable motility, DNA stability is impaired, which could adversely affect fertility rates. This data suggests that semen cryopreservation during transitional periods should be avoided, though field studies evaluating the translation of these results into satisfactory pregnancy rates are warranted.

A comparison between the semen and sperm parameters from the captive-bred Vervet monkey (Chlorocebus aethiops) and Rhesus monkey (Macaca mulatta).

It is generally accepted that non-human primates (NHP) represent the model of choice for integrative studies of testicular function and endocrine control. However, there are many species-specific differences that necessitate identification prior to the selection of an appropriate model for these studies. In an NHP breeding facility, this opportunity of selection is usually presented during breeding periods when it is crucial to determine which individuals should be maintained as breeders. With reference to adult males and their use in breeding programs and reproductive studies, it is therefore imperative to document the normal semen and sperm values, expected seasonal changes and the variabilities found within samples and among individuals. The comparison of closely related species that differ by breeding seasonality will, therefore, highlight their value in reproductive research. Semen samples were obtained by rectal probe electroejaculation (RPE). The seminal and sperm characteristics of captive-bred Vervet monkeys (Chlorocebus aethiops) (n=10) and Rhesus monkeys (Macaca mulatta) (n=10) were evaluated and compared. Parameters such as semen volume, pH, sperm concentration, and sperm motility were analyzed by means of a computer-aided sperm analysis (CASA) system. Large variations in semen and sperm parameter values indicated differences between species and samples. Monthly variations were observed for the Vervet regardless of breeding and conceptions that occurred throughout the year. In contrast, Rhesus seminal characteristics indicated a clear seasonal trend. Non-human primates have long provided as research models for studying complexities of human reproductive biology. The baseline values reported from this study can be applied as guidelines during the selection of male individuals for reproductive studies. Of further interest is the comparative data on semen and sperm parameters between two congeneric species that differ by seasonal versus aseasonal breeding.

IN VITRO EFFECTS OF ACINETOBACTER BAUMANNII AND SELECTED NATURAL BIOMOLECULES ON RABBIT SPERMATOZOA MOTILITY

The aim of this study was to assess the potential efficiency of selected biologicallyactive substances on the motility behaviour of rabbit spermatozoa subjected to invitro induced A. baumannii contamination. The semen samples used for A.baumannii detection were collected from 10 New Zealand white male rabbits andthe presence of the bacterium was confirmed using MALDI-TOF MassSpectrometry. For the in vitro experiments rabbit spermatozoa were re-suspendedin PBS, containing mineral supplements, BSA and glucose in the presence of 3x105CFU A. baumannii and diverse concentrations of selected biomolecules (resveratrol- RES, quercetin - QUE, curcumin - CUR, epicatechin - EPI, isoquercitrin - ISO).The sperm motility was assessed using the computer-aided sperm analysis at 0h,2h, 4h and 6h. A. baumannii significantly decreased the sperm motility (P&lt;0.001)at Time 2h and maintained this negative impact throughout the in vitro culture.Meanwhile, the motility at Time 2h was significantly higher in the samples subjectedto A. baumannii together with 10 μmol/L RES (P&lt;0.01); 5, 10 and 50 μmol/L QUE(P&lt;0.001); 1 μmol/L CUR (P&lt;0.05); 10, 50 and 100 μmol/L EPI (P&lt;0.01) as well as 50μmol/L (P&lt;0.05) and 100 μmol/L ISO (P&lt;0.001) in comparison to the control exposedto the bacterium exclusively. After 4h, the motility remained significantly higher in thegroups co-treated with the inoculum and 10 μmol/L RES (P&lt;0.05), 50 μmol/L QUE(P&lt;0.05) as well as 50 μmol/L EPI (P&lt;0.05) when compared to the positive control.Nevertheless, none of the biomolecules was effective against the rapid decline ofsperm motility caused by A. baumannii during later stages of the experiment (Time6h). Based on these results, one can conclude that RES, QUE and EPI exhibitantibacterial properties providing a selective advantage to spermatozoa in thepresence of A. baumannii, particularly during short-term rabbit semen handling.

Correlations of Sperm Mitochondrial Membrane Potential with Semen Parameters and Male Obesity

Background: To investigate the correlations of sperm mitochondrial membrane potential (MMP) with semen parameters and body mass index (BMI) in males with obesity. Methods: Semen samples were obtained by masturbation after 3–7 days of sexual abstinence from males who visited semen collect room of Shanghai Ninth People’s Hospital. Conventional semen analyses were performed by computer-aided sperm analysis (CASA), and sperm morphology was analyzed by modified Papanicolaou staining. Spermatozoa were stained by JC-1 to evaluate MMP through flow cytometry. Results: Sperm MMP of asthenozoospermia group (41.24% ± 9.71%) was significantly lower than that in control group (56.68% ± 11.13%). MMP was negatively correlated with BMI (r = −0.25, P < 0.01), but positively correlated with total sperm motility (r = 0.63, P < 0.01), motility of progressive sperm (r = 0.64, P < 0.01), and normal sperm morphology rate (r = 0.37, P < 0.01). In addition, MMP showed no significant correlations with age, volume of semen, sperm concentration, sperm count, and other indexes. Conclusions: Sperm MMP is an important index in the evaluation of sperm function, and detection of MMP may provide references for the diagnosis and treatment of male infertility. Key words: Body Mass Index; Conventional Semen Analyses; JC-1; Mitochondrial Membrane Potential

Characteristic and mechanism of immobilization effect of Staphylococcus aureus on human spermatozoa

ObjectiveThis study was designed to investigate the impacts of Staphylococcus aureus isolated from sperm of male infertility patients, and explore the mechanism of the spermatozoa immobilization attributed to S. aureus. MethodsS. aureus MJ015 and MJ163, the representative strains of immobilization positive and negative group respectively, were obtained from semen of infertile men. Computer-aided sperm analysis (CASA) were performed to measure sperm motility. Transmission electron microscopy (TEM) was utilized to assess morphological alterations of spermatozoa. Two-dimensional gel electrophoresis (2-DE) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) were undertaken to analyse the difference between the secretory proteins of MJ015 and MJ163. ResultsA highly significant decline in motility of spermatozoa after incubating with cultured supernatant of MJ015 by sperm motility measurements, which was not observed when co-cultured with the supernatant of MJ163. TEM illustrated that the culture supernatant of MJ015 contributed to apparently ultrastructural impairment and inhibitory impacts on sperm motility. Various proteins expressed by two samples were identified. Data processing and database search preliminarily establish a link between four differential proteins and spermatozoal immobilization ability. ConclusionsOur data manifested that the clinical isolates of S. aureus have a key role on the motility and morphology of sperm. A better correlation between four identified differentially expressed proteins and the marked decline of the motility of spermatozoa was established.

Functional deficit of sperm and fertility impairment in men with antisperm antibodies

Autoimmune reactions against the sperm cells play an ambiguous role in fertility impairment. The objective of this study was to characterize functional deficit of sperm conditioned by antisperm immune response in normozoospermic men. This was a multi-centric, cross-sectional, case–control study. The study subjects were 1060 infertile normozoospermic men and 107 fertile men. The main outcome measures were clinical examination, semen analysis including MAR test for antisperm antibodies (ASA), computer-aided sperm analysis, acrosome reaction (AR) detected with flow cytometry, DNA fragmentation measured with sperm chromatin dispersion, reactive oxygen species (ROS) assessed using the luminol-dependent chemiluminescence method. 2% of the fertile men had MAR-IgG ≥ 50%, but all subjects with MAR-IgG > 12% were outliers; 16% infertile men had MAR-IgG ≥ 50% (p < 0.0001). There was a direct correlation between the infertility duration and MAR-IgG (R = 0.3; р < 0.0001). The ASA-positive infertile men had AR disorders 2.1 times more frequently (р < 0.02), predominantly inductivity disorders. We found signs of hyperactivation proportionate to the ASA level (p < 0.001). DNA fragmentation was more highly expressed and was 1.6 and 1.3 times more frequent compared with the fertile and the ASA-negative patients, respectively (p < 0.001 and p < 0.05). We found signs of oxidative stress (OS): ROS generation by washed ASA-positive spermatozoa was 3.7 times higher than in the fertile men (p < 0.00001) and depended on the ASA levels (R = 0.5; p < 0.0001). The ASA correlation with ROS generation in native sperm was weak (R = 0.2; р < 0.001). We concluded that autoimmune reactions against spermatozoa are accompanied by a fertility decrease in normozoospermia. This results from AR and capacitation disorders and DNA fragmentation. The pathogenesis of sperm abnormalities in immune infertility is associated with

The in Vitro Effect of Taurine on Boar Spermatozoa Quality

The aim of this in vitro study was to evaluate the effects of taurine (TAU) supplementation on boar spermatozoa motility, viability, acrosome integrity and morphology. Eighteen boar semen samples were diluted with the Androhep PlusTM extender containing no TAU (control) or supplemented with 1.5 mM, 7 mM, 12.5 mM TAU and cultured at 4 °C for 18 days. Sperm motility was evaluated using the computer-aided sperm analysis (CASA) system. Furthermore, the samples were fixed and assessed for the occurrence of morphological abnormalities using phase contrast microscopy. Fluorescent dyes SYBR – 14 and propidium iodide were used to determine the sperm viability. Acrosome integrity was examined using PNA – Alexa Fluor 647 and flow cytometry. A gradual decrease of the semen quality was detected in all experimental groups over the course of the study. CASA revealed no selective advantage of TAU supplementation on the spermatozoa motility (p &gt; 0.05). TAU administration showed to be ineffective in preserving spermatozoa viability as well as acrosome integrity as measured by flow cytometry (p &gt; 0.05). Under the conditions of this study, no significant positive effect of TAU was recorded following its administration to the Androhep PlusTM boar semen extender with respect to spermatozoa quality.

English

Successful artificial insemination depends on maintaining longevity of semen in the reproductive tract, and semen characteristics among others. The study compared efficacy of Androhep extender supplemented with 75 mM taurine, 5 m M-cysteine, or 200 mg/l alpha-tocophero on liquid preservation of Kolbroek boar semen characteristics over 24 and 48 h after storage at 17°C. Total motility was evaluated using computer aided sperm analyzer (CASA), viability using SYBR/propidium iodidemembrane function using HOST test and lipid peroxidation using the MDA test. Statistical analysis was done using PROC GLM procedure of SAS (1996). Data were analyzed using analysis of variance (ANOVA). Untreated samples showed higher sperm quality in terms of membrane integrity, functionality and motility. All antioxidant treated spermatozoa did not show increased longevity (p>0.05) after 48 h of storage compared with untreated samples. Supplementation of Androhep extender with antioxidants did not improve sperm survivability of kolbroek liquid preserved semen. Therefore, the antioxidants were ineffective in improving longevity of Kolbroek liquid preserved semen after 48 h storage at 17°C. Further studies are required to find the effective antioxidant concentration to elicit sperm protection for Kolbroek boar semen liquid preservation. Key words: Semen characteristics, longevity, lipid peroxidation.

Application of computer-aided sperm analysis (CASA) for detecting sperm-immobilizing antibody.

Since the 1970s, anti-sperm antibodies have been studied as a pathogenic factor contributing to infertility. The complement-dependent sperm-immobilization test (SIT) and quantitative SIT have been used as effective tools for detecting anti-sperm antibodies in clinical settings. These tests have been carried out traditionally by manually counting the number of motile sperm through eye estimation. In this study, we developed a novel method using computer-aided sperm analysis. The results were compared with those obtained by the traditional method. The results were identical and 25 of 78 samples tested were positive and 53 samples were negative for sperm-immobilizing (SI) antibodies based on both methods. For SI-positive samples, the values of SI50 obtained using the two methods correlated closely with high co-efficiency. Using the novel method, manually counting the number of motile spermatozoa becomes unnecessary. The novel method presented here will increase the objectivity and convenience of using the SIT as a clinical indicator.

CASA: tracking the past and plotting the future.

The human semen sample carries a wealth of information of varying degrees of accessibility ranging from the traditional visual measures of count and motility to those that need a more computational approach, such as tracking the flagellar waveform. Although computer-aided sperm analysis (CASA) options are becoming more widespread, the gold standard for clinical semen analysis requires trained laboratory staff. In this review we characterise the key attitudes towards the use of CASA and set out areas in which CASA should, and should not, be used and improved. We provide an overview of the current CASA landscape, discussing clinical uses as well as potential areas for the clinical translation of existing research technologies. Finally, we discuss where we see potential for the future of CASA, and how the integration of mathematical modelling and new technologies, such as automated flagellar tracking, may open new doors in clinical semen analysis.

Current perspectives of CASA applications in diverse mammalian spermatozoa.

Since the advent of computer-aided sperm analysis (CASA) some four decades ago, advances in computer technology and software algorithms have helped establish it as a research and diagnostic instrument for the analysis of spermatozoa. Despite mammalian spermatozoa being the most diverse cell type known, CASA is a great tool that has the capacity to provide rapid, reliable and objective quantitative assessment of sperm quality. This paper provides contemporary research findings illustrating the scientific and commercial applications of CASA and its ability to evaluate diverse mammalian spermatozoa (human, primates, rodents, domestic mammals, wildlife species) at both structural and functional levels. The potential of CASA to quantitatively measure essential aspects related to sperm subpopulations, hyperactivation, morphology and morphometry is also demonstrated. Furthermore, applications of CASA are provided for improved mammalian sperm quality assessment, evaluation of sperm functionality and the effect of different chemical substances or pathologies on sperm fertilising ability. It is clear that CASA has evolved significantly and is currently superior to many manual techniques in the research and clinical setting.

Implementation of novel statistical procedures and other advanced approaches to improve analysis of CASA data.

Computer-aided sperm analysis (CASA) produces a wealth of data that is frequently ignored. The use of multiparametric statistical methods can help explore these datasets, unveiling the subpopulation structure of sperm samples. In this review we analyse the significance of the internal heterogeneity of sperm samples and its relevance. We also provide a brief description of the statistical tools used for extracting sperm subpopulations from the datasets, namely unsupervised clustering (with non-hierarchical, hierarchical and two-step methods) and the most advanced supervised methods, based on machine learning. The former method has allowed exploration of subpopulation patterns in many species, whereas the latter offering further possibilities, especially considering functional studies and the practical use of subpopulation analysis. We also consider novel approaches, such as the use of geometric morphometrics or imaging flow cytometry. Finally, although the data provided by CASA systems provides valuable information on sperm samples by applying clustering analyses, there are several caveats. Protocols for capturing and analysing motility or morphometry should be standardised and adapted to each experiment, and the algorithms should be open in order to allow comparison of results between laboratories. Moreover, we must be aware of new technology that could change the paradigm for studying sperm motility and morphology.

Evaluation of sperm motility with CASA-Mot: which factors may influence our measurements?

Computer-aided sperm analysis (CASA) is now routinely used in IVF clinics, animal breeding centres and research laboratories. Although CASA provides a more objective way to evaluate sperm parameters, a significant number of factors can affect these measurements. This paper classifies these factors into four categories: (1) sample and slide (e.g. preincubation time, type of specimen and type of chamber slide); (2) microscope (e.g. light source and microscope stage); (3) hardware and software, including the settings of each system; and (4) user-related factors. We review the effects of the different factors in each category on the measurements made and emphasise the need to take measures to standardise evaluations. The take-home message of the present article is that there are several commercial and useful CASA systems, and all are appropriate for routine analysis. Non-commercial systems may also be good choices when the user needs to adapt the device to specific experimental conditions. In both cases (commercial and non-commercial), it is important that standard protocols are put in place for evaluation, as well as methods to validate the system.

Effect of counting chamber depth on the accuracy of lensless microscopy for the assessment of boar sperm motility.

Sperm motility is one of the most significant parameters in the prediction of male fertility. Until now, both motility analysis using an optical microscope and computer-aided sperm analysis (CASA-Mot) entailed the use of counting chambers with a depth to 20µm. Chamber depth significantly affects the intrinsic sperm movement, leading to an artificial motility pattern. For the first time, laser microscopy offers the possibility of avoiding this interference with sperm movement. The aims of the present study were to determine the different motility patterns observed in chambers with depths of 10, 20 and 100µm using a new holographic approach and to compare the results obtained in the 20-µm chamber with those of the laser and optical CASA-Mot systems. The ISAS®3D-Track results showed that values for curvilinear velocity (VCL), straight line velocity, wobble and beat cross frequency were higher for the 100-µm chambers than for the 10- and 20-µm chambers. Only VCL showed a positive correlation between chambers. In addition, Bayesian analysis confirmed that the kinematic parameters observed with the 100-µm chamber were significantly different to those obtained using chambers with depths of 10 and 20µm. When an optical analyser CASA-Mot system was used, all kinematic parameters, except VCL, were higher with ISAS®3D-Track, but were not relevant after Bayesian analysis. Finally, almost three different three-dimensional motility patterns were recognised. In conclusion, the use of the ISAS®3D-Track allows for the analysis of the natural three-dimensional pattern of sperm movement.

Sperm motility in fish: technical applications and perspectives through CASA-Mot systems.

Although a relatively high number of sperm quality biomarkers have been reported over the years in several fish species, sperm motility is nowadays considered the best biomarker for fish spermatozoa. The first scientific reports focusing on fish sperm motility date from a century ago, but the objective assessment allowed by computer-aided sperm analysis (CASA-Mot) systems was not applied to fish species until the mid-1980s. Since then, a high number of sperm kinetic parameters from more than 170 fish species have been reported in more than 700 scientific articles, covering a wide range of topics, such as sperm physiology, sperm storage, broodstock management, the phenomenon of sperm competition, ecotoxicology and understanding the life cycle of the species. The sperm kinetic parameters provided by CASA-Mot systems can serve as powerful and useful tools for aquaculture and ecological purposes, and this review provides an overview of the major research areas in which fish sperm motility assessment by a CASA-Mot system has been used successfully.

Fish sperm motility analysis: the central role of the flagellum.

Motility analysis of spermatozoa relies on the investigation of either head trajectories or flagellum characteristics. Those two sets of parameters are far from being independent, the flagellum playing the role of motor, whereas the head plays a passive role of cargo. Therefore, quantitative descriptions of head trajectories represent a simplification of the complex pattern of whole sperm cell motion, resulting from the waves developed by the flagellum. The flagellum itself responds to a large variety of signals that precisely control its axoneme to allow activation, acceleration, slowing down or reorientation of the whole spermatozoon. Thus, it is obvious that analysis of flagellum characteristics provides information on the original source of movement and orientation of the sperm cell and presents additional parameters that enrich the panoply of quantitative descriptors of sperm motility. In this review, we briefly describe the methodologies used to obtain good-quality images of fish spermatozoa (head and especially flagellum) while they move fast and the methods developed for their analysis. The paper also aims to establish a link between classical analyses by computer-aided sperm analysis (CASA) and the descriptors generated by fish sperm flagellum analysis, and emphasises the information to be gained regarding motility performance from flagellum motion data.

CASA in invertebrates.

Sperm movement has been described in several phyla of invertebrates. Yet, sperm motility has only been quantified using computer-aided sperm analysis (CASA-Mot) in externally fertilising species (broadcast spawners) of two phyla, molluscs and echinoderms. In the present study we quantified in detail the nature of the sperm tracks, percentage motility groupings and detailed kinematics of rapid-, medium- and slow-swimming spermatozoa in the oyster Crassostrea gigas and four species never previously studied by CASA-Mot, namely the molluscs Choromytilus meridionalis, Donax serra and Haliotis midae and the echinoderm Parechinus angulosus. A feature common to all these species are the helical tracks, the diameter of which seems to be species specific. Using CASA-Mot, the behaviour of spermatozoa was also studied over time and in the presence of egg water and Ca2+ modulators such as caffeine and procaine hydrochloride. For the first time, we show that hyperactivation can be induced in all species in the presence of egg water (sea water that was mixed with mature eggs and then centrifuged) and/or caffeine, and these hyperactivated sperm tracks were characterised using CASA-Mot. We relate the different patterns of sperm motility and behaviour to reproductive strategies such as broadcast spawning and spermcasting, and briefly review studies using CASA-Mot on other invertebrates.

CASA-Mot technology: how results are affected by the frame rate and counting chamber.

For over 30 years, CASA-Mot technology has been used for kinematic analysis of sperm motility in different mammalian species, but insufficient attention has been paid to the technical limitations of commercial computer-aided sperm analysis (CASA) systems. Counting chamber type and frame rate are two of the most important aspects to be taken into account. Counting chambers can be disposable or reusable, with different depths. In human semen analysis, reusable chambers with a depth of 10µm are the most frequently used, whereas for most farm animal species it is more common to use disposable chambers with a depth of 20µm . The frame rate was previously limited by the hardware, although changes in the number of images collected could lead to significant variations in some kinematic parameters, mainly in curvilinear velocity (VCL). A frame rate of 60 frames s-1 is widely considered to be the minimum necessary for satisfactory results. However, the frame rate is species specific and must be defined in each experimental condition. In conclusion, we show that the optimal combination of frame rate and counting chamber type and depth should be defined for each species and experimental condition in order to obtain reliable results.

Improvement of Post-Thaw Sperm Kinematics and DNA Integrity of Cross-Bred Bovine Sperm by Incorporating DGC as Selection Method Prior to Cryopreservation

The aim of this study was to assess post-thaw sperm quality following initial sperm selection using density gradient centrifugation (DGC) prior to cryopreservation. Ejaculates from four mature Charolais cross Kedah-Kelantan bulls were collected using artificial vagina at IBVK Pahang, Malaysia. The ejaculates were aliquoted into 3 groups: non-cryopreserved group (NC); control group of cryopreserved sperm without DGC (ND) and treatment group of sperm undergoing DGC sperm selection before cryopreservation (CDGC). Prior to analysis, samples from both cryopreserved groups were thawed at 37 °C for 30 sec. All samples were analysed for kinematics parameters, viability and compromise in DNA integrity (evaluated as DNA Fragmentation Index, DFI). All kinematics parameters were analysed using computer aided sperm analysis (CASA). Results indicated significant (p &lt; 0.05) kinematics parameter changes for all parameters of velocity (VCL, VSL, VAP) and progression (WOB, LIN, ALH and BCF). Unfortunately, changes in spermatozoa straightness were insignificant (STR) F(2, 68) = 1.004, p = 0.371. Spermatozoa viability had increased by 26.2% (p &lt; 0.01) following the treatment. DFI revealed the treatment group recorded a significant reduction in DFI value (0.17% fragmented DNA). In conclusion, DGC sperm selection prior to cryopreservation reduced the effects of cryodamage and showed an improvement in post-thaw sperm quality, thus reducing the occurrence of asthenozoospermia in populations of frozen-thawed cross-bred bovine spermatozoa.