Post-thaw Sperm Motility Research Articles

Cryoprotectant effects of natural honey on spermatozoa quality of pre-freezing and frozen-thawed boar semen.

Natural honey has been successfully used in the preservation of mammalian gametes because of its beneficial properties. The objectives of this study were to determine the inclusion level of honey in extender for improving boar semen quality before freezing and to investigate the effects of honey inclusion in extender and freezing media on post-thaw quality of frozen-thawed boar semen samples. Ejaculates from six terminally crossbred boars were collected using the gloved-hand technique for two experiments. Experiment 1 was a randomized block design, evaluating four inclusion levels of honey in boar semen extender [Control (0H)-Androhep Plus or Androhep Plus with 0.25%, 0.50%, and 0.75% honey (0.25H, 0.50H, and 0.75H respectively)]. Ejaculates were pooled, aliquoted according to treatments, and cooled for 24 h at 17 ºC. The results of this experiment were used to determine inclusion levels in exp. 2. Experiment 2 was a 2 x ×3 factorial design, evaluating the inclusion of honey in boar semen extender and freezing media. Semen samples from individual boars were cooled in extender with or without honey (C0: Androhep Plus; C1: Androhep Plus + 0.25% honey). After 24 h, semen samples were evaluated, diluted in lactose-egg yolk (LEY) media, and one of three freezing media types; F0: 93% LEY + 6% glycerol + 1% Equex-STM Paste (ESP); F1: 93% LEY + (3% glycerol and 3% honey) + 1% ESP; and F2: 93% LEY + 6% glycerol + (0.5% ESP and 0.5% honey). Samples were frozen in 0.5 mL straws using a controlled-rate freezer and stored in liquid nitrogen. In exp. 1, 0.25H and 0.50H improved motility (P = 0.033) and progressive motility (P = 0.001) of cooled boar semen. Nevertheless, 0.25H was selected for exp. 2. In exp. 2, post-thaw motility and progressive motility were highest (P < 0.05) in C0F2 but not different from C1F2. Morphologically normal cells and acrosomes were higher with all inclusion levels of honey (P < 0.05). In conclusion, 0.25% and 0.50% inclusion of honey in Androhep Plus improves motility and progressive motility of cooled boar semen samples after 24 h. Supplementing Androhep Plus with 0.25% honey maintains higher normal sperm cells and acrosomes of cryopreserved boar semen. Replacing 50% Equex-STM paste with honey in freezing media improves post-thaw sperm motility, progressive motility, percentage of normal sperm, and acrosome of cryopreserved boar semen.

Does Antioxidant Mitoquinone (MitoQ) Ameliorate Oxidative Stress in Frozen-Thawed Rooster Sperm?

In this study, we aimed to determine the benefit of mitoquinone (MitoQ) in rooster semen extenders on sperm quality, motility parameters, antioxidant capacities, and apoptotic changes in post-thawed rooster semen. A total of 85 ejaculates from 18 roosters were collected and then divided into five equal aliquots and cryopreserved in extenders with 1.0% soy lecithin nanoparticles that contained various concentrations of MitoQ (0 nM (M0), 50 nM (M50), 100 nM (M100), 150 nM (M150), and 200 nM (M200)). By using a computer-assisted semen analyzer, sperm motility parameters were assessed after freeze thawing. The M150 group had significantly higher percentages of total motility, progressive motility, viability, acrosome membrane integrity, and mitochondrial activity than the other groups (p &lt; 0.05). Compared to other groups, M100 and M150 groups produced a higher percentage of plasma membrane integrity and ATP contents (p &lt; 0.05). Additionally, the lowest levels of ROS and MDA in spermatozoa were observed in M150 group (p &lt; 0.05), whereas the highest levels of ROS and MDA were observed in sperm in the controls or the M200 group (p &lt; 0.05). Significantly higher values of SOD, GPx, and Cas-3 were found in the M150 group compared to other groups (p &lt; 0.05). Overall, these results demonstrate that MitoQ at 150 nM not only ameliorates post-thawed sperm quality and motility parameters by restoring ATP levels and preventing membrane damage, but also improves redox balance and antiapoptotic activities.

Curcumin Supplementation in Semen Extender Improves Post-Thaw Quality and Antioxidant Capacity of Gir Bull Spermatozoa

This investigation was carried out on semen of three Gir bulls at Cattle Breeding Farm, Junagadh (India) for a period of 6 months. The aim was to assess the effect of different concentrations of Curcumin, viz., 0 µM (Control), 5 µM, 10 µM, and 15 µM in Andromed extender on sperm quality along with oxidative stress parameters of cryopreserved semen. Semen ejaculates (n=30, 10/bull) with &gt;70% initial motility were included in the study. Soon after initial evaluation, the semen was extended in Andromed extender @ 80 million sperm per ml and was divided into four equal aliquots. The Curcumin stock solution (10 mM) was added in these aliquots to get a final concentration of 0, 5, 10, and 15 µM and sperm motility was checked. It was soon filled and sealed in medium French straws (0.5 ml), equilibrated at 4°C for 4 h and frozen in LN2 vapour using conventional wide mouth freezer. Thawing of straws was done next day in water bath at 37°C for 30 seconds, and various sperm quality parameters including oxidative markers were assessed. The results showed that supplementation of Curcumin to freezing extender improved semen quality and antioxidant capacity. The post-thaw sperm motility, sperm viability, HOST reactive sperm and acrosomal integrity in semen extended with 15 µM Curcumin concentration (57.33±0.67, 73.20±1.05, 64.77±1.56 and 72.43±1.38 %, respectively) were significantly (p&lt;0.05) higher and sperm abnormality (18.00±0.41%) was significantly (p&lt;0.05) lower as compared to those of control extender and the 5 µM Curcumin group. Mean malondialdehyde (MDA) level in post-thaw seminal plasma did not differ significantly (p&gt;0.05) among the groups, while total antioxidants activity (TAC) with 15 µM Curcumin (476.36±16.52 μmol/L) was significantly (p&lt;0.05) higher as compared to control and 5 µM Curcumin group. It is concluded that Curcumin @ 15 µM in semen extender improves post-thaw sperm quality and antioxidant capacity of cryopreserved Gir bull semen.

EPICATECHIN IMPROVES FROZEN SPERM VITALITY BY ITS ANTIOXIDANT AND CRYOPROTECTIVE ACTIONS

This study served to elucidate the effects of three selected doses of epicatechin (EPI; 25, 50 or 100 μmol/L) on the motion and oxidative profile of bovine spermatozoa subjected to the process of cryopreservation. Furthermore, we focused on the impact of EPI on the expression patterns of heat shock proteins 90 and 70 as well as the BAX and Bcl-2 proteins involved in the process of apoptosis. The experimental samples were compared with a native control as well as specimens cryopreserved in the absence of any supplement. Our dada indicates that particularly 50 and 100 μmol/L EPI assured a higher preservation of the sperm motility (p&lt;0.001) in comparison with the cryopreserved control. The best protection of the sperm DNA (p&lt;0.05), lipids (p&lt;0.05) and proteins (p&lt;0.01) were observed in the samples exposed to 100 μmol/L EPI in comparison to untreated frozen samples. The presence of higher EPI concentrations in the cryopreservation medium led to significant stabilization of the heat shock protein 90 (p&lt;0.01 in case of 50 μmol/L EPI; p&lt;0.001 with respect to 100 μmol/L EPI) as well as a higher maintenance of the BAX:Bcl-2 ratio (p&lt;0.001) when compared to the untreated frozen control. In summary, we may assume that EPI exhibits antioxidant properties which enable the molecule to prevent excessive damage to biomolecules essential for the cell survival and offer an additional layer of protection to spermatozoa exposed to low temperatures, all of which may be translated into a higher post-thaw sperm motility and activity.

Effects of mitoquinone (MitoQ) supplementation during boar semen cryopreservation on sperm quality, antioxidant status and mitochondrial proteomics

Mitochondria are the most important organelles and the main reactive oxygen species producers in spermatozoa. The objective of this study was to investigate the effects of mitochondria-targeted antioxidant mitoquinone (MitoQ) supplementation in boar semen extender during cryopreservation on sperm quality, antioxidant status and the changes of sperm mitochondrial proteomic profile. Semen collected from 10 Large White boars was cryopreserved in lactose-egg yolk extender supplemented with various concentrations of MitoQ (0, 5, 10, 20 and 40 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The comprehensive mitochondrial proteomic profiling was performed on spermatozoa in the control and MitoQ10 groups. Supplementation with 10 μM of MitoQ resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed boar sperm were also elevated in the MitoQ10 group. Excessive MitoQ (40 μM) supplementation induced a reduction of sperm motility parameters, sperm quality and antioxidant status and the abundance of GLUT3 and 8 proteins. A total of 189 proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05, and 33 of them were dramatically (FC > 1.5) regulated by MitoQ. These DEPs are mainly involved in sperm motility, energy metabolism and oxidative phosphorylation. Our data suggest that the beneficial effect of MitoQ on cryopreserved boar semen is achieved by regulating sperm antioxidant capacity and the mitochondrial proteins related to motility and metabolism.

Effect of Dimethylacetamide Concentration on Motility, Quality, Antioxidant Biomarkers, Anti-Freeze Gene Expression, and Fertilizing Ability of Frozen/Thawed Rooster Sperm.

Simple SummaryAlthough sperm cryopreservation has a great potential for application in the poultry industry, it is still very limited at the economical scale due to the low fertility of frozen/thawed avian spermatozoa. Dimethylacetamide (DMA) has been widely employed as a cryoprotectant for the cryopreservation of chicken spermatozoa, but with variable results in motility and fertility. To determine the appropriate concentration of DMA in the chicken semen freezing extender, in vitro sperm quality and motility analyses and in vivo field trials based on the fertility of the frozen/thawed rooster sperm were conducted in the present study. The antioxidant biomarkers and novel anti-freeze-associated genes were also analyzed in the post-thawed sperm. Results conclude that DMA is preferably used at a low concentration to obtain applicable results in sperm motility and fertility after thawing without harming the quality and antioxidant system defense. The current study could highlight the possible molecular mechanisms behind the obtained fertility results of DMA-frozen sperm, especially when artificial insemination is implemented in the poultry industry.Sperm cryopreservation is of great importance for the poultry industry but still needs to be optimized. The high susceptibility of poultry sperm to cryodamage leads to low fertility rates after cryopreservation. Therefore, the present study aimed at evaluating the effect of including a cryoprotectant, dimethylacetamide (DMA), in the chicken semen freezing extenders at a final concentration of 3%, 6%, or 9% on the post-thawed sperm motility, quality, antioxidant biomarkers, anti-freeze gene expression, and fertilizing ability. Results showed that the total motile sperm, progressivity, and viability were quadratically increased (p < 0.05) in the 6% DMA group. The antioxidant enzyme activity and lipid peroxidation were negatively (p < 0.05) affected by the increase in DMA concentration. Furthermore, some anti-freeze-associated genes such as heat shock protein 70 (HSP70) and ras homolog family member A (RHOA) were linearly and quadratically down-regulated (p < 0.05) with the high concentration of DMA. Finally, the fertility and hatchability rates did not indicate statistical differences between DMA groups. It can be concluded that using the low concentration of 3–6% DMA in the freezing semen extender is preferable to obtain acceptable results in the post-thawed sperm quality and fertility.

Effect of Antioxidants on Cryopreserved Turbot (Scophthalmus maximus) Spermatozoa Quality and DNA Damage

The effects of several antioxidants added to cryopreservation extenders on turbot (Scophthalmus maximus) sperm post-thaw motility, fertilization, sperm characteristics, and DNA damage were investigated. The following antioxidants were used in conjunction with the conventional extenders: rosmarinic acid (RA) (25, 50, and 100 μg/ml); uric acid (UA) (25, 50, and 100 μg/ml); and taurine (TA) (25, 50, and 100 μg/ml). On sperm samples that had been diluted by the extenders at a 1:9 ratio, cryopreservation was carried out. The results demonstrated that the post-thaw motility of sperm was higher in 25 μg/ml RA (77.41±2.90%), followed by 50 μg/ml RA (74.86±3.2%), 50 and 100 μg/ml UA (69.56±3.30% and 70.74±2.51%, respectively) which significantly differentiated from conventional extender. The highest hatching rate (70.67±4.67%) was attained at UA 50 μg/ml, while the highest fertilization rate (81.67±2.52%) was observed in RA 25 μg/ml. At UA 50 μg/ml, the DNA fragmentation rate was lowest (5.07±1.57%). In conclusion, antioxidants had a protective impact on sperm of turbot that had been frozen and thawed.

Cryopreservation of rabbit semen: impacts of permeable and non-permeable mixture of cryoprotectant, male group individuality, freezing rate, semen package size and antioxidant bovine serum albumin on rabbit semen freezability

In the present study, three experiments were designed to identify the most appropriate technique for freezing rabbit semen. Experiment 1 aimed to determine the optimal levels of dimethyl sulfoxide (DMSO) contents in freezing medium and their effects on individual bucks. Semen ejaculates for each buck (n=15 bucks) were mixed and split into three portions for extension with a freezing medium containing varying concentrations of DMSO (0.75, 1.0, and 1.4 M). Diluted semen samples were packaged in 0.25 mL straws and suspended above liquid nitrogen (LN) for 10 min, then dipped in LN. A few days after freezing, post-thaw semen evaluation was assessed, and according to the results, six bucks and an extender containing 0.75 M of DMSO were used for experiments 2 and 3. In experiment 2, the pooled semen from 6 bucks was divided into two portions for packaging in two straw sizes (0.25 and 0.50 mL). Each straw size was divided into five groups and suspended at different heights above LN (2, 4, 6, 8, and 10 cm) for 10 minutes before being preserved in LN. In experiment 3, the pooled semen was divided into four portions for dilution with freezing medium containing different concentrations of bovine serum albumin (BSA; 0, 2.5, 5.0, and 7.5 mg/mL). Semen samples were packaged in a 0.50 mL straw and suspended 10 min, 4 cm above LN for freezing. Pre-freezing and post-thawing, semen samples were evaluated for semen quality. Results showed that the extender containing 0.75 M DMSO had higher significant values for post-thaw sperm motility, longevity, acrosome integrity and sperm plasma membrane permeability. Bucks’ individuality had significant effects on post-thaw motility, acrosome and sperm plasma membrane integrity. A significant interaction was recorded between DMSO concentrations and bucks’ individuality on sperm longevity. Semen package sizes had no significant effects on the evaluated parameters. Semen was frozen at 2 and 4 cm above LN had significantly better post-thaw quality. BSA at concentrations 5 and 7.5 mg/mL improved recovery rates of acrosome integrity and sperm membrane permeability. DMSO 0.75 M and freezing 4 cm above LN seem to be more adequate for rabbit semen cryopreservation. The appropriate level of DMSO differs between bucks, as the post-thaw sperm longevity is affected. BSA enhanced acrosome and sperm membrane integrity. Results obtained will need further investigation to be confirmed in the field.

Beneficial Effect of Proline Supplementation on Goat Spermatozoa Quality during Cryopreservation.

Simple SummarySperm suffers damage from reactive oxygen species stress (ROS) during cryopreservation. Supplementation of antioxidants is suggested to reduce sperm cryodamage induced by ROS. Proline has been regarded as a powerful antioxidant. However, there is no information on the role proline plays during goat sperm cryopreservation. Thus, this study aims to investigate the effects of proline during goat sperm cryopreservation procedures. In the present study, supplementation of 2 mM proline significantly enhanced post-thaw goat sperm quality by regulating sperm redox homeostasis. Proline supplementation protects goat sperm against ROS stress through PRODH-mediated metabolism.Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.

Melatonin Supplementation for the Cryopreservation of Canine Sperm.

Antioxidants can be used in sperm cryopreservation protocols to reduce oxidative stress that occurs due to the cryopreservation process. The aim of this study was to evaluate the effects of melatonin supplementation on quality and oxidative stress parameters in cryopreserved canine sperm. Eighteen sperm ejaculates were collected from 6 Frenchie Bulldog males (3 collections per male). Sperm motility parameters, membrane integrity, and sperm morphology were analyzed before the cryopreservation process. The extender used in cryopreservation was composed of Tris-egg yolk and ethylene glycol 5% was added as a cryoprotectant. The cryoprotective medium was supplemented with 1.0, 1.5, 2.0, 2.5, and 3.0 mM melatonin, and the control group (without melatonin). Post-thaw sperm was evaluated as described for fresh sperm and oxidative stress parameters (lipid peroxidation, catalase, and superoxide dismutase). Post-thaw sperm motility parameters, membrane integrity, sperm morphology, and oxidative stress parameters did not differ (p > 0.05) among the control group and samples supplemented with melatonin. The results of this study showed that melatonin supplementation had no positive or negative effect on the parameters evaluated. Thus, it is suggested that different concentrations of melatonin be tested to assess its effectiveness as an antioxidant in the cryopreservation process in canine sperm.

Caffeine Supplementation in Semen Extender Protects Buffalo Spermatozoa from Cryodamage

Caffeine is widely known for its phosphodiesterase enzymes inhibiting, and its powerful reactive oxygen species scavenging property. This study aimed to evaluate the effect of caffeine supplementation @ 0 mM, 1 mM, 3 mM and 5 mM in AndroMed® extender on cryopreservation of Jaffarabadi buffalo semen. Twenty-four semen ejaculates (6/bull) with &gt;70% initial sperm motility were obtained from four mature Jaffarabadi bulls using AV. The ejaculates were split-diluted in an egg-yolk-free AndroMed® extender supplemented with different concentrations of caffeine and were cryopreserved using a standard protocol. The semen samples were evaluated for sperm quality parameters as well as oxidative stress parameters, viz., lipid peroxidation, and Glutathione-S-transferase (GST) enzyme activity in seminal plasma at pre-freeze (after equilibration) and post-thaw stage. The levels of caffeine had a significant effect on all these parameters, except sperm abnormalities, at both pre-freeze and post-thaw stages. Supplementation of caffeine in semen extender at 1 mM and 3 mM concentration showed a significant (p&lt;0.05) increase in post-thawed sperm motility (62.04 ± 0.84, 61.25 ± 1.01%), viability (64.21 ± 0.88, 64.25 ± 0.84%), acrosome integrity (58.08 ± 1.08, 57.30 ± 0.93%) and plasma membrane integrity (52.75 ± 0.89, 52.71 ± 0.74%) and a significant (p&lt;0.05) decrease in oxidative damage as evident by lower lipid peroxidation (MDA 7.62 ± 0.41, 7.80 ± 0.70 μM) and GST enzyme activity (31.15 ± 1.36, 29.54 ± 0.54 nmol CDNB/mL/min) as compared to control and 5 mM caffeine. The study concludes that the post-thaw quality of frozen semen of Jaffarabadi buffalo bulls improves significantly with decreased oxidative stress if the AndroMed® extender is supplemented with 1 and 3 mM concentration of caffeine over control.

Effect of Betaine and Raffinose in Cryopreservation Medium on Fertility in Kadaknath Chicken

BACKGROUND: Kadaknath is an important indigenous chicken with black pigmentation and cryopreserved semen reputably had low fertility. OBJECTIVE: The aim of this study was to evaluate the effects of betaine and raffinose in semen extenders on post thaw semen parameters and fertility. MATERIALS AND METHODS: Semen was cryopreserved in 4% dimethyl sulfoxide (DMSO) with betaine supplemented at 0.1, 0.2 and 0.4 M or raffinose supplemented at 1, 5 and 10 mM. Post thaw semen parameters and fertility were evaluated. RESULTS: Betaine at higher concentrations significantly (p &lt; 0.05) inhibited the post thaw sperm motility, live sperm and MTT dye reduction and a declining trend in the fertility with increasing betaine. Inclusion of raffinose had no effect on the post thaw in vitro semen parameters, however, the fertility was significantly (p &lt; 0.05) higher in the 10 mM raffinose supplemented group. CONCLUSION: Betaine has negative effect on post thaw semen parameters and raffinose at 10 mM concentration improves the fertility from cryopreserved semen.

Asthenozoospermic stallions tend to have high acrosome reacted spermatozoa as evidenced by dual fluorescent staining assay

Acrosome intactness of spermatozoa is the critical factor for establishing sperm reservoir in oviduct and for fertilizing an oocyte. However, frozen thawed spermatozoa tend to show higher proportion of acrosome reacted spermatozoa thereby compromising the fertility. Conventional staining techniques identify only sperm acrosome integrity and not precisely the acrosome reaction status. In this context, the current study was conducted to assess the acrosome status of cryopreserved spermatozoa using Fluorescein isothiocyanate conjugated peanut agglutinin and propidium iodide (FITC-PNA+PI) in stallions with varying sperm motility. Stallions were classified into high- (≥45%) and low-motile group (≤30%) based on their post-thaw sperm motility. The proportion of live acrosome intact (LAI) spermatozoa was significantly higher in high-motile group as compared to low-motile group. A significant positive correlation was observed between LAI and post-thaw sperm motility. In conclusion, the present study showed that FITC-PNA+PI combination could be used for rapid and accurate assessment of acrosome reaction status of stallion spermatozoa, and the proportion of LAI population in cryopreserved stallion semen had a strong correlation with sperm motility.

High sperm concentration during cryopreservation decreases post-thaw motility percentage without compromising in vitro fertilization outcomes in common carp

To avoid mechanical compression of spermatozoa during the cryopreservation, a high dilution ratio with the cryoprotective medium is applied. Due to this, the sample volume increases, which entails increased cryobank capacity. Moreover, the high volume of cryomedias does not allow to fertilize of large volumes of eggs in practical artificial reproduction of common carp aquaculture. The current study demonstrated that reasonable lowering of dilution rates might still be effective for carp sperm cryobanking —elevation of spermatozoa concentration up to 13 × 109 spz mL−1 resulted in a significant decrease in post-thaw sperm motility percentage to 20% compared with 39% in 0.5 × 109 spz mL−1. Nevertheless, no significant differences in sperm kinetic parameters (VCL and LIN) were found in this case. The fertilization outcome (embryo development and hatching rates) was similar after applying thawed sperm samples with “optimal” (2.2–2.4 × 109 spz mL−1) and “sub-optimal” (11.0–13.0 × 109 spz mL−1) concentrations (sperm/egg ratio at fertilization was in the range of 0.3–4.5 × 105 spz/egg). Thus, applying a low dilution rate such as one part of sperm to one of the cryoprotective mediums is favorable for decreasing cryo-storage space and the sperm volume needed to fertilize big egg numbers. The experiment also shows that the 4.5 × 105 spz/egg ratio is not sufficient for good fertilization, and it is necessary to use higher sperm concentrations per egg or improve the method of fertilization.

Expression of TXNRD1, HSPA4L and ATP1B1 Genes Associated with the Freezability of Boar Sperm.

Cryopreservation is associated with increased oxidative stress, which is responsible for sperm damage. We analyzed the effect of cryopreservation on mRNA and protein expression of thioredoxin reductase 1 (TXNRD1), heat shock protein family A (HSP 70) member 4 like (HSPA4L) and sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1) genes in boar sperm with different freezability. Boars were classified as having good and poor semen freezability (GSF and PSF, respectively), according to the assessment of post-thaw sperm motility. Total RNA was isolated from fresh pre-freeze (PF) and frozen-thawed (FT) sperm from five boars of the GSF and PSF groups, respectively. Quantification of TXNRD1, HSPA4L and ATP1B1 gene expression was performed by RT-qPCR analysis. Proteins extracted from sperm were subjected to Western blotting and SDS-PAGE analyses. Poor freezability ejaculates were characterized by significantly higher relative mRNA expression levels of TXNRD1 and HSPA4L in FT sperm compared with the fresh PF sperm. Furthermore, the relative mRNA expression level of ATP1B1 was significantly higher in the fresh PF sperm of the GSF group. Western blotting analysis revealed significantly higher relative expression of TXNRD1 protein in the fresh PF sperm of the GSF group, while HSPA4L protein expression was markedly increased in FT sperm of the PSF group. Electrophoretic and densitometric analyses revealed a higher number of proteins in the fresh PF and FT sperm of the PSF and GSF groups, respectively. The results of this study indicate that ATP1B1 mRNA expression in the fresh PF sperm is a promising cryotolerance marker, while the variations of TXNRD1 and HSPA4L protein expression in the fresh PF or FT sperm provide useful information that may help to elucidate their biological significance in cryo-damage.

Optimizing cryopreservation of sea cucumber (Apostichopus japonicus) sperm using a programmable freezer and computer-assisted sperm analysis

The sea cucumber Apostichopus japonicus has high nutritional, medicinal, and economic value. However, factors such as overexploitation, climate change, and environmental pollution have resulted in serious germplasm degradation in both farmed and wild A. japonicus, and it has been listed as endangered on the IUCN (International Union for Conservation of Nature) Red List of Threatened Species. Cryopreservation is an important method to protect germplasm resources and solve the problem of germplasm degradation. Using a programmable freezer and computer-aided sperm analysis, we comprehensively studied and screened the factors that affect the post-thaw motility of A. japonicus sperm during cryopreservation. Based on our results, we propose the following optimal cryopreservation procedure for A. japonicus sperm: cryo-diluent composition of 12.5% dimethyl sulfoxide and 0.1 mol/L glucose, with filter-sterilized (the filter mesh size: 0.45μm) natural seawater (NSW) as the extender; 1:5 mixing ratio of sperm and cryo-diluent; cooling rate and thawing temperatures of 10°C/min and 20°C, respectively. The post-thaw motility of sperm treated using the optimal procedure was &amp;gt; 65%, the fertilization rate (in the blastocyst stage) was nearly 80%, and the hatching rate (in the early auricularia larva stage) was &amp;gt; 65%. Additionally, frozen sperm that had been cryopreserved for 1 year retained a considerable post-thaw motility and fertilization rate compared to recently cryopreserved sperm. We detected obvious differences in sperm freezability among individual A. japonicus, and cryopreservation caused some damage to the sperm structure. In conclusion, our optimized procedure make large-scale cryopreservation of A. japonicus sperm possible, and our results provide valuable information that could be applied to research and conservation of A. japonicus.

Green Tea Extract in the Extender Improved the Post-Thawed Semen Quality and Decreased Amino Acid Mutation of Kacang Buck Sperm.

This study was the first to combine the addition of antioxidants to a skim milk-egg yolk (SM-EY) extender and different equilibration periods to obtain higher quality post-thawed Kacang buck semen. This study aimed to determine the effects of green tea extract (GTE) on the quality of frozen Kacang goat sperm equilibrated for one and two hours. The pool of Kacang buck ejaculate was equally divided into four portions and was diluted in an SM-EY extender that contained four doses of 0, 0.05, 0.10, and 0.15 mg of GTE/100 mL for T0, T1, T2, and T3 groups, respectively. The aliquots were treated for an equilibration period of 1-2 h before further processing as frozen semen. Post-thawed semen quality was evaluated for sperm quality. The Sanger method was used for DNA sequencing, and the amino acid sequence was read using MEGA v.7.0. The post-thawed semen of the T2 group that was equilibrated for one hour had the highest semen quality. Pre-freezing motility had the highest determination coefficient compared to post-thawed sperm motility. This study is the first to report amino acid mutation due to freeze-thawing. The frequency of amino acid mutations revealed that T2 was the least mutated amino acid. Glycine, valine, leucine, serine, and asparagine strongly correlated to post-thawed sperm motility. It can be concluded that a combination of 0.1 mg GTE/100 mL extender as an antioxidant and one-hour equilibration period resulted in the best post-thawed Kacang buck semen quality.

Effects of Heterologous Bovine Seminal Plasma-Supplemented Egg Yolk-Based Extender on Cryosurvivability of Pantja Buck Semen.

The present study was conducted to observe the effects of removal of seminal plasma of Pantja buck semen and supplementation of bovine seminal plasma (BSP) in the extender before cryopreservation. In a preliminary experiment, different levels of BSP were supplemented (1, 3, 5, 7, and 9% v/v) in egg yolk (7.5% egg yolk)-tris (EYT) extender and used for cryopreservation of Pantja buck semen. Results in terms of motility, viability, plasma membrane integrity, acrosome integrity, and lipid peroxidation showed that 5% BSP was suitable for maintaining Pantja buck semen quality during cryopreservation. In the final experiment, pooled semen from four Pantja bucks was split into three aliquots (I, II, and III). Aliquot I was directly diluted in EYT extender and grouped as the control (C); aliquot II and III were washed separately with TALP solution and diluted as D1 (Washed semen with EYT extender) and D2 (Washed semen with EYT extender containing 5% BSP), respectively. Seminal attributes (sperm individual motility, viability, plasma membrane integrity, acrosome integrity, and total morphological abnormalities) were assessed at the postdilution, postequilibration, and post-thawing stages. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) concentration, and glutathione peroxidase (GSH-Px) activity were measured at post-thaw. Washed semen significantly improved (p < 0.05) seminal parameters at post-thaw compared with unwashed semen (control). A significant difference (p < 0.05) was observed in seminal attributes between freezing stages and between dilution groups. Significantly higher (p < 0.05) post-thaw sperm motility, viability, plasma membrane integrity, acrosome integrity, and GSH-Px activity, and significantly lower (p < 0.05) MDA concentration and extracellular release of enzymes (ALT, AST) were observed in group D2 compared with control and D1. The results of the present study demonstrated that cryopreservation of washed Pantja buck semen diluted with 5% BSP-supplemented EYT extender can improve post-thaw semen quality.

Saccharides Influence Sperm Quality and Expressions of Motility and Fertilization-Associated Genes in Cryopreserved Sperm of Pacific Abalone, Haliotis discus hannai.

Pacific abalone, Haliotis discus hannai, is a highly commercial seafood in Southeast Asia. The present study aimed to determine the influence of saccharides and vitamins on post-thaw sperm quality, ATP content, fertilization capacity, hatching capacity, and mRNA content of motility and fertilization-associated genes of Pacific abalone. Sperm cryopreserved using saccharides improved the post-thaw sperm quality including motility, acrosome integrity (AI), plasma membrane integrity (PMI), and mitochondrial membrane potential (MMP). However, vitamins (l-ascorbic acid) did not result in any significant improvement in sperm quality. Sperm cryopreserved using saccharides also improved ATP content, DNA integrity, and mRNA content of motility and fertilization-associated genes of post-thaw sperm than sperm cryopreserved without saccharides. Among sperm cryopreserved using different saccharides, post-thaw sperm quality indicators (except PMI) and mRNA content of motility and fertilization-associated genes did not show significant differences between sperm cryopreserved using 3% sucrose (S) combined with 8% dimethyl sulfoxide (DMSO) and sperm cryopreserved using 1% glucose (G) combined with 8% ethylene glycol (EG). However, sperm cryopreserved using 3% S + 8% DMSO showed higher post-thaw sperm quality (motility: 58.4 ± 2.9%, AI: 57.1 ± 3.2%, PMI: 65.3 ± 3.3%, and MMP: 59.1 ± 3.2%), ATP content (48.4 ± 1.8 nmol/ml), and % DNA in tail (2.09 ± 0.20%) than sperm cryopreserved using other saccharides. When sperms were cryopreserved using 3% S + 8% DMSO, the mRNA content of motility (heat shock protein 70, HSP70; heat shock protein 90, HSP90; protein kinase A, PKA-C; axonemal protein 66.0, Axpp66.0; and tektin-4) and fertilization-associated (sperm protein 18 kDa, SP18 kDa) genes were higher than in sperm cryopreserved using other saccharides. However, changes in the mRNA contents of these genes were insignificant between sperm cryopreserved using 3% S + 8% DMSO and 1% G + 8% EG. Taken together, these results indicate that cryopreservation using 3% S + 8% DMSO can improve post-thaw sperm quality and mRNA contents better than other examined cryoprotectants. The present study suggests that 3% S + 8% DMSO is a suitable cryoprotectant for sperm cryopreservation and molecular conservation of this valuable species.

Cryopreservation of Sperm from an Endangered Snake with Tests of Post-Thaw Incubation in Caffeine.

Simple SummaryCryopreservation of sperm from reptiles to aid the recovery of endangered species continues to be a challenge. In this study, we tested the cryoperformance of a cryoprotective agent (CPA) mixture to cryopreserve sperm from the endangered Louisiana pinesnake (Pituophis ruthveni). The mixture contained Lake’s buffer with 10% N,N-dimethyl formamide (DMF), 2% methanol, 5% clarified egg yolk, (v/v% final concentration) and was tested against 16 experimental mixtures containing variable concentrations and mixtures of diluents, extenders, CPAs, and additives. In addition, we investigated the effects of post-thaw incubation on sperm motility in TL HEPES supplemented with 10% fetal bovine serum (H10) alone or supplemented with caffeine. We found that the majority of our test additives did not significantly improve the post-thaw motility or viability of sperm. The best performing experimental CPA mixture contained Lake’s buffer with 10% DMF, 2% methanol, and 5% clarified egg yolk with the addition of 5 mg/mL bovine serum albumin (BSA), and post-thaw incubation in both H10 and H10 with caffeine showed improved forward motility. Cryopreservation of sperm from the Louisiana pinesnake improved with the addition of BSA to our base CPA mixture, and post-thaw incubation in H10 improved with caffeine.Cryopreservation of sperm to preserve the genetic diversity of declining populations is a promising technique to aid in the recovery of endangered species such as the Louisiana pinesnake (Pituophis ruthveni). However, this technique has been performed on only a handful of snake species and with limited success. Here, we tested a cryoprotective agent (CPA) mixture containing Lake’s buffer with 10% N,N-dimethyl formamide (DMF), 2% methanol, 5% clarified egg yolk, (v/v% final concentration) against 16 other CPA-treatment mixtures. These contained either Lake’s buffer or TEST egg yolk buffer as the base diluent with a penetrating or non-penetrating CPA on the post-thaw recovery of sperm motility and viability. We also investigated the effect of post-thaw incubation treatment in TL HEPES supplemented with 10% fetal bovine serum (H10) alone or with caffeine on post-thaw motility parameters. Sperm from 16 Louisiana pinesnakes was cryopreserved, and the effectiveness of the CPA treatment mixtures and post-thaw treatments was determined based on measurements of sperm motility and viability. Sperm cryopreservation significantly reduced initial post-thaw sperm quality for all of the extender treatments. Viability of sperm was best maintained when cryopreserved in an CPA treatment mixture containing Lake’s buffer with 10% DMF, 2% methanol, and 5% clarified egg yolk with the addition of 5 mg/mL bovine serum albumin (BSA). For several extender mixtures a similar percent of post-thaw motility was observed, but no forward motility returned in any post-thaw samples prior to incubation in dilution treatments. Following incubation in both post-thaw treatments, the percent of forward motility and the index of forward progressive movement improved significantly. Post-thaw dilution with H10 containing caffeine improved motility parameters over H10 alone, suggesting further investigation of post-thaw treatment in caffeine could be beneficial. Although, cryopreservation of sperm from the Louisiana pinesnake continues to present a challenge, post-thaw dilution and the addition of BSA to CPA mixtures provides areas for improving cryopreservation methods for this endangered species.