Higher Sperm Motility Research Articles

Standardized cryopreservation protocol of European perch (Perca fluviatilis) semen allows to obtain high fertilization rates with the use of frozen/thawed semen

The general aim of this study was to develop an efficient and standardized cryopreservation procedure for European perch semen. The specific aims of this study were: to test the effects of (i) final glucose concentration, (ii) final sperm concentration in the extended semen and (iii) the storage time of post-thaw semen on sperm motility parameters. Moreover, the effects of cryopreservation of semen on fertilization rates were tested at sperm:egg ratios ranging between 50,000:1 and 500,000:1 (iv), as well as the fertilization of 25 g portions of eggs (v). The range of optimal sperm concentrations in straws with high post-thaw sperm motility (66–73%) was quite wide, from 1.0 to 6.0 × 109 spermatozoa ml−1. The final glucose concentration of 0.30–0.34 M in 7.5% methanol produced the highest results of sperm motility (76 ± 5%) after cryopreservation. Storage time of post-thaw semen negatively influenced sperm motility parameters. The fertilization rates while using cryopreserved semen were high (79%) and did not differ from fresh semen (85%). Similar results (73–77%) were obtained between sperm:egg ratio ranged from 50,000:1 to 500,000:1. Moreover, cryopreserved semen was successfully used for fertilization as much as 12,500 eggs (25 g). However, semen has to be used for fertilization within 2 min after thawing. In our opinion, standardizing the procedure of semen cryopreservation presented in this study is a prerequisite for the development of repeatable procedures and the future implementation of cryopreserved semen in hatchery practice. Further studies should focus on extension of time of post-thaw sperm motility, determination of optimal sperm:egg ratio at the conditions of large-scale fertilization and scaling up procedure to fertilize 50–150 g of eggs (one ribbon) at single application.

Effects of different extenders and additives on liquid storage of Awassi ram semen

The aim of this study was to evaluate the effects of different extenders and additives on ram semen during liquid storage and to demonstrate the role of oxidative stress parameters on this process. In the present study, ejaculates taken by artificial vagina twice a week from 4 rams during the breeding season were used. They were mixed and used if motility and viability were above 70% and there was a 95% intact acrosome. The semen specimens were diluted by Tris-citrate-glucose (TRIS), Tris-TES (TEST), HEPES-buffered Tyrode lactate (TL-HEPES), and Dulbecco?s phosphate-buffered saline (PBS) extenders supplemented with different additives [centrifuged egg yolk, Equex-STM, bovine serum albumin (BSA), and ethylenediaminetetraacetic acid (EDTA)]. The specimens were stored at 4 °C for up to 96 h and evaluated for motility, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), and oxidative stress parameters. At the end of the 96-h storage, the highest sperm motility was 64.2 ± 3.7% (P < 0.05) and significant loss of sperm motility and membrane integrity were not detected in extender TEST-3, but the MMP rate significantly declined. Acrosome integrity was not affected by storage time or extender types. BSA and EDTA decreased lipid peroxidation (LPO) and total oxidant status (TOS), but did not positively affect motility or membrane integrity. As a result, TRIS, TEST, and TL-HEPES-3 were observed to provide better protection for ram semen during liquid storage at 4 °C than other extenders. The role of oxidative stress and MMP are considerable in liquid storage.

Cryopreservation of epididymal and ejaculated bull spermatozoa

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

The addition of docosahexaenoic acid (DHA) and antioxidants (glutathione peroxidase and superoxide dismutase) in extenders to epididymal sperm cryopreservation in bulls.

SummaryThe cryopreservation of epididymal sperm is an important technique that allows genetic material to be preserved, even post mortem. However, cryopreservation leads to increased oxidative stress and impaired sperm viability. Polyunsaturated fatty acid (PUFA) supplementation may improve certain sperm characteristics, but it also makes sperm more susceptible to oxidative stress, therefore adding antioxidants that counteract oxidative stress has become an option. In this context, this study aimed to evaluate the effect of the interaction between docosahexaenoic acid (DHA) and antioxidants on the quality after the cryopreservation of epididymal bull sperm. Twenty epididymides were collected after slaughter, and epididymal sperm was cryopreserved with bovine extender supplemented with docosahexaenoic acid (DHA), glutathione peroxidase (GPx) and superoxide dismutase (SOD). We verified an improvement in motility in the group that was treated only with DHA 5 µM and a concentration-dependent effect on susceptibility to lipid peroxidation that was associated with DHA concentration (1 µM, 5 µM or 10 µM). Moreover, treatment with DHA (5 µM) and SOD (20 IU/ml) resulted in higher sperm motility. Thus, the association between DHA (5 µM) and SOD (20 IU/ml) appears to be an option for increased epididymal sperm features in bulls.

Effects of Green Tea (Camellia sinensis) Extract Supplementation at Different Dilution Steps on Boar Sperm Cryopreservation and in vitro Fertilization

We evaluated the effects of green tea extract (GTE) supplementation at different dilution steps on boar sperm freezing and in vitro fertilization. Sperm intracellular hydrogen peroxide (H₂O₂), motility, viability, acrosome integrity and morphology were determined. In addition, sperm IVF parameters (penetration and monospermy) and glutathione (GSH) levels of presumptive zygotes (PZs) were evaluated. Semen was diluted in lactose egg yolk (LEY) and cooled at 5℃ for 3 h (first dilution step) and then diluted in LEY with 9% glycerol and maintained at 5℃ for 30 min (second dilution step). Four experimental groups were compared: first and second dilution steps without GTE (control), first dilution step with GTE (Step 1), second dilution step with GTE (Step 2) and first and second dilution step with GTE (Step 1+2). The spermatozoa were frozen in nitrogen vapor. Higher sperm motility, viability and acrosome integrity after thawing were observed in Step 1, Step 2 and Step 1+2 groups compared with the control (P 〈 0.05). Lower H₂O₂ level was observed in Step 1+2 compared with control and Step 1 (P 〈 0.05). For IVF, matured oocytes were co-cultured with spermatozoa frozen according to the experimental groups. GSH levels of PZs were significantly higher in Step 2 and Step 1+2 than in control and Step 1 (P 〈0.05) without a significant difference in IVF parameters. In conclusion, supplementation with GTE in both first and second dilution steps during the freezing process resulted in better boar sperm cryopreservation and might be beneficial for further embryo development.

Optimisation of sodium and potassium concentrations and pH in the artificial seminal plasma of common carp Cyprinus carpio L.

The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n = 5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg−1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0–9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.

Cryopreserved rainbow trout semen can be used for the fertilization of up to 8000 eggs in a single application

Here we aimed to test the fertilizing ability of cryopreserved semen of rainbow trout (Oncorhynchus mykiss) during the fertilization of between 500 and 8000 eggs, thus establishing the optimal number of eggs to use in a single application of cryopreserved semen. The cryopreservation procedure resulted in a high post-thaw sperm motility (~70%). The fertilization rates ranged from 29 to 92% and did not differ depending on the number of eggs used. However, we did detect high and significant variability in fertilization rate among the pools of cryopreserved semen. Our results show that the cryopreserved semen of rainbow trout can be used to fertilize up to 8000 eggs in a single application. Cryopreserved sperm can be potentially implemented into breeding programs based on the crossing of selected males with individual females after confirmation of the rearing performance of larvae. Further studies should now focus on the cause of the between male variability in fertilizing ability observed in this study.

Does natural honey act as an alternative to antibiotics in the semen extender for cryopreservation of crossbred ram semen?

Antibiotics are added to semen extenders to take care of heavy microbial load, however, their continuous use poses a constant threat of developing antibiotic resistance by the common microbes present in the semen. Our hypothesis was that natural honey, having antibacterial activity and rich in fructose could replace the use of antibiotics and fructose in the semen extender. Twenty-four ejaculates from six crossbred rams were obtained and extended with tris-based extender without (control) and with honey at 2.5% (T1), 5% (T2) and 7% (T3). Sperm quality was measured in terms of percentage sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa. The semen samples at post-thaw were also evaluated for total viable count (colony forming units/ml). At post-thaw, control exhibited significantly (P<0.05) higher sperm motility in comparison to T2 and T3. The percent of live sperm count, intact acrosome and HOST reacted spermatozoa were significantly higher (P<0.05) for control than all other treatment groups at post-thaw. Among treatment groups, T1 maintained significantly higher (P<0.05) percentage of live sperm count, intact acrosome and HOST reacted spermatozoa than T2 and T3. The total viable count at post-thaw was significantly lower (P<0.05) for control than all the treatment groups. In conclusion, honey cannot be used as an alternative to antibiotics to take care of heavy microbial load in semen, however, levels up to 2.5% may be supplemented to semen as an energy source.

Effects of negative pressure on boar semen quality during liquid storage at 17°C

This study aimed to investigate the influence of negative pressure on boar semen quality during liquid preservation at 17°C. Semen samples from ten large white boars were collected and pooled, divided into four equal parts, and diluted with Modena containing 0.4% (w/v) of bovine serum albumin. The semen samples were placed in a closed container with valve, and a negative pressure was applied for 2–5 minutes using a vacuum pump with a barometer. The control group had no treatment, the P200 group was treated at 200 mbar, the P400 group at 400 mbar, and the P800 group at 800 mbar. During liquid preservation, sperm motility, total antioxidant capacity, and semen H2O2 content were analyzed every 24 h. The effective survival time of boar semen during preservation was evaluated. The results indicated that a suitable negative pressure decreased the effects on reactive oxygen species on boar sperm quality during liquid preservation compared with the control group. Among all the groups, the 400 mbar negative pressure group had the highest sperm motility, total antioxidant capacity, and the percentage of spermatozoa with high mitochondrial membrane potential. The P400 group also had semen H2O2 content than the other groups. A suitable negative pressure improves sperm quality by reducing oxidative stress and the respiratory metabolism of sperm, and the optimum negative pressure is 400 mbar.

Impact of Eurycoma longifolia extract on DNA integrity, lipid peroxidation, and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.

Optimal sperm concentration in straws and final glucose concentration in extender are crucial for improving the cryopreservation protocol of salmonid spermatozoa

Ensuring the precise conditions during cryopreservation relies on constant sperm concentration in straws with constant cryoprotectant concentrations, which are essential for standardizing cryopreservation protocols. The aim of our study was to test the effect of different sperm concentrations in straws with constant extender concentration on sperm motility parameters of cryopreserved semen of brown trout (Salmo trutta m. fario), sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar). At the same time, the usefulness of different straw sizes (0.25 and 0.5ml) for cryopreservation of the semen of these species was examined. Additionally, the effect of the final glucose concentration at a constant sperm concentration on post-thaw sperm motility was evaluated. The final sperm concentration in straws at a constant cryoprotectant concentration influenced the post-thaw sperm motility parameters of salmonids. The optimal sperm concentration with high post-thaw sperm motility was 2.0×109 spermatozoa ml−1 for brook trout, 3.0×109 spermatozoa ml−1 for brown trout and sea trout and 4.0×109 spermatozoa ml−1 for Atlantic salmon. The use of 0.5ml straws led to higher or similar values of post-thaw sperm motility than the 0.25ml straws. The final glucose concentration of 0.15M produced the highest results of sperm motility after cryopreservation for all tested species. Our results demonstrated that the influence of sperm concentration in straws on cryopreservation success is species-specific within salmonids. On the other hand, the effect of glucose concentration did not appear to be species-specific. In our opinion, standardization of the semen cryopreservation protocol presented in this study is a prerequisite for the development of a repeatable procedure and hence the future implementation of cryopreserved semen in hatchery practice.

Effect of trehalose- and sucrose-based extenders on equine sperm quality after vitrification: Preliminary results

There has been a lack of research into equine sperm vitrification to date, but studies of other species suggest it may have significant potential. To evaluate the impact of various cryoprotectant agents (CPA) and vitrification on equine sperm quality, a controlled study was carried out. A total of 12 ejaculates were subjected to exposure to CPA and vitrification. Sperm was diluted in a range of CPA: fresh, control (BSA), sucrose (0.15M, 0.3M and 0.5M), trehalose (0.15M, 0.3M and 0.5M) and the combination of sucrose and trehalose (M1: 0.15M sucrose+0.5M trehalose; M2: 0.5M sucrose+0.15M trehalose). Sperm motility, viability, acrosome integrity and DNA fragmentation were assessed at the time of CPA exposure and after vitrification. The exposure of spermatozoa to various concentrations of sucrose and/or trehalose significantly reduced sperm motility, with lower concentrations resulting in higher sperm motility. Sperm viability and DNA fragmentation did not vary after exposure to CPA, but acrosome integrity fell significantly when spermatozoa were exposed to CPA with high osmolality. When spermatozoa were vitrified, motility values were significantly higher than those obtained during the exposure. Low concentrations of sucrose (0.15M and 0.3M) and trehalose (0.15M) showed the best progressive sperm motility. The vitrification-warmed procedure significantly reduced sperm viability and acrosome integrity, but DNA did not vary with any of CPA used. Equine sperm vitrification demonstrates a low capacity for preserving sperm motility, and extenders containing trehalose or sucrose at lower concentrations are associated with a better protective effect on sperm motility. After vitrification, acrosome and plasma membranes were severely impaired, while the DNA structure was maintained. Equine spermatozoa partially recover the motility after vitrification, but there is a need for further studies into the preservation of sperm membranes.

Impact of holding and equilibration time on post-thaw quality of shipped boar semen

Cryopreservation of boar semen is of growing interest for breeding companies. Overnight-shipping of pre-diluted ejaculates to specialized laboratories offers a practicable method, but requires fine-tuned protocols. In this study, the impact of holding post shipping at 17°C for 2 or 24h (n=10 samples) and of equilibration in lactose-egg yolk extender without glycerol at 5°C for 2, 4, 24 or 48h (n=11 samples) before freezing was investigated. Sperm-rich fractions of ejaculates from 21 mature Pietrain boars were collected at a single boar stud. After pre-dilution (1+1, v:v) with Beltsville thawing solution, samples were sent to the laboratory. Temperature profiles during transport and initial equilibration time were recorded. Semen quality post-thaw (PT) was evaluated using CASA and flow cytometry. Holding of 2h after shipping resulted in higher sperm motility (P=0.013) and beat cross frequency (BCF; P=0.047) compared to 24h. Differences between both groups vanished with prolonged incubation at 38°C PT. Equilibration at 5°C for 4h yielded the highest motility and BCF, whereas the equilibration for 48h impaired sperm motility. Membrane integrity, mitochondrial activity and DNA fragmentation index were not affected by any protocol modification. In conclusion, processing of pre-diluted boar semen shipped overnight within 2h after arrival at the laboratory is preferred to 24h of additional holding at 17°C. Extending the equilibration period in lactose-egg yolk extender without glycerol at 5°C from 2h to 4h before freezing is recommended.

Biological properties of mouse spermatozoa separated by using the microfluidic sperm sorter

The microfluidic sperm sorter (MFSS) is a novel device that is able to separate spermatozoa with high motility and low DNA fragmentation from raw semen without washing and centrifugation process. However, some biological properties and effect on embryonic development of spermatozoa separated by the MFSS are as yet unexplained. The purpose of this study was to compare and investigate the biological properties and the developmental ability between spermatozoa separated by the MFSS and the swim-up (SU) procedure. Motility parameters and mitochondrial membrane potential (MMP) of spermatozoa prepared by MFSS and SU were studied. Cleavage and blastocyst rate were compared between embryos with spermatozoa separated by MFSS and those with spermatozoa processed by SU. Spermatozoa derived from C57BL/6J mouse were divided into two aliquots. One aliquot was processed by the MFSS and separated into high motility (HM) and low motility (LM) spermatozoa; the second aliquot was centrifuged (1800rpm, 10min) and processed by the swim-up procedure for 20-30 min (SU). Sperm motility parameters of three groups were analyzed by using the sperm motility analyzing system (SMAS). MMP was evaluated with the flow cytometer after JC-1 staining. In addition, spermatozoa of each group were injected into ovulated MII oocytes. After 3.5 hours from ICSI, a part of fertilized oocytes derived from each group was stained immunohistochemically with gamma H2AX antibody to detect DNA damage, and the others were cultured until to the blastocyst stage. By the SMAS analysis, the motile sperm rate was 90.5%, 47.8%, and 89.5% for HM, LM, and SU, respectively. The average speed of HM was significantly higher than that of LM and SU (HM: 116.8±8.6 μm/sec, LM: 68.2±8.2, SU: 83.1±4.1, p<0.05). MMP of spermatozoa in HM and SU were significantly higher than that of LM. After ICSI, there was no significant difference in the rate of pronuclear formation between each group. By immunohistochemical staining, high positive rate of gamma H2AX was observed in male pronucleus of embryos with LM spermatozoa. In addition, the blastocyst rate of embryos with HM was significantly higher than that of LM and was comparable to that of SU (HM: 84.3%, LM: 56.1%, SU: 79.7%, p<0.05). In this study, we demonstrated that spermatozoa with high sperm motility and developmental ability were able to separate by using the MFSS. By optimizing the MFSS system, the most appropriate spermatozoa for IVF/ICSI would be provided efficiently.

Antioxidant effects of lycopene on bovine sperm survival and oxidative profile following cryopreservation

Reactive oxygen species overgeneration as a side effect of semen cryopreservation may lead to lipid peroxidation, protein degradation, DNA fragmentation and cell death, resulting in a decrease of sperm survival and fertilisation ability. Lycopene has been proposed as a potential supplement to semen extenders because of its antioxidant properties. The aim of this study was to evaluate the effects of lycopene on the structural integrity, functional activity and selected oxidative stress parameters of cryopreserved bovine sperm. Thirty bovine ejaculates were split into two aliquots and diluted with a commercial semen extender supplemented with 1.5 mmol/l lycopene or containing no supplement (control), cooled down to 4 °C, frozen and kept in liquid nitrogen. Prior to experiments, frozen straws were thawed at 37 °C for 20 s. Lycopene addition resulted in a higher sperm motility (P &amp;lt; 0.001), progressive motility (P &amp;lt; 0.001) and all secondary motion characteristics (P &amp;lt; 0.001 with respect to the average path velocity, linear velocity, velocity of curvilinear motion, beat cross frequency, path straightness and linearity; P &amp;lt; 0.01 in the case of the amplitude of lateral head displacement). Furthermore, lycopene exhibited protective effects on the sperm membrane (P &amp;lt; 0.05) and acrosomal (P &amp;lt; 0.01) integrity in comparison to control. An assay for metabolic function revealed that lycopene supplementation to the cryopreservation medium resulted in a higher preservation of the sperm mitochondrial activity (P &amp;lt; 0.001). Reactive oxygen species production as well as intracellular superoxide generation were decreased following lycopene addition (P &amp;lt; 0.01 in the case of reactive oxygen species; P &amp;lt; 0.001 with respect to superoxide production). Finally, the presence of lycopene led to a decrease in protein carbonyl production (P &amp;lt; 0.01), lipid peroxidation (P &amp;lt; 0.001) as well as oxidative DNA damage (P &amp;lt; 0.05) when compared to control. In conclusion, lycopene exhibited significant reactive oxygen species-trapping and antioxidant properties which may prevent oxidative damage to frozen-thawed sperm, and, thus, enhance the post-thaw vitality of male reproductive cells in cattle breeding.

Antioxidants protect proteins' anchorage to the bilayer by improving plasma membrane integrity of ram spermatozoa during liquid preservation in a soya lecithin-based diluent.

Antioxidants are known to prevent the reactive oxygen species (ROS)-mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid-protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll® . Sperm pellet was resuspended in soya lecithin-Tris-fructose diluent (400×106 cells/ml) containing either antioxidants (100IU/ml catalase+10mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3-5°C for 72hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo-osmotic swelling test (HOST) were performed at 0, 24, 48 and 72hr. Sperm proteins extracted with 0.5% Triton X-100 were resolved by SDS-PAGE and quantified using Quantity One software (Bio-Rad, USA). The rapid motility, linearity and straight-line velocity (VSL) were found significantly (p<.05) higher in the antioxidant-treated group compared to the control at 48hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant-treated sample compared to the control both at 48 and at 72hr. Overall, the proteins P1 (106.09kDa), P2 (87.00kDa) and P4 (51.14kDa) were lower (p<.05) in the sperm extract of antioxidant-treated group compared to the control. The content of P4 (51.14kDa) in sperm extract was found to increase (p<.05) earlier (48 vs. 72hr) in the control group compared to the antioxidant-treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72hr of storage.

IN VITRO EFECTS OF SELECTED BIOLOGICALLY ACTIVE COMPOUNDS ON RABBIT SPERMATOZOA MOTILITY BEHAVIOUR

The aim of this study was to evaluate the in vitro effects of selected pure naturally occuring biologically active compounds (resveratrol-RES, quercetin-QUE, curcumin-CUR, epicatechin-EPI, isoquercitrin-ISO) on rabbit sperm motility using the IDENT staining method and the computer-aided sperm (CASA) analysis. Semen samples were collected regularly from 10 male rabbits, pooled, the seminal plasma was removed and the sperm fraction was exposed to different concentrations (1, 5, 10, 50 and 100 μmol/L) of chosen biomolecules. At culture times of 0h, 2h, 4h, 6h and 8h, the spermatozoa motility was assessed using the TOX IVOS II. CASA system and the IDENT fluorescent staining method. The motility assessment revealed different behavior patterns, specific and unique to each of the studied biomolecules. After 8h of in vitro culture, the highest sperm motility was detected in experimental groups subjected to 10 μmol/L RES (P

Standardization of spermatozoa concentration for cryopreservation of rainbow trout semen using a glucose-methanol extender

Determination of exact sperm concentrations in straws is necessary for standardizing protocols of fish cryopreservation. The aim of this study was to test the effect of final sperm concentration in straws on the post-thaw quality of rainbow trout semen in conditions of constant cryoprotectant concentration (0.15M glucose and 7.5% methanol). In addition, the utility of 0.5-ml (at two different thawing times of 10s and 13s) and 0.25-ml straws for the cryopreservation of rainbow trout semen was tested. A shorter thawing time (10s) of rainbow trout semen cryopreserved in 0.5-ml straws significantly improved the motility of sperm in thawed semen (62.7±5.0%) compared to a 13-s thawing time (57.4±9.4%). Dilution of semen to sperm concentrations of 0.5–1.0×109 spermatozoa ml−1 and cryopreservation in 0.5-ml straws resulted in higher sperm motility (60, 62 and 66% for 0.5; 0.75 and 1.0×109 sperm ml−1, respectively) in comparison to 0.25-ml straws containing the same sperm concentrations (43, 48 and 46%, respectively). However, sperm motility was reduced to similar values in semen thawed from 0.25-ml and 0.5-ml straws containing higher final sperm concentrations of 2.0–3.0×109 spermatozoa ml−1. Our results clearly demonstrate that cryopreserved sperm quality is related to final sperm concentration and straw volume. This study establishes a detailed and practical procedure for rainbow trout sperm cryopreservation using 0.5-ml straws and adjusting sperm concentrations in the straw to 0.5–1.0×109 spermatozoa ml−1, while maintaining constant concentrations of cryoprotectant.

Effect of season on semen quality parameters in Murrah buffalo

Seasonal influence on frozen semen quality in Murrah buffalo breeding bulls was determined. Frozen semen samples of 6 Murrah buffalo bulls were collected and semen frozen in 4 different seasons, viz. winter (Dec-Feb), spring (mid Feb-Apr), summer (May-Jun) and rainy (Jul-Aug) were assessed. Samples (12) of each bull, in a season, were evaluated for sperm motility, viability and acrosome integrity. Motility and other kinematics of spermatozoa during incubation (37°C) at 0, 30, 60, 90 and 120 min of thawing were assessed with computer assisted semen analyzer. Post-thaw sperm total motility and viability differed significantly among the seasons, the highest was in winter. Sperm plasma membrane integrity, acrosome integrity, progressive motility, rapid motility and other CASA evaluated parameters did not differ significantly among the seasons. Higher values of plasma membrane integrity (PMI), progressive motility, rapid motility, average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), beat cross frequency (BCF), linearity (LIN) and straightness (STR) were obtained in winter season as compared to other seasons. Post-thaw motility at 0 min and 60 min of post-thaw incubation varied significantly between seasons and higher sperm motility was sustained for a longer period in semen cryopreserved in winter followed by rainy season, summer and spring. It can be concluded from this study that buffalo bull semen produced and frozen during winter season resulted in higher sperm motility, viability and postthaw longer survivability in comparison to other seasons.

Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between High- and Low-motile sperm populations

BackgroundSmall RNAs present in bovine ejaculate can be linked to sperm abnormalities and fertility disorders. At present, quality parameters routinely used in semen evaluation are not fully reliable to predict bull fertility. In order to provide additional quality measurements for cryopreserved semen used for breeding, a method based on deep sequencing of sperm microRNA (miRNA) and Piwi-interacting RNA (piRNA) from individual bulls was developed.To validate our method, two populations of spermatozoa isolated from high and low motile fractions separated by Percoll were sequenced, and their small RNAs content characterized.ResultsSperm cells from frozen thawed semen samples of 4 bulls were successfully separated in two fractions. We identified 83 miRNAs and 79 putative piRNAs clusters that were differentially expressed in both fractions. Gene pathways targeted by 40 known differentially expressed miRNAs were related to apoptosis. Dysregulation of miR-17-5p, miR-26a-5p, miR-486-5p, miR-122-5p, miR-184 and miR-20a-5p was found to target three pathways (PTEN, PI3K/AKT and STAT).ConclusionsSmall RNAs sequencing data obtained from single bulls are consistent with previous findings. Specific miRNAs are differentially represented in low versus high motile sperm, suggesting an alteration of cell functions and increased germ cell apoptosis in the low motile fraction.