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Effects of hyaluronic acid on sperm parameters, mitochondrial function and apoptosis of spermatozoa in Simmental bulls with good and poor freezing ability
Abstract
Bulls with varying freezability exhibit substantial variation in semen characteristics after cryopreservation. Sperm freezability is positively correlated with membrane cholesterol content, membrane integrity, mitochondrial activity and antioxidant content. The purpose of this study was to determine the optimal concentration of hyaluronic acid (HA) in bull sperm with different cryotolerances. Simmental bulls ( n = 10) semen samples were taken and categorized based on their progressive motility (PM) after freeze‐thawing: Group I, consisting of bulls ( n = 5) with progressive sperm motility ≥45%, was considered good freezability ejaculates (GF), and Group II, including bulls ( n = 5) with progressive sperm motility ≤30%, was considered poor freezability ejaculates (PF) bulls. Semen samples were diluted with a Tris‐egg‐yolk‐glycerol (TEYG) extender containing various concentrations of HA: without HA (control), 1 mM HA, 2 mM HA and 4 mM HA. After the freeze‐thaw process, sperm kinematics, plasma membrane and acrosome integrity, mitochondrial activity and apoptotic status were evaluated. The addition of 1 mM HA to the diluent of bulls with GF increased PM and linearity (LIN) compared to the control group ( p < 0.05). Normal morphology was improved after thawing in the samples treated with 1 and 2 mM HA in the GF and PF bulls respectively. The membrane and acrosome integrity of GF bulls treated with 1 mM HA was significantly ( p < 0.05) greater than that of the control groups. Adding 1 mM HA to the extender of bulls with GF and PF improved the proportion of viable cells compared with the highest concentration (4 mM) of HA. The mitochondrial activity of PF bulls treated with 1 and 2 mM HA was significantly ( p < 0.05) greater than that of the controls and 4 mM HA. Finally, it can be concluded that adding low doses of HA (1 mM) to the TEYG extender of GF and PF bulls ameliorated the post‐thaw semen quality.
... Based on their postthaw sperm characteristics, bulls (n = 5) with progressive sperm motility (PM) £30% were selected and considered low-freezeability bulls. 16 Semen samples obtained from the five bulls were pooled and subsequently divided into four groups. Group 1 served as the control group and received the extender without exosomes. ...
... Simultaneous readings for FL 1 (530/30 nm) and FL 2 (575/24 nm) were obtained. 16 ...
Introduction: Sperm cryopreservation is a useful storage technique in artificial insemination. Nanoparticles and nanovesicles such as exosomes are widely used in sperm cryopreservation procedures to alleviate cold-induced injury inflicted during sperm freezing. Objective: The objective of the present study was to examine the impact of varying concentrations of exosomes derived from seminal plasma added to a freezing extender on the quality of post-thawed bull sperm. Methods: Five Holstein bulls were chosen based on their samples having less than 30% progressive motility. After exosome extraction, semen samples from bulls (n = 5) with progressive sperm motility ≤30% were collected, diluted with different exosome concentrations (0, 25, 50, and 100 μg/mL), and aspirated into 0.5 mL straws. After the freeze-thaw process, sperm total and progressive motility, viability, morphology, plasma membrane integrity, mitochondrial activity, and apoptosis status were assessed. Furthermore, the expression levels of annexin (ANX1), dystrophy-associated Fer-1-like protein (DYSF), fibronectin 1 (FN1), and reactive oxygen species modulator 1 (ROMO1) were evaluated via real-time polymerase chain reaction (PCR). Results: Adding different concentrations of exosomes (25, 50, and 150 μg/mL) significantly increased the progressive motility, viability, and membrane integrity of sperm compared with the control group (p < 0.05). For the apoptosis index, treatment with 100 μg/mL exosomes significantly increased the percentage of live cells (p < 0.05), while the percentage of necrotic cells decreased significantly (p < 0.05) compared with 25 μg/mL exosome. The results of quantitative PCR showed that the expression levels of ANX1 were significantly (p < 0.05) upregulated at 50 μg/mL exosome, and the expression of ROMO1, FN1, and DYSF were downregulated upon treatment with different exosome concentrations. Conclusions: In conclusion, supplementing the freezing diluent with exosome-derived seminal plasma could preserve the quality parameters of the post-thaw semen of the bull with low freezeability and could be used as a helpful method for reproductive programs.
... The freezing resulted in the significant decrease in sperm motility parameters (Asadpour et al., 2024), however, the mechanism of cryodamage is still unclear. Mitochondria were known as the "power plants" of cells and played a critical role in cellular homeostasis and sperm motility (Lu et al., 2013;Maryam et al., 2018). ...
Spermatozoa cryopreservation has been widely used for animal genetic conservation. Despite advances in chicken semen cryopreservation, the mechanism of spermatozoa cryodamage remains to be revealed. The cryopreservation process induces motion parameter decreased, structure damaged, proteomic and antioxidant system remodeled in spermatozoa. Mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2) is part of the malate aspartate shuttle, which is ubiquitous in mitochondria and is associated with cellular metabolism regulation. Thus, this study is the first to investigate GOT2 biological role in chicken spermatozoa during freezing process. The results showed that the sperm total motility, straight-line velocity (VSL) and mitochondrial membrane potential (MMP) of the frozen group were significantly lower than these of the fresh group (P < 0.05). The fresh sperm mitochondrial membrane was continuous and mitochondrial matrix was dense and homogeneous. However, after the freezing-thawing, the density of the matrix was reduced, and the mitochondria appeared slightly swollen and membrane damaged. In chicken sperm, the GOT2 protein was localized in the head and the midpiece of spermatozoa where mitochondria are located by immunostaining analysis. This was consistent with the subcellular localization prediction. GOT2 protein was more abundant in the fresh sperm than in the frozen sperm, which indicated that freezing may lead to sperm mitochondrial damage, reduced GOT protein expression, and affected sperm motility and fertility. The protein-protein interaction prediction of GOT2 protein indicated that its ten most confident interactors were predominantly mitochondria-related proteins. The binding ability was higher between GOT2 protein and two mitochondria-targeted antioxidants, SkQ1 and Mito-TEMPO, respectively. In conclusion, GOT2 played an important role in chicken spermatozoa, which was possibly associated with the regulation of mitochondria function and spermatozoa metabolism. Moreover, it may be a potential cryodamage improvement target for spermatozoa. However, the underlying mechanism of GOT2 in spermatozoa cryopreservation needs further exploration.
Many cellular processes in spermatozoa, including apoptosis and motility, are regulated by miRNA. Different miRNAs and molecular pathways are involved in asthenozoospermia (AS) conditions, which are thought to be one of the causes of infertility with reduced sperm motility. Thirty-two semen samples from four Holstein bulls with normozoospermia (NS), total motility ≥ 70%, and progressive motility ≥ 60%, and 32 semen samples from four bulls with AS, total motility ≤ 40%, and progressive motility ≤ 32% were used to investigate the function of apoptosis-related miRNAs in the AS group. Samples were then aspirated into a 0.5 mL straw after dilution with a Tris-egg yolk extender and frozen at -196°C. After freezing, semen samples were thawed for 2 weeks at 37°C and sperm kinematic parameters, plasma membrane integrity, acrosome integrity, DNA fragmentation, apoptosis status, and expression of apoptosis-related miRNAs (miR-2114, miR-296-3p, miR-455-3p, and miR345-3p) were evaluated. Our results showed that the functional and flow cytometric parameters of the NS group were significantly better than those of the AS group. In the NS group, miR-455-3pp and miR-2412 were upregulated, while miR-345-3p was downregulated compared with the AS group. In the AS group, miR-296-39, miR-2412, and miR-345-3p levels were strongly correlated with membrane integrity, DNA fragmentation, and apoptosis status. The findings demonstrated that the selected miRNAs based on bioinformatic analysis in AS and NS samples had a substantial association with functional and flow cytometry indicators and may be involved in regulating apoptosis and motility in AS samples.
In this study, it was hypothesized that the addition of an appropriate concentration of Y-27632 (a ROCK inhibitor) to the freezing extender prevents cryopreservation-induced apoptosis and improves embryonic development after in vitro fertilization (IVF). Semen samples were collected from five fertile Simmental bulls using an artificial vagina twice a week for 4 weeks. Selected samples were pooled and diluted with Tris-egg-yolk-glycerol (TEYG) extender containing different concentrations of Y-27632 (0, 10, 20, 30, and 40 μM) and then frozen in liquid nitrogen. After thawing, computer-assisted semen analysis (CASA), plasma membrane integrity, and acrosome intactness were evaluated in terms of morphological abnormalities, intracellular generation of reactive oxygen species (ROS), DNA fragmentation, phosphatidylserine (PS) externalization, and apoptotic-related gene expression. Finally, groups of frozen and thawed spermatozoa were used for bovine oocyte IVF. The results show that the semen extender at a concentration of 20 μM Y-27632 effectively improved total motility (TM), curvilinear velocity (VCL), as well as the plasma membrane and acrosome integrity compared to the control group (p < 0.05). Intracellular ROS levels were significantly (p < 0.05) lower in samples treated with 30 μM Y-27632 compared to the control specimen. Furthermore, supplementation of the semen extender with 20 μM Y-27632 resulted in more viable spermatozoa compared with the control group (p < 0.05). According to qRT-PCR results, the expression levels of BAX and CASPASE-9 genes in samples treated with 30 μM Y-27632 were significantly downregulated, while the expression of BCL2 was increased compared to the control (p < 0.05). The results of IVF demonstrated that the treatment of frozen–thawed spermatozoa with 20 μM Y-27632 increased blastocyst rates compared to the control group (p < 0.05). In conclusion, the addition of 20 μM Y-27632 into the freezing extender can improve the functionality and the fertilizing capacity of frozen spermatozoa due to its antioxidative and anti-apoptotic properties.
Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.
Background:
The purpose of this research was to compare the functional parameters of frozen-thawed Iranian Azari buffalo spermatozoa with imported semen samples of Italian Mediterranean buffalo (IMB) after the thawing process and 4 hours of incubation.
Materials and methods:
In this experimental study, a total of twenty-four ejaculates from four Iranian Azari buffalo bulls were collected. Semen samples were diluted in AndroMed extender at a concentration of 50×106 spermatozoa/ ml. The diluted samples were filled in 0.5 ml straws and were frozen in a programmable freezer. For imported semen samples, twenty-four samples of four IMB were used, which were diluted in AndroMed extender and frozen by the same procedure. Frozen-thawed sperm motion patterns, mitochondrial activity, membrane integrity, DNA integrity, reactive oxygen species (ROS), and apoptosis status were evaluated immediately after thawing and 4 hours of incubation.
Results:
Post-thawed sperm motility, progressive motility (PM), mitochondrial activity, membrane integrity were significantly higher in imported semen samples in compare with Iranian Azari buffalo. After 4 hours of incubation, sperm velocity patterns were superior in Iranian Azari semen samples. Moreover, the percentage of sperm cells with un-damaged DNA was higher in Iranian semen samples compared to imported samples at the time 0 of incubation. Following 4 hours of incubation, a significant increase in intracellular ROS level leads to reduced membrane integrity, mitochondrial activity, and DNA integrity in both buffalo breeds. At time 4, Iranian samples showed significantly lower apoptosis and higher dead spermatozoa compared to imported semen samples.
Conclusion:
Our study showed that the post-thawed quality of Iranian Azari buffalo semen was comparable with imported samples after 4 hours of incubation. Further investigations are recommended to assess the in vitro and in vivo fertility rate of both buffalo breeds.
Artificial insemination of livestock has been a staple technology for producers worldwide for over sixty years. This reproductive technology has allowed for the rapid improvement of livestock genetics, most notably in dairy cattle and pigs. This field has experienced continuous improvements over the last six decades. Though much work has been carried out to improve the efficiency of AI, there are still many areas which continue to experience improvement, including semen analysis procedures, sperm selection techniques, sperm sexing technologies, and semen storage methods. Additionally, the use of AI continues to grow in beef cattle, horses, and small ruminants as the technology continues to become more efficient and yield higher pregnancy rates. In this review, AI trends in the various livestock species as well as cutting edge improvements in the aforementioned areas will be discussed at length. Future work will continue to refine the protocols which are used for AI and continue to increase pregnancy rates within all livestock species.
Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. In some farm animals, the use of cryopreserved sperm has so many benefits for which relevance has become more evident in recent decades. Values for post-thaw sperm quality, however, are variable among species and within individuals of the same species. There is no standardized methodology for each of the stages of the cryopreservation procedure (andrological examination, semen collection, dilution, centrifugation, resuspension of the pellet with the freezing medium, packaging, freezing and post-thaw sperm evaluation), which also contributes to differences among studies. Cryotolerance markers of sperm and seminal plasma (SP) have been evaluated for prediction of ejaculate freezability. In addition, in previous research, there has been a focus on supplementing cryopreservation media with different substances, such as enzymatic and non-enzymatic antioxidants. In most studies, inclusion of these substances have led to improved post-thaw sperm quality and fertilizing capacity as a result of minimizing the adverse effects on sperm structure and function. Another approach is the use of different cryoprotectants. The aim with this review article is to provide an update on sperm cryopreservation in farm animals. The main detrimental effects of cryopreservation are described, including the negative repercussion on reproductive performance. Furthermore, the potential use of molecular biomarkers to predict sperm cryotolerance is discussed, as well as the addition of substances that can mitigate the harmful impact of freezing and thawing on sperm.
Molecular studies of the causes of male infertility revealed a significant contribution of oxidative stress. When excessive amounts of reactive oxygen species (ROS) are produced or antioxidant activity fails, the equilibrium between oxidation and reduction is disrupted, causing oxidative stress (OS). High levels of ROS can have an adverse effect on sperm function through the initiation of DNA damage, lipid peroxidation, loss of membrane integrity and increased permeability, inactivation of cellular enzymes, and cell apoptosis. In addition to endogenous factors such as immature sperm, leukocytes, and varicocele, potential causes of excessive ROS can also be found exogenously in males with testicular hyperthermia or exposed to environmental toxicity. To maintain the optimal functioning of sperm cells, it is, therefore, necessary to balance the redox potential, i.e., to balance ROS by antioxidants. The purpose of this review is to present the antioxidant defense systems in semen.
Background:
Asthenozoospermia is a usual male infertility factor, characterized by decreased semen quality. It has been revealed that antioxidants improve sperm function, enhance endogenous antioxidant activities, and protect spermatozoa against oxidative damage during cryopreservation. This aimed to evaluate the effects of vitamin D on sperm kinematics and apoptosis in the semen of bulls with normozoospermia and asthenozoospermia after the freeze-thaw process. For this purpose, 32 semen samples of four Holstein bulls (normozoospermic, progressive motility > 70 %) and 32 semen samples of four bull (asthenozoospermic progressive motility < 40 %) were collected and pooled separately (normozoospermic and asthenozoospermic). Samples were then diluted into four equal aliquots of extender containing different vitamin D concentrations (0, 5, 10, and 50 ng/mL) and aspirated into a 0.5 mL straw.
Results:
The percentages of sperm progressive motility and viability were significantly higher (P < 0.05) in 50 ng/mL of vitamin D in normozoospermic group. Sperm kinematics parameters including curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) were significantly higher in the high dose (50 ng/mL) vitamin D-treated group compared to the low dose vitamin D-treated group (5ng/mL) in normozoospermic bull semen samples. The supplementation of the semen extender with different concentrations of vitamin D could not increase the rate of acrosome integrity in normozoospermic bulls compared to the control group (P < 0.05). In the asthenozoospermic group, 10 ng/mL vitamin D-treated group could increase the rate of plasma membrane integrity compared to 5 ng/mL vitamin D-treated group (P < 0.05). The percentages of early-apoptosis (P = 0.049) and late-apoptosis (P = 0.005) were significantly higher in the asthenozoospermic than the normozoospermic group.
Conclusions:
The present study revealed that a high dose (50 ng/mL) of vitamin D protected normozoospermic bulls' sperms from the freezing procedure and lead to higher quality of frozen-thawed bull sperm.
The study evaluated the relation between the oxidative stress index (OSI) in porcine seminal plasma (n = 76) with sperm resilience and in vivo fertility (farrowing rate and litter size of 3137 inseminated sows) of liquid-stored artificial insemination (AI) semen doses. The OSI was assessed as the ratio of advanced oxidation protein products to Trolox-equivalent antioxidant capacity, both measured using an automated analyzer. Sperm motility (computer-assisted sperm analyzer) and viability (flow cytometry) were evaluated in semen AI-doses at 0 and 72 h of storage at 17 °C. Sperm resilience was defined as the difference between storage intervals. Semen AI-doses were hierarchically clustered as having high, medium and low seminal OSI (p < 0.001) with those of low displaying higher resilience (p < 0.01). Boars were hierarchically clustered into two groups (p < 0.001) as having either positive or negative farrowing rate and litter size deviation; the negative one showing higher seminal OSI (p < 0.05). In sum, seminal OSI was negatively related to sperm motility and the in vivo fertility of liquid-stored boar semen AI-doses, with the receiver operating characteristic curve presenting seminal OSI as a good predictive biomarker of in vivo fertility of AI-boars (area under the curve: 0.815, p < 0.05).
Mitochondria, fundamental organelles in cell metabolism, and ATP synthesis are responsible for generating reactive oxygen species (ROS), calcium homeostasis, and cell death. Mitochondria produce most ROS, and when levels exceed the antioxidant defenses, oxidative stress (OS) is generated. These changes may eventually impair the electron transport chain, resulting in decreased ATP synthesis, increased ROS production, altered mitochondrial membrane permeability, and disruption of calcium homeostasis. Mitochondria play a key role in the gamete competence to facilitate normal embryo development. However, iatrogenic factors in assisted reproductive technologies (ART) may affect their functional competence, leading to an abnormal reproductive outcome. Cryopreservation, a fundamental technology in ART, may compromise mitochondrial function leading to elevated intracellular OS that decreases sperm and oocytes’ competence and the dynamics of fertilization and embryo development. This article aims to review the role played by mitochondria and ROS in sperm and oocyte function and the close, biunivocal relationships between mitochondrial damage and ROS generation during cryopreservation of gametes and gonadal tissues in different species. Based on current literature, we propose tentative hypothesis of mechanisms involved in cryopreservation-associated mitochondrial dysfunction in gametes, and discuss the role played by antioxidants and other agents to retain the competence of cryopreserved reproductive cells and tissues.
Sperm cryopreservation is a powerful tool for the livestock breeding program. Several technical attempts have been made to enhance the efficiency of spermatozoa cryopreservation in different farm animal species. However, it is well-recognized that mammalian spermatozoa are susceptible to cryo-injury caused by cryopreservation processes. Moreover, the factors leading to cryo-injuries are complicated, and the cryo-damage mechanism has not been methodically explained until now, which directly influences the quality of frozen–thawed spermatozoa. Currently, the various OMICS technologies in sperm cryo-biology have been conducted, particularly proteomics and transcriptomics studies. It has contributed while exploring the molecular alterations caused by cryopreservation, identification of various freezability markers and specific proteins that could be added to semen diluents before cryopreservation to improve sperm cryo-survival. Therefore, understanding the cryo-injury mechanism of spermatozoa is essential for the optimization of current cryopreservation processes. Recently, the application of newly-emerged proteomics and transcriptomics technologies to study the effects of cryopreservation on sperm is becoming a hotspot. This review detailed an updated overview of OMICS elements involved in sperm cryo-tolerance and freeze-thawed quality. While also detailed a mechanism of sperm cryo-injury and utilizing OMICS technology that assesses the sperm freezability potential biomarkers as well as the accurate classification between the excellent and poor freezer breeding candidate.
The accurate prediction of male fertility is of major economic importance in the animal breeding industry. However, the results of conventional semen analysis do not always correlate with field fertility outcomes. There is evidence to indicate that mammalian fertilization and subsequent embryo development depend, in part, on the inherent integrity of the sperm DNA. Understanding the complex packaging of mammalian sperm chromatin and assessment of DNA integrity could potentially provide a benchmark in clinical infertility. In the era of assisted reproduction, especially when in-vitro fertilization or gamete intrafallopian transfer or intracytoplasmic sperm injection is used, assessment of sperm DNA integrity is important because spermatozoa are not subjected to the selection process occurring naturally in the female reproductive tract. Although sperm DNA integrity testing measures a significant biological parameter, its precise role in the infertility evaluation in farm animals remains unclear. In this review, the earlier findings on sperm DNA integrity in relation to male fertility are compiled and analyzed. Furthermore, the causes and consequences of sperm DNA damage are described, together with a review of advances in methods for detection of sperm DNA damage, and the prognostic value of sperm DNA quality on male fertility.
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.
Sperm cryopreservation is an important tool for storing genetic traits and assisted reproduction techniques. Several studies have developed semen cryopreservation protocols. However, the sperm proteome is different between ejaculated and epididymal spermatozoa and little is known about cryopreservation effects on epididymal spermatozoa. Therefore, our study aimed to (i) investigate the differences of sperm parameters based on the freezing tolerance of spermatozoa and (ii) identify potential markers to predict the freezability of bull epididymal spermatozoa. Our preliminary study demonstrated that spermatozoa from individual bulls differ in cryopreservation freezability. We categorized spermatozoa into high freezing-tolerant spermatozoa and low freezing-tolerant spermatozoa group based on sperm motility after freezing/thawing. We evaluated several sperm functional parameters, including sperm motility/motion kinematics, sperm speed parameters, viability, mitochondrial activity, and capacitation status. Our results demonstrated that motility, sperm speed parameters, viability, and mitochondrial membrane potential had significant differences between the two groups but motion kinematics and capacitation status did not. In addition, the concentration of three proteins - glutathione s-transferase mu 5, voltage-dependent anion-selective channel protein 2, and ATP synthase subunit beta, differed between both groups. Thus, our research highlighted differences in bull epididymal spermatozoa freezability upon cryopreservation and these proteins might be useful markers to select high freezing-tolerant epididymal spermatozoa.
Cryopreservation of semen and artificial insemination have an important, positive impact on cattle production, and product quality. Through the use of cryopreserved semen and artificial insemination, sperm from the best breeding bulls can be used to inseminate thousands of cows around the world. Although cryopreservation of bull sperm has advanced beyond that of other species, there are still major gaps in the knowledge and technology bases. Post-thaw viability of sperm is still low and differs significantly among the breeding bulls. These weaknesses are important because they are preventing advances both in fundamental science of mammalian gametes and reproductive biotechnology. Various extenders have been developed and supplemented with chemicals to reduce cryodamage or oxidative stress with varying levels of success. More detailed insights on sperm morphology and function have been uncovered through application of advanced tools in modern molecular and cell biology. This article provides a concise review of progress in the cryopreservation of bull sperm, advances in extender development, and frontiers using diverse techniques of the study of sperm viability. This scientific resource is important in animal biotechnology because with the advances in discovery of sperm fertility markers, there is an urgent need to improve post-thaw viability and fertility of sperm through enhanced cryopreservation for precision agriculture to produce food animals to ensure food security on the global scale.
Background: Cryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination (AI) centres. However, it is well-known that cryopreservation procedures induce changes in the water phase in both intra and extracellular compartments, which alter the content and localisation of several proteins and ends up curtailing the structural integrity of functional sperm (i.e., cryoinjuries). Alterations and deficiencies of sperm-oocyte binding proteins during gamete recognition are one of the causes of reproductive failure both in vitro and in vivo. In this sense, characterisation of cryopreservation effects upon oocyte-binding proteins of sperm, such as IZUMO1 and GSTM3, is essential when assessing the impact of this technique in swine reproduction.
Results: Cryopreservation was found to induce changes in the localisation of IZUMO1 and GSTM3 in boar sperm. However, the relative content of both proteins was not altered after thawing. Furthermore, whereas IZUMO1 content was found not to be related to the cryotolerance of boar sperm, GSTM3 content was observed to be higher in poor (PFE) than in good (GFE) freezability ejaculates in both pre-frozen (1.00 INT·mm2 ± 0.14 INT·mm2 vs. 0.72 INT·mm2 ± 0.15 INT·mm2; P < 0.05) and post-thawed (0.96 INT·mm2 ± 0.20 INT·mm2 vs. 70 INT·mm2 ± 0.19 INT·mm2; P < 0.05) samples. Moreover, GSTM3 levels were found to be higher in those spermatozoa that exhibited low mitochondrial activity, high reactive oxygen species (ROS) production, and high membrane lipid disorder post- thaw (P < 0.05).
Conclusions: The difference in GSTM3 content between GFE and PFE, together with this protein having been found to be related to poor sperm quality post-thaw, suggests that it could be used as a cryotolerance marker of boar spermatozoa. Furthermore, both IZUMO1 and GSTM3 relocate during cryopreservation, which could contribute to the reduced fertilising capacity of frozen-thawed boar sperm.
The bioflavonoid quercetin is a component of food with numerous biological effects, but its function in reproductive processes and mechanisms in various species remain unclear. The aim of this study was to examine the effect of quercetin on ovarian cells isolated from ovaries of two phytophagous mammalian species (i.e. pigs and cattle). There was analysis of the effect of quercetin (0, 1, 10, and 100 ng/mL) on cultured granulosa cells of pigs and cattle. Proliferation (PCNA) and apoptosis (bax) markers and release of progesterone (P4), testosterone (T), estradiol (E2), and IGF-I were quantified using quantitative immunocytochemistry, enzyme immunoassay, or radioimmunoassay. Treatments with quercetin reduced PCNA and bax accumulation and decreased P4 release from both granulosa cells of pigs and cattle. In cells of pigs, treatment with quercetin reduced T output, however, in cells of cattle quercetin increased T release. In cells of pigs, quercetin reduced IGF-I release. In cells of cattle, quercetin at smaller doses (1 or 10 ng/mL), promoted and at a large dose (100 ng/mL) reduced IGF-I secretions. There was no substantial E2 release from granulosa cells of pigs or cattle. These observations are the first to indicate there is a direct action of quercetin on basic ovarian cell functions (proliferation, apoptosis, and hormones release) which can be species-specific.
For over 30 years, sperm morphology assessment has been one of the most common tests in evaluation of fertility. This review examines the clinical relevance of sperm morphology assessment in the diagnosis of infertility and in assisted reproductive technology, as well as its analytical reliability. Publications on the pathophysiology, the analytical reliability of the test and its clinical relevance in diagnosis and in Assisted Reproductive Technology (ART) were evaluated. This review compared and discussed study methodologies and results, including patient characteristics, preparation, smear staining methods and classification systems. The assessment of the percentage of some abnormalities such as for example thin head, amorphous head, or bent or asymmetrical neck is of little clinical use, and their pathophysiology is not well explained as most are physiological traits. Some studies have highlighted correlations between the percentage of normal forms and functional sperm abnormalities, as well as correlations with ability to conceive in vivo and, in some situations, with the success of intra-uterine insemination (IUI) or conventional IVF. However, except in the case of some specific sperm defects (easy to detect with 99 or 100% of spermatozoa affected) and which are often linked to genetic disorders (globozoospermia, macrocephaly, decapitated sperm syndrome and fibrous sheath dysplasia), sperm morphology assessment has very poor sensitivity and specificity in the diagnosis of infertility. Moreover, there is very little evidence that indices of multiple sperm defects [sperm deformity index (SDI), teratozoospermia index (TZI), and multiple abnormalities index (MAI)] are relevant. Above all, many publications report a major lack of analytical reliability of this test, mainly in assessment of the details of sperm abnormalities. Many questions arise concerning how and when sperm morphology should be assessed, and how to interpret the thresholds of normal forms. Questions are raised on the real clinical impact of this test.
Cr(VI) exposure could produce kinds of intermediates and reactive oxygen species, both of which were related to DNA damage. Hyaluronan (HA) has impressive biological functions and was reported to protect corneal epithelial cells against oxidative damage induced by ultraviolet B, benzalkonium chloride, and sodium lauryl sulfate. So the aim of our study was to investigate HA protection on human corneal epithelial (HCE) cells against Cr(VI)-induced toxic effects. The HCE cell lines were exposed to different concentrations of K 2 Cr 2 O 7 (1.875, 3.75, 7.5, 15.0, and 30 μ M) or a combination of K 2 Cr 2 O 7 and 0.2% HA and incubated with different times (15 min, 30 min, and 60 min). Our data showed that Cr(VI) exposure could cause decreased cell viability, increased DNA damage, and ROS generation to the HCE cell lines. But incubation of HA increased HCE cell survival rates and decreased DNA damage and ROS generation induced by Cr(VI) in a dose- and time-dependent manner. We report for the first time that HA can protect HCE cells against the toxicity of Cr(VI), indicating that it will be a promising therapeutic agent to corneal injuries caused by Cr(VI).
The present study aimed to determine the localisation of aquaglyceroporins 3 (AQP3) and 7 (AQP7) in bull spermatozoa and their relationship with the sperm cell’s resilience to withstand cryopreservation (i.e. cryotolerance). A total of 18 bull ejaculates were cryopreserved and their sperm quality analysed before and after freeze–thawing. The presence and localisation of AQP3 and AQP7 was determined through immunoblotting and immunocytochemistry. AQP3 was found in the mid-piece and AQP7 in the mid-piece and post-acrosomal region of bull spermatozoa. Immunoblotting showed specific signal bands at 30 and 60 kDa for AQP3 and at 25 kDa for AQP7. Neither the relative abundance of AQP3 and AQP7 nor their localisation patterns was altered by cryopreservation but individual differences between bull ejaculates were found in immunoblots. In order to determine whether these individual differences were related to sperm cryotolerance, bull ejaculates were classified as having good (GFE) or poor freezability (PFE) on the basis of their sperm quality after thawing. While the relative abundance of AQP3 before cryopreservation did not differ between ejaculates with GFE and PFE, the abundance of AQP7 was higher in GFE than in PFE ejaculates. This finding was further confirmed through principal component and linear regression analyses. In conclusion, the relative abundance of AQP7 in fresh semen may be used as a marker to predict bull sperm cryotolerance.
Despite ongoing research in a number of species, the efficiency of embryo production by nuclear transfer remains low. Incomplete epigenetic reprogramming of the nucleus introduced in the recipient oocyte is one factor proposed to limit the success of this technique. Nonetheless, knowledge of reprogramming factors has increased-thanks to comparative studies on reprogramming of the paternal genome brought by sperm on fertilization-and will be reviewed here. Another valuable model of reprogramming is the one obtained in the absence of sperm fertilization through artificial activation-the parthenote-and will also be introduced. Altogether the objective of this review is to have a better understanding on the mechanisms responsible for the resistance to reprogramming, not only because it could improve embryonic development but also as it could benefit therapeutic reprogramming research.
Objective:
In 2014, enrolled 20 patients who applied to the Unit of Assisted Reproduction Techniques, Konya Necmettin Erbakan University. Based on the presence of hyaluronic acid (HA) in the oocyte-cumulus cell complex, sperm attached to HA in vivo were modeled in vitro. Available healthy sperm obtained in the swim-up procedure using HA were investigated.
Materials and methods:
This observational cohort study, a routine analysis was conducted on the ejaculation samples obtained from 20 patients. We divided each sample into two groups and the swim-up method was applied. Human serum albumin (HSA, 0.5%) was added to samples from the first group. HA (10%) was added to samples from the second group. We determined the floating linear and non-linear sperm concentrations of both groups annexin V was used to determine the rate of apoptosis of these sperm.
Results:
Following swim-up, linear and non-linear sperm concentrations were higher in the group that contained HA compared to the group with HSA. However, there was a significantly higher apoptosis rate in the HSA group compared to the HA group.
Conclusion:
The addition of HA to the medium in the swim-up procedure positively affected sperm parameters. Thus, healthier sperm cells were obtained without DNA damage and with high motility.
The ejaculated spermatozoon, as an aerobic cell, must fight against toxic levels of reactive oxygen species (ROS) generated by its own metabolism but also by other sources such as abnormal spermatozoa, chemicals and toxicants, or the presence of leukocytes in semen. Mammalian spermatozoa are extremely sensitive to oxidative stress, a condition occurring when there is a net increase in ROS levels within the cell. Opportunely, this specialized cell has a battery of antioxidant enzymes (superoxide dismutase, peroxiredoxins, thioredoxins, thioredoxins reductases, and glutathione s-transferases) working in concert to assure normal sperm function. Any impairment of the antioxidant enzymatic activities will promote severe oxidative damage which is observed as plasma membrane lipid peroxidation, oxidation of structural proteins and enzymes, and oxidation of DNA bases that lead to abnormal sperm function. Altogether, these damages occurring in spermatozoa are associated with male infertility. The present review contains a description of the enzymatic antioxidant system of the human spermatozoon and a reevaluation of the role of its different components and highlights the necessity of sufficient supply of reducing agents (NADPH and reduced glutathione) to guarantee normal sperm function.
Contents
A soybean lecithin‐based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen–thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1 mg ml ‐1 in a Tris‐based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH‐PX) activity, lipid peroxidation and acrosomal status after freezing–thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity‐related parameters, ALH, BCF, lipid peroxidation, GSH‐PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1 mg ml ‐1 HA), total motility (only with 1.5% lecithin), viability (1 mg ml ‐1 HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality‐related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.
Subfertility problems are encountered frequently in the cattle and buffalo bulls commercially maintained for semen production in dairy farms and under field conditions for natural insemination. Reports are scarce on the incidence of subfertility in breeding bulls, especially in India. The objective of the present study was to assess the incidence of the male reproductive anomalies leading to disposal of bovine bulls at GADVASU dairy farm, Ludhiana, Punjab (India). Data on frequency of various subfertility and disposal pattern of bulls maintained at the dairy farm, GADVASU, were collected for 12 yrs (1999 to 2010) and compiled from different record registers. Percentage of bulls that produced freezable semen (out of reserved ones) was less in cattle (25.641%) as compared to that of buffalo (30.4%). Various subfertility traits like poor libido and unacceptable seminal profile were found to be the significant reasons (p
The viscous nature of alpaca semen limits its use in cryopreservation and other assisted reproductive technologies. The cause and source of this viscosity is unknown although it has been postulated, but never proven, that glycosaminoglycans (GAGs) secreted by the bulbourethral gland are responsible. The present study investigated the concentration and composition of GAGs in alpaca seminal plasma, testes, bulbourethral gland and prostate gland and compared them to those in the ram to determine the relationship between seminal plasma GAGs and viscosity and to identify the source of seminal plasma GAGs. Alpaca seminal plasma contained more GAGs than ram (P < 0.001) and the predominant GAG, keratan sulfate, was correlated with viscosity (P = 0.05, R² = 0.2635). The alpaca bulbourethral gland contained most GAGs compared with prostate or testis (P < 0.001). In the ram, the prostate contained most GAGs. These findings suggest that GAGs, particularly keratan sulfate, may be the cause of seminal plasma viscosity in alpacas, and that the seminal plasma GAGs originate from the bulbourethral gland.
This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze–thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20 s in a water bath for the evaluation.
Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen using three commercial semen extenders. Sperm motility, membrane integrity, acrosome integrity and apoptosis were evaluated before and after freezing for each aliquot. In addition, the percentage of ASA-bound sperm was evaluated post-thaw. In Experiment 2, semen from 22 stallions was frozen immediately after collection a single formulation of semen extender. Sperm motility and ASA binding were evaluated post-thaw. The results of both experiments showed similar findings. The frequency of ASA-positive samples was higher among stallions with poor sperm cryosurvival (Exp. 1 and 2 = 6/11, 54.5%) than for good sperm cryosurvival (Exp. 1 = 0/16, 0%; Exp. 2 = 1/11, 9.1%). The percentage of IgG- and IgA-bound sperm was also higher in stallions with poor sperm cryosurvival in both experiments (P < 0.05). Post-thaw sperm motility, velocity and distance parameters were lower in ASA-positive than ASA-negative stallions (P < 0.005). No effect of the semen extender used was observed. In addition, stallions with ASAs had a higher percentage of apoptotic sperm than stallions without ASAs. The presence of sperm-bound ASAs was associated with poor cryosurvival for stallion spermatozoa. Thus, it may be beneficial to evaluate stallions for binding of ASAs prior to freezing to offer and indicator of the prognosis for cryosurvival.
This study aimed to evaluate the comparative effects of taxifolin hydrate and trehalose on the quality of frozen-thawed ram spermatozoa for the first time. Ejaculates collected from six mature rams were pooled, and divided to eight equal aliquots to extend them with different concentrations of glycerol (%5 and %3), taxifolin hydrate (10, 100, and 500 μM), and trehalose (60 mM) as eight groups (G5T0, G5T10, G5T100, G5T500, G3T0, G3T10, G3T100, and G3T500). After freeze-thawing process of cryopreservation, microscopic and oxidative stress parameters, and gene expression levels were investigated for understanding of possible impacts of taxifolin hydrate and trehalose. The study showed that G3T10 resulted in the highest post-thawed viability and mitochondrial activity. Moreover, all extenders with taxifolin hydrate reduced DNA fragmentation in comparison to G5T0, but DNA damage was prevented at the highest rate in presence of G5T10. The level of LPO significantly decreased in the groups G5T500 and G3T100, and the expression levels of NQO1, GCLC, and GSTP1 genes significantly increased in the groups G5T100, G5T500, G3T10, and G3T100 compared to the group G5T0. Finally, co-supplementation of tris-based extender having 3% glycerol with 60 mM trehalose and 10 μM taxifolin hydrate in cryopreservation extender may be recommended to improve the quality of post-thawed ram spermatozoa. However, further in vivo and in vitro studies are suggested to evaluate fertility rates of frozen-thawed ram spermatozoa co-supplemented with trehalose and taxifolin hydrate.
A groundbreaking contribution to the literature now in its revised and expanded second edition, this textbook offers a comprehensive review of diagnostic and treatment techniques for male infertility. This state-of-the-art, evidence-based textbook incorporates new multidisciplinary and complementary medicine approaches to create a first-of-its-kind guide to treatment strategies for male infertility and beyond. While this new edition is primarily designed as a reference for students and residents in reproductive medicine and andrology, it will be equally useful as well for professionals in urology, reproductive endocrinology, embryology, and research fields who are interested in the role that antioxidants play in male infertility.
World-renowned experts in these areas have been selected to participate in this work. Careful selection of the highest quality content will span the whole range of topics in the area of male infertility, providing a complete review of well-established and current diagnostic and treatment techniques for male infertility. The incorporation of 20 new chapters will enhance the book’s appeal by including the most recent advances brought to the male infertility arena. Additionally, this edition incorporates new features, including bulleted key points, review criteria and select video clips demonstrating some of the most fascinating male infertility treatment modalities. A dedicated new section on current guidelines on male infertility will enlighten readers on how to most optimally manage male infertility clinical scenarios.
Covering all aspects of diagnosis and management, ART, lifestyle factors and associated conditions for male infertility, Male Infertility: Contemporary Clinical Approaches, Andrology, ART and Antioxidants will be a readily accessible, high quality reference for medical students and residents, and will be of significant value to professionals working in the various fields treating this condition as well.
Semen freezability is positive correlated with the cholesterol content in the sperm cell. Freeze-thawing mainly cause temperature chock and change on media osmolarity, which can modify plasma membrane lipids content and sperm conformation, resulting in decreased fertility. Therefore, the aim of this study is to investigate the effect of adding cholesterol-loaded cyclodextrin (CLC) to the cryopreservation process of ram semen with low freezability. For that, two experiments were performed using 5 ejaculates of 6 rams, totalizing 30 samples. For experiment 1 the following treatments were tested: in natura (IN), Tris solution (CON), CLC + Tris solution (CLC), and pure methyl-β-cyclodextrin + Tris solution (MCD). For experiment 2 treatments CON and CLC were tested in samples subdivided into three freezability classes: high (n = 10), intermediate (n = 10) and low (n = 10). Freezability classes were based on the variation of sperm motility between IN and CON groups from the first experiment. Sample analyzes included sperm motility, sperm morphology, plasma and acrosome membrane integrity, mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and fluidity of plasma membrane. Results showed that CLC treatment was more efficient in maintaining sperm motility, integrity of plasma membrane, integrity of acrosome, and mitochondria membrane potential. In addition, CLC treatment in the groups with low and intermediate freezability showed improvement on progressive motility and percentage of rapid cells. In contrast, no difference was noted between CLC and CON treatments in the high freezability group. Therefore, the addition of CLC to semen extender improved sperm cryopreservation, especially in rams with low freezability.
Sperm cryopreservation facilitates the storage and transport of germplasm for its use in artificial insemination (AI) and other advanced reproductive technologies. The cryopreservation process can damage sperm and compromise functionality. Several cryobiological studies have found that the physical and biological factors that affect sperm survival at low temperatures during the cryopreservation process often involve the integrity of sperm membrane. In this review, the behavior of the sperm membrane against cooling, cold shock, ice crystal formation, oxidative stress, osmotic changes, reorganization of the lipid bilayer and addition of cryoprotective agents (CPA) is discussed. In addition, the phenomenon of reactive oxygen species (ROS) and its relationship with the cryopreservation process is also described. Semen cryopreservation techniques have progressed slowly in past years and the current performance, measured as post‐thawed survival, is not very different compared to past decades. Recent advances in understanding the structure of the cell membrane, its function and metabolism have driven to new conservation systems, including lyophilization and vitrification. However, none of these technologies is commercially available, although its future appears very promising.
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A ground-breaking contribution to the literature, Male Infertility: Contemporary Clinical Approaches, Andrology, ART & Antioxidants offers a comprehensive review of well-established, current diagnostic and treatment techniques for male infertility. This state-of-the-art, evidence-based resource incorporates new multidisciplinary and complementary medicine approaches to create a first-of-its-kind guide to treatment strategies involving antioxidants for male infertility. Designed as an easily accessible practical reference for daily use, Male Infertility: Contemporary Clinical Approaches, Andrology, ART & Antioxidants provides a high quality guide for urologists, reproductive endocrinologists, embryologists, andrologists, biologists and research scientists interested in the role that antioxidants play in male infertility. © Springer Science+Business Media, LLC 2012. All rights reserved.
During semen processing for cryopreservation, most seminal plasma is usually removed, and components with protective effects on sperm may be missing after thawing and within the female reproductive tract. The present study evaluated the effect of postthaw addition of autologous seminal plasma on motion characteristics of stallion sperm with fair (n = 4) or poor (n = 3) freezability. Therefore, pure seminal plasma (group SP1), seminal plasma combined with fresh semen extender (group SP2), or seminal plasma mixed with freezing extender (group SP3) were used to fill 0.5 mL straws and frozen similar to stallion semen. Postthawing, semen samples (n = 42) were diluted either with semen extender (group FT) or with seminal plasma (n = 126) of groups SP1 to SP3 to 25 × 10⁶ sperm/mL. In fair freezer stallions, total and progressive motilities were higher in group FT than in group SP1 (P <.05), but there was no difference in poor freezing stallions among groups (P >.05). However, comparing individual stallions, positive effects of seminal plasma on total or progressive motility were detected in two stallions. Curvilinear velocity increased in groups SP2 and SP3 in fair freezer stallions and in all groups with seminal plasma compared with group FT in poor freezer stallions (P <.05). Although straightness was higher in groups SP2 and SP3 compared with group FT in fair freezer stallions (P <.05), there was no difference among groups in stallions with poor freezability (P >.05). Average lateral head displacement did not change among groups of fair freezer stallions (P >.05) but was higher in groups SP2 and SP3 than in group FT in poor freezer stallions (P <.05). Beat cross frequency was higher in all groups diluted with seminal plasma postthawing in fair freezer stallions (P <.05), but only in group SP1 than in group FT in poor freezer stallions (P <.05). The addition of autologous seminal plasma to frozen-thawed semen can improve motion characteristics of stallions with fair and poor freezability. This is a valuable additional protocol for laboratories dealing with cryopreservation of stallion semen and for veterinarians working with fair or poor freezer stallions.
The cryopreservation of spermatozoa was introduced in the 1960s as a route to fertility preservation. Despite the extensive progress that has been made in this field, the biological and biochemical mechanisms involved in cryopreservation have not been thoroughly elucidated to date. Various factors during the freezing process, including sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for poor sperm quality post-thaw. Little is known regarding the new aspects of sperm cryobiology, such as epigenetic and proteomic modulation of sperm and trans-generational effects of sperm freezing. This article reviews recent reports on molecular and cellular modifications of spermatozoa during cryopreservation in order to collate the existing understanding in this field. The aim is to discuss current freezing techniques and novel strategies that have been developed for sperm protection against cryo-damage, as well as evaluating the probable effects of sperm freezing on offspring health.
Response surface methodology (RSM) is a commonly used system to optimize cryoprotectants of biocontrol strains when they are subjected to preparations. Various kinds of cryoprotectants and centrifugal conditions were tested to improve the survival of biocontrol agents after freeze-drying. To determine the optimum levels of incorporation of three cryoprotectants (glucose, trehalose and xylitol) in the freeze-drying process of strain Bacillus amyloliquefaciens B1408, a range of experiments based on Box-Behnken Design (BBD) were conducted. The results indicated that the suitable centrifugation conditions were 5000 r/min,10 min and the optimum concentrations of cryoprotectants were glucose 1.00%, trehalose 4.74% and xylitol 1.45%. The proven survival rate of cells after freeze-drying was 91.24%. These results convincingly demonstrated that freeze-drying could be used to preparation of biocontrol strain B1408. This study provides a theoretical basis for commercial possibilities and formulation development.
Beneficial effects of Hyaluronic acid (HA) has not been yet assessed for cryopreservation of rooster sperm. This study was conducted to evaluate the effects of different concentrations of HA (0, 1, 2, 4 and 8 mM) in Beltsville extender on the cryopreservation of rooster sperm. Semen samples were collected from six Ross broiler breeders (24-week) using abdominal massage, then divided into five equal aliquots and cryopreserved in Beltsville extender that contained different concentrations of HA. Motion characteristics, morphology, membrane functionality, viability, acrosome integrity, lipid peroxidation and fertility potential of sperm were assessed after thawing. HA at concentration of 2 mM (HA2) resulted in the highest (P < 0.05) total motility (55.3 ± 1.1%) and progressive motility (25.2 ± 0.8%) compared to the other groups. HA8 produced the lowest significant (P < 0.05) percentage of total (38.6 ± 1.1%) and progressive (14.7 ± 0.8%) motility. High significant percentage of membrane functionality were observed in HA1 and HA2 (43.2 ± 1.0 and 46.1 ± 1.0%, respectively) compared to HA4 (40.1 ± 1.0%) and HA8 (32.5 ± 1.0%). Moreover, HA1 and HA2 produced the higher percentage of acrosome integrity (54.8 ± 1.2 and 57.5 ± 1.2, respectively) compared to other groups. HA1 and HA2 reduced (P < 0.05) malondialdehyde formation (3.66 ± 0.08 and 3.75 ± 0.08 nmol/ml) compared to other groups. Fertility rate and hatching rate obtained from artificial insemination were significantly higher in HA1 (63.7 and 54.7%) and HA2 (67.5 and 57.7%) compared to control group (40 and 37%). Our results showed that supplementation of Beltsville extender with 1 and 2 mM HA significantly improved the quality of rooster sperm after freeze thawing.
The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Wharton's jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs.
Sperm is highly differentiated cell that can be easily obtained and purified. Mature sperm is considered to be transcriptionally and translationally silent and incapable of protein synthesis. Recently, a large number of proteins have been identified in sperm from different species by using the proteomic approaches. Clinically, sperm proteins can be used as markers for male infertility due to different protein profiles identified in sperm from fertile and infertile male animals. Recent evidences have shown that the conditions of sperm preservation in vitro can also change the sperm protein profiles. This paper reviews the recent scientific publications available to address sperm proteome and their relationship with sperm cryopreservation, capacitation, fertilization, and separation of X and Y sperm. Future directions in the application of sperm proteomics to develop or optimize reproductive technologies in mammals are also discussed.
This study investigated the effects of long-term extenders on post-thaw sperm quality characteristics following different holding times (HT) of boar semen at 17° and 10°C. Sperm rich fractions, collected from 5 boars, were diluted in Androhep® Plus (AHP), Androstar® Plus (ASP), Safecell® Plus (SCP) and TRIXcell® Plus (TCP) extenders. The extended semen samples were held for 2h at 17°C (HT 1) and additionally for 24h at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre-freeze and frozen-thawed semen. The Vybrant Apoptosis Assay Kit was used to analyze the proportions of viable and plasma membrane apoptotic-like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing-thawing. Differences in the pre-freeze semen were more marked in the sperm motion patterns between the HTs. Pre-freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO-PRO-1-/PI-) among the extenders. Post-thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP- and ASP-extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing-thawing. In most of the extenders, the incidence of frozen-thawed spermatozoa with apoptotic-like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 h in long-term preservation extenders modulates post-thaw sperm quality characteristics in an extender-dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen. The full paper is available at the below-mentioned link (http://onlinelibrary.wiley.com/doi/10.1111/rda.12751/epdf).
This study aimed to evaluate the effects of supplementing freezing and thawing media with hyaluronic acid (HA) on the quality parameters of frozen–thawed boar spermatozoa. Boar semen samples were collected from seven mature Yorkshire boars once a week using the gloved hand technique; these samples were frozen–thawed in the extender with added HA. Boar sperm was cryopreserved in the extender with HA added at concentrations of 0 (used as control), 4, 6, 8, 8 and 12 mg/L, and their effects on the quality of frozen–thawed boar sperm were evaluated. HA addition to the extender significantly improved sperm motility, sperm membrane integrity, mitochondrial activity, acrosomal integrity, superoxide dismutase and catalase activity, but decreased sperm malondialdehyde level (p < 0.05). Therefore, HA could be a promising cryoprotectant for boar sperm.
The objective was to examine if there are relationships between alterations in sperm viability, ROS synthesis and DNA integrity induced by cryopreservation of bovine sperm. Four ejaculates were collected from each of six bulls. Each ejaculate was diluted and divided into two aliquots; one was incubated for 24 h at 37 °C, and the other frozen, thawed and incubated for 24 h at 37 °C. Analyses of quality of sperm were performed after 0, 3, 6, 12, and 24 h of incubation. Progressive motility was determined with computer assisted sperm analysis. Percentages of plasma membrane- and acrosome-intact sperm (%PMAI), sperm with a high mitochondrial membrane potential (%HMMP), sperm showing a high degree of DNA fragmentation (%DFI), and their ROS content were assessed with dichlorofluorescein-diacetate (DCFH), dihydrorhodamine (DHR), diaminofluorescein diacetate (DAF-2 DA), and mitochondrial superoxide indicator (MITOSOX) using flow cytometry. While all other sperm parameters showed alterations (P < 0.05) during the 24h incubation time, %DFI stayed constant (P > 0.05, 0.91±0.23) in non-frozen sperm. Cryopreservation induced changes of all sperm parameters (P < 0.05). In contrast to all other sperm parameters, DFCH-fluoroescence indicating the synthesis of H2O2 showed a similar exponential rise (P < 0.05) like the %DFI values in frozen sperm. In conclusion, changes of DNA integrity in frozen sperm seem to be related to synthesis of H2O2, but not to sperm viability and synthesis of other reactives oxygen species.
The aim of the present study was to investigate the effects of a crude extract of Garcinia kola on male sexual function after subchronic and chronic treatment periods at different sublethal doses. Adult male Wistar rats were treated orally with 100, 200 and 400 mg kg(-1) of a 70% ethanolic extract of G. kola daily for 56 days. Sexual behaviour studies were performed on days 28 and 50. At termination on day 56, organ weights, sperm count, reproductive hormone levels and testicular histology were assessed. Subchronic and chronic treatment of normal male rats with G. kola extract resulted in overall increase in components of libido, erection and ejaculation in treated rats - with lower doses being more efficient than the higher dose. There was a slight reduction in some components of sexual behaviour with prolonged time of treatment. G. kola treatment at all doses resulted in increased testicular weights, increased sperm count with no change in motility and increased serum testosterone levels with no change in gonadotropin levels. Gross testicular histology was not affected by treatment. We conclude that G. kola seed extract possesses potent aphrodisiac activity in male albino rats with resultant increase in sperm count and testosterone levels.
© 2015 Blackwell Verlag GmbH.
The main aim of this work was to evaluate how supplementing freezing and thawing media with reduced glutathione (GSH) and/or L-acid ascorbic (AC) affected the quality parameters of frozen-thawed boar spermatozoa. With this purpose, semen samples of 12 ejaculates coming from 12 boars were used. Each ejaculate was split into seven aliquots to which 5 mM GSH and 100 μM AC were added separately or together at two different steps of freeze-thawing. Various sperm parameters (levels of free-cysteine residues in sperm nucleoproteins, sperm viability, acrosome-membrane integrity, intracellular peroxide and superoxide levels (ROS), and total and progressive motility) were evaluated before freezing and 30 and 240 min after thawing. Both GSH and AC significantly improved boar sperm cryotolerance when they were separately added to freezing and thawing media. However, the highest improvement was recorded when both freezing and thawing media were supplemented with 5 mM GSH plus 100 μM AC. This improvement was observed in sperm viability and acrosome integrity, sperm motility and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AC to both freezing and thawing extenders significantly decreased intracellular peroxide levels and had no impact on superoxide levels. According to our results, we can conclude that supplementation of freezing and thawing media with both GSH and AC has a combined, beneficial effect on frozen-thawed boar sperm that is greater than that obtained with the separate addition of either GSH or AC.
Activated neutrophils (PMNs), the ROS/RNS released by PMNs and the derived inflammatory processes are involved in the pathogenesis and progression of human inflammatory airway diseases. Similar diseases are also present in horses which suffer from recurrent airway obstruction (RAO), exercise-induced pulmonary haemorrhage (EIPH) and inflammatory airway diseases (IAD). Hyaluronic acid (HA) plays numerous roles in modulating inflammatory processes. The aim of this study was to examine whether a preparation of HA (MW 900 000 Da) interferes with ROS/RNS during the course of equine PMN respiratory bursts, and to establish the lowest concentration at which it still has antioxidant activity by means of luminol-amplified chemiluminescence (LACL). Electron paramagnetic resonance (EPR) spectroscopy was also used to investigate the direct antiradical activity of HA. The hydroxyl radical was significantly scavenged in a concentration-dependent manner at HA concentrations ranging from 2.5 to 0.16 mg/mL. Superoxide anion, Tempol radical and the ABTS•+were significantly inhibited at concentrations ranging from 2.5 to 0.62 mg/mL. The LACL of stimulated equine neutrophils showed that HA induced a statistically significant concentration–effect reduction from 5 mg/mL to 1.25 mg/mL. These findings were confirmed also when l-Arg was added to investigate the inhibition of the resulting peroxynitrite anion. Our findings indicate that, in addition to the human use, HA can also be used to antagonize the oxidative stress generated by free radicals in horses peripheral blood mononuclear cells (PBMCs). In order to achieve therapeutic concentrations, a direct aerosol administration to horses with horse respiratory diseases can be considered, as this route of application is also recommended in human medicine.
The aim of this study was to compare the effectiveness of antioxidants including cysteamine (2.5, 7.5 mm), hyaluronan (0.25, 1 mg ml(-1) ) and fetuin (5, 10 mg ml(-1) ) in the freezing of Brown Swiss bull semen. The best percentages of CASA motilities were achieved with 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine. For sperm morphology, 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine had better protective effects (P < 0.001). The results of hypo-osmotic swelling test showed that the percentage values of membrane integrity in all the groups, excluding that supplemented with 5 mg ml(-1) of fetuin, were higher than those of the control group (P < 0.001). Results obtained for the DNA damage of sperm cells demonstrated that 0.25 mg ml(-1) of hyaluronan, and 2.5 and 7.5 mm of cysteamine led to lower rates of spermatozoa with damaged DNA, compared with the control group (P < 0.001). The maintenance of superoxide dismutase and glutathione peroxidase antioxidant activities following freeze-thawing with 2.5 and 7.5 mm of cysteamine and 10 mg ml(-1) of fetuin was demonstrated to be at a higher level in comparison with the control group (P < 0.001). Malondialdehyde formation was found to be lower in the groups supplemented with 0.25 mg ml(-1) of hyaluronan and 7.5 mm of cysteamine after the freeze-thawing process (P < 0.001).
Addition of hyaluronan, a nonsulfated glycosaminoglycan, to fresh and frozen thawed human semen results in substantial retention of motility over time. Hyaluronan also has been reported to preserve postthaw viability and maintain membrane stability of boar spermatozoa. Therefore, experiments were designed to investigate the use of a commercially available hyaluronan (Map-5, Bioniche Animal Health, Inc., Athens, GA) in freezing extender for cryopreservation of equine spermatozoa. In experiment 1, aliquots from ejaculates were supplemented before freezing with one of four levels of hyaluronan: 100 μg/mL, 200 μg/mL, 400 μg/mL, and 1000 μg/mL along with an untreated control. No differences in sperm motility, assessed by computer-assisted sperm motility analysis (CASA), were found for any treatment at times 0, 30, or 60 minutes postthaw. Decreases in motility were noted in the highest hyaluronan group (1,000 μg/mL) after 90 and 120 minutes of incubation. Sperm viability, as assessed using SYBR-14/propidium iodide staining, was decreased (P < .05) when treated with 1,000 μg/mL compared with the control (37.1% and 46.1%, respectively). Motility parameters tended to remain elevated in those ejaculates treated with 200 μg/mL at various time points. Experiment 2, therefore, further investigated the effects of hyaluronan at 200 μg/mL on motility parameters and acrosome integrity and zona pellucida binding. Total (TM) and progressive (PM) motility of treated sperm immediately after thawing and at 60 minutes post-thaw were higher compared with control (P < .05). A tendency (P < .1) to maintain TM at 90 and 120 minutes post-thaw also was noted. No differences were noted for the mean number of spermatozoa bound to bovine oocytes for control or treated sperm (22 ± 14 vs 25 ± 17, respectively). Acrosome integrity also was unchanged between the two groups based on fluorescein isothiocyanate (FITC)−peanut agglutinin (PNA)/propidium iodide staining. All samples contained
An osmotic resistance test (ORT) was developed for bull semen. Raw semen was incubated for 40–60 min at 35°C in a fructose/sodium citrate solution with an osmolarity of 150 mOsm kg−1. Semen processed in skim milk, egg yolk and glycerol diluent was mixed 1:4 with a similar solution at 100 mOsm kg−1. On examination with a phase contrast microscope (× 400), cells with intact membranes were swollen. Some of these showed residual activity, i.e. a non-progressive flickering of the mid-piece. The proportion (%) of cells showing this activity was taken as the test result. Repeatability was 0.85 for raw semen and 0.93 for post-thaw. Over 401 ejaculates from 80 bulls (three straws per ejaculate), 51.2% of variation was due to bull, 33.7% to ejaculate within bull and 15.1% to straw within ejaculate. Five or six ejaculates from yearling Holstein/Friesian bulls were used for a fertility trial. Each was ORT tested post-thaw and two groups of ejaculates formed for each bull according to ORT result. Friesian/Holstein cows were inseminated and 49-day non-return rates (NRR) obtained for each group. Raw ORT correlated with post-thaw ORT (r=0.56), but not with NRR (r=0.08). Over a series of 48 bulls, the post-thaw ORT:NRR correlation was 0.37, but after removing bull effects a correlation of 0.79 was obtained. As the method is simple and rapid, it shows promise as a routine assessment technique in commercial AI laboratories.
This study was conducted to determine the effect of antioxidants on standard semen parameters, lipid peroxidation and fertility of Boer goat semen after cryopreservation. Ejaculates from four bucks were collected, evaluated and pooled at 37°C. The pooled semen was diluted with Tris citric acid fructose for washing. Semen samples, which were diluted with a Tris-based extender containing the antioxidant ascorbic acid (8.5mg/ml), butylated hydroxytoluene (2mM), cysteine (5mM) and hypotaurine (10mM) and an extender without antioxidant supplementation were cooled to 4°C and frozen in 0.25 straws with programmable freezer and finally stored in liquid nitrogen. Data (10 replicates) were analyzed by one-way analysis of variance. Mean (±SEM) progressive motility was significantly higher in ascorbic acid than other supplement groups and control samples (P>0.05). Best values were observed in ascorbic acid followed by BHT, cysteine, and hypotaurine. Antioxidant supplementation in extender showed significant (P<0.05) better values than the control group for sperm membrane integrity, acrosome integrity and viability. The ability of antioxidants to reduce the lipid peroxidation (LPO) after freeze thawing was measured by the formation of malondialdehyde (MDA) using the thiobarbituric acid method. Results showed that addition of antioxidants significantly reduced the rate of LPO in comparison to control (P<0.05). Ascorbic acid exhibited better values (1.27±0.28), than butylated hydroxytoluene, cysteine and hypotaurine 1.32±0.42, 2.27±0.16 and 2.38±0.17 respectively, which are significantly better than control (3.52±0.54). Higher pregnancy rate was observed with ascorbic acid followed by butylated hydroxtolune, hypotaurine and cysteine. However, differences in the fertility rate were non-significant with hypotaurine, cysteine and control groups.
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Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen–thawed buffalo spermatozoa quality parameters such as sperm viability by SYBR‐14/propidium iodide staining; mitochondrial function by JC‐1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange; and acrosome reaction (AR) by FITC‐PNA staining. Semen samples from five Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4% to 43.6%. A consistent rate (55.1 ± 10.8%) of sperm cells showed high mitochondrial membrane potential (Δψ high ), with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the five buffalo bulls; moreover, data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %‐DFI, ‐DFI, SD‐DFI, were 11.2 ± 8.6, 153.3 ± 24.6 and 81.6 ± 21.2, respectively. Regarding AR, the percentage of acrosome‐reacted live (ARL) and acrosome‐reacted dead (ARD) spermatozoa was 0.3 ± 0.2 and 15.3 ± 5.5, respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca ²⁺ ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL (10.8%) and ARD population (22.0%). This study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted in sperm functional parameters sensitive enough for the diagnosis of frozen‐thawed semen fertilizing potential.