Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen

The aim of this work was to assess the cryoprotective effects of dimethylformamide (DMF) for freezing goat semen, using an objective analysis by computer-assisted sperm analysis (CASA). Twenty-one ejaculates (seven per animal) were collected from three stud bucks with the aid of an artificial vagina and immediately evaluated for gross and microscopic characteristics. The semen was diluted in two steps with a Tris–egg yolk extender containing 6% glycerol or 6% DMF, frozen in 0.50-mL straws, and stored in liquid nitrogen. Samples were accessed for sperm morphology, sperm membrane structural and functional integrity, and by CASA, immediately after thawing. There were differences (P < 0.05) between glycerol and DMF with regard to subjective progressive motility (23.9 ± 2.2% vs. 16.6 ± 2.0%), objective progressive motility (3.5 ± 0.4% vs. 1.8 ± 0.3%), linearity (53.9 ± 1.6% vs. 48.1 ± 1.4%) and amplitude of lateral head (2.3 ± 0.1 vs. 2.9 ± 0.1 mm), which confirmed the efficiency of glycerol. In conclusion, dimethylformamide could be used as an alternative cryoprotectant for goat semen freezing. However it was showed that no benefits were derived by using dimethylformamide to replace glycerol at an equal 6% concentration.     

Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters

In this study, we examined different computer-assisted sperm analysis (CASA) systems (CRISMAS, Hobson Sperm Tracker, and Image J CASA) on the exact same video recordings to evaluate the differences in sperm motility parameters related to the specific CASA used. To cover a wide range of sperm motility parameters, we chose 12-second video recordings at 25 and 50 Hz frame rates after sperm motility activation using three taxonomically distinct fish species (sterlet: Acipenser ruthenus L.; common carp: Cyprinus carpio L.; and rainbow trout: Oncorhynchus mykiss Walbaum) that are characterized by essential differences in sperm behavior during motility. Systematically higher values of velocity and beat cross frequency (BCF) were observed in video recordings obtained at 50 Hz frame frequency compared with 25 Hz for all three systems. Motility parameters were affected by the CASA and species used for analyses. Image J and CRISMAS calculated higher curvilinear velocity (VCL) values for rainbow trout and common carp at 25 Hz frequency compared with the Hobson Sperm Tracker, whereas at 50 Hz, a significant difference was observed only for rainbow trout sperm recordings. No significant difference was observed between the CASA systems for sterlet sperm motility at 25 and 50 Hz. Additional analysis of 1-second segments taken at three time points (1, 6, and 12 seconds of the recording) revealed a dramatic decrease in common carp and rainbow trout sperm speed. The motility parameters of sterlet spermatozoa did not change significantly during the 12-second motility period and should be considered as a suitable model for longer motility analyses. Our results indicated that the CASA used can affect motility results even when the same motility recordings are used. These results could be critically altered by the recording quality, time of analysis, and frame rate of camera, and could result in erroneous conclusions.     

Additional value of computer assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination

In order to obtain a more standardised semen motility evaluation, Varkens KI Nederland has introduced a computer assisted semen analysis (CASA) system in all their pig AI laboratories. The repeatability of CASA was enhanced by standardising for: 1) an optimal sample temperature (39 °C); 2) an optimal dilution factor; 3) optimal mixing of semen and dilution buffer by using mechanical mixing; 4) the slide chamber depth, and together with the previous points; 5) the optimal training of technicians working with the CASA system; and 6) the use of a standard operating procedure (SOP). Once laboratory technicians were trained in using this SOP, they achieved a coefficient of variation of < 5% which was superior to the variation found when the SOP was not strictly used. Microscopic semen motility assessments by eye were subjective and not comparable to the data obtained by standardised CASA. CASA results are preferable as accurate continuous motility dates are generated rather than discrimination motility percentage increments of 10% motility as with motility estimation by laboratory technicians. The higher variability of sperm motility found with CASA and the continuous motility values allow better analysis of the relationship between semen motility characteristics and fertilising capacity. The benefits of standardised CASA for AI is discussed both with respect to estimate the correct dilution factor of the ejaculate for the production of artificial insemination (AI) doses (critical for reducing the number of sperm per AI doses) and thus to get more reliable fertility data from these AI doses in return.     

Effects of dithioerythritol on ram semen after the freeze-thawing process

The aim of this study was to evaluate the effects of dithioerythritol added to cryopreservation extender on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities of Merino ram sperm. Semen samples from 5 mature Merino rams (1 and 2 years of age) were used in the study. Semen samples, which were diluted with a Tris-based extender containing 0.5, 1, and 2 mM dithiothreitol and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37 °C for 20 s in a water bath for evaluation.The addition of dithioerythritol at 0.5 and 2 mM doses led to higher percentages of subjective motility (62.9 ± 4.2% and 63.6 ± 1.8%) compared to control (52.0 ± 4.9%, P < 0.05). As regards CASA motility, dithioerythritol 0.25 and 2 mM (60.2 ± 4.5% and 59.6 ± 1.2%) groups were higher from that of control (44.2 ± 8.7%, P < 0.05). For the CASA progressive motility, 0.25, 0.5 and 2 mM doses of dithioerythritol (22.0 ± 2.1%, 21.7 ± 2.5% and 24.0 ± 1.2%) had increasing effect in comparison to control (15.0 ± 2.5%). Dithioerythritol at 1 and 2 mM doses for ALH provided higher values compared to the control (P < 0.001) following the freeze–thawing process. Supplementation with dithiothreitol did not significantly affect the integrities of sperm membrane and acrosome, and mitochondrial activities. No significant differences were observed in biochemical parameters among the groups (P > 0.05). Findings of this study showed that dithioerythritol supplementation in semen extenders, was of greater benefit to sperm motility of frozen–thawed ram sperm.     

Infertility in a beef bull due to a failure in the capacitation process

The objective of this case report was to identify the cause of apparent idiopathic infertility in a Red Angus (beef) bull. Semen was collected by electroejaculation and submitted to a series of assays, including evaluation of sperm motility by computer-assisted sperm analysis (CASA), sperm morphology and DNA integrity, semen cryopreservation, AI, IVF, induction of the acrosome reaction, and determination of the level of sperm proteins associated with bull fertility potential. Total (92 ± 2%) and progressive (79 ± 4%) sperm motility; sperm concentration (1647 ± 429 × 10⁶ sperm/mL); proportions of morphologically normal sperm (83 ± 6%) and DNA integrity (96 ± 2), and acrosome-intact sperm (64 ± 4%) exceeded minimum acceptable values. Frozen sperm had good total (58.7 ± 6.7%) and progressive (43.9 ± 9.2%) motility immediately after thawing. However, AI of 16 heifers resulted in no pregnancies and blastocyst production rate (following IVF using sperm from this infertile bull) was nearly identical to that produced using dead sperm (a control of parthenogenesis; 2 ± 2 and 2 ± 3%; respectively P < 0.05). Treatment with a calcium ionophore (A23187) failed to induce the acrosome reaction in sperm from the infertile bull (P < 0.05). Evaluation of several proteins associated with the fertility potential of bulls revealed that the level of Binder Sperm Protein-1 (BSP1), known to be associated with the capacitation process, was much greater on sperm from the infertile bull compared to that of his sire. In conclusion, we inferred that the idiopathic infertility in this bull was caused by a failure to complete the capacitation process.     

Dimethylformamide is no better than glycerol for cryopreservation of canine semen

The objective of this study was to compare the effects of dimethylformamide (DMF) and glycerol in canine (Canis lupus familiaris) semen cryopreservation based on postthaw motility and velocity evaluated by computer-assisted sperm analysis (CASA) and the effects on subjective progressive motility, percentage of live sperm, and plasma membrane functional integrity. The semen was diluted in two steps with an egg-yolk Tris extender containing 6% glycerol or DMF, frozen in 0.25-mL straws, and stored in liquid nitrogen. Immediately after thawing, samples were accessed for subjective sperm motility, sperm membrane functional integrity, percentage of live sperm, and evaluation by CASA. There were differences (P < 0.05) between glycerol and DMF with regard to subjective progressive motility (43.1% vs. 21.5%), objective progressive motility (11.8% vs. 6.2%), velocity average pathway (31.1 vs. 23.1 μm/sec), and amplitude of lateral head (3.3 vs. 3.9 μm), which confirmed the efficiency of glycerol. In conclusion, objective analysis performed by CASA confirmed that no benefits were derived by using DMF to replace glycerol for cryopreservation of canine semen.     

Motility and fertility of the subtropical freshwater fish streaked prochilod (Prochilodus lineatus) sperm cryopreserved in powdered coconut water

Streaked prochilod (Prochilodus lineatus) is a freshwater fish inhabiting many South American rivers. The objective was to determine the effectiveness of coconut water (ACP™), combined with methylglycol, as a freezing medium for streaked prochilod sperm. A secondary objective was to compare a computer-assisted sperm analyzer (CASA) system versus subjective microscropic examination as a means of assessing sperm motility. As a control, glucose and methylglycol was used, according to our previous study. Sperm diluted in each medium was loaded into 0.5 mL straws, frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 °C). Half of the samples were evaluated for sperm motility, both subjectively and with CASA; the remainder were evaluated for fertility. There was no difference (P > 0.05) between subjective or CASA assessment of post-thaw sperm motility. Although sperm motility was higher in sperm cryopreserved in ACP™ (85%) than in glucose (75%), cryopreservation in either extender yielded similar fertilization rates (46-48%) and sperm velocities. There were positive correlations (r = 0.56-0.8) between all sperm velocities and fertilization rate. In conclusion, streaked prochilod sperm cryopreserved in glucose or ACP™ and methylglycol was fertile, and thus could be used for research or commercial settings. Furthermore, although the CASA system provided objective data regarding sperm motility, in the present study, subjective evaluation of sperm motility was practical and a good indication of sperm quality; it could readily be done by well-trained personnel under field or laboratory conditions.     

Incorporation of ascorbic acid and α-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm

Despite the overwhelming application of sperm cryopreservation in aquaculture and broodstock management, its detrimental effects on sperm quality must be taken into account. Imbalance of reactive oxygen species is considered one of the main triggers of cell damage after cryopreservation, because the spermatozoa antioxidant system is decimated during this process, mainly because the natural antioxidants present in seminal plasma diminish when sperm is diluted in extenders. It has been demonstrated that the addition of antioxidants to the extender improves the quality of thawed sperm. Thus, the aim of the present work was to evaluate the status of the antioxidant system in cryopreserved sea bass sperm, and the possibility of enhancing this system to reduce oxidation of the membrane compounds by extender supplementation with vitamins. To do this, sperm from European sea bass (Dicentrarchus labrax) was cryopreserved using an extender control (NAM), supplemented with 0.1 mm α-tocopherol or 0.1 mm ascorbic acid. Sperm motility (computer assisted sperm analysis (CASA) parameters), viability (SYBR Green/propidium iodide (PI)), lipid peroxidation (malondialdehyde (MDA) levels) and protein oxidation (DNPH levels) were analyzed, as well as the status of the sperm antioxidant system by determining glutathione peroxidase, glutathione reductase and superoxide dismutase (GPX, GSR and SOD) activity. The results demonstrated that extenders containing vitamins significantly increased sperm motility. Total motility, velocity and linearity increased from 31.2 ± 3.0 μm/sec, 18.3 ± 1.7 μm/sec and 46.9 ± 2.0% in extender containing 0.1 mm α-tocopherol or 30.6 ± 3.9 μm/sec, 19.5 ± 1.6 μm/sec and 47.9 ± 2.2% in extender containing 1 mm ascorbic acid respect to the extender control (20.7 ± 3.3 μm/sec, 13.8 ± 1.7 μm/sec and 37.3 ± 4.1%). However, viability and levels of lipid peroxidation and protein oxidation were not affected by the presence of these antioxidants, suggesting that membrane impairment could be more associated to osmotic shock or membrane destabilization than oxidative damage. The increased activity of both GPX and GSR after cryopreservation showed that the antioxidant system of sea bass sperm must play an important role in preventing oxidation of the membrane compounds. In conclusion, the addition of α-tocopherol and ascorbic acid to the extender media, together with the antioxidant system of the spermatozoa improved sea bass sperm motility, which is one of the impairment parameters most affected by cryopreservation.     

Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining

The Zebrafish has gained increased popularity as an aquatic model species in various research fields, and its widespread use has led to numerous mutant strains and transgenic lines. This creates the need to store these important genetic materials as frozen gametes. Sperm cryopreservation in zebrafish has been shown to yield very low post-thaw survival and many protocols suffer from great variability and poor reproducibility. The present study was intended to develop a freezing protocol that can be reliably used to cryopreserve zebrafish sperm with high post-thaw survival. In particular, our study focused on cooling protocol optimization with the aid of cryomicroscopy. Specifically, sperm suspended in 8% DMSO or 4% MeOH were first incubated with live/dead fluorescent dyes (SYBR14/PI) and then cooled at various rates from 4 °C to different intermediate stopping temperatures such as −10, −20, −30 and −80 °C before rewarming to 35 °C at the rate of 100 °C/min. %PI-positive (dead) cells were monitored throughout the cooling process and this screening yielded an optimal rate of 25 °C/min for this initial phase of freezing. We then tested the optimal cooling rate for the second phase of freezing from various intermediate stopping temperatures to −80 °C before plunging into liquid nitrogen. Our finding yielded an optimal intermediate stopping temperature of −30 °C and an optimal rate of 5 °C/min for this second phase of freezing. When we further applied this two-step cooling protocol to the conventional controlled-rate freezer, the average post-thaw motility measured by CASA was 46.8 ± 6.40% across 11 males, indicating high post-thaw survival and consistent results among different individuals. Our study indicates that cryomiscroscopy is a powerful tool to devise the optimal cooling conditions for species with sperm that are very sensitive to cryodamage.     

Cryopreservation of giant grouper Epinephelus lanceolatus (Bloch, 1790) sperm

The grouper, Epinephelus lanceolatus, is a vulnerable species of high economic value. An effective protocol was developed for the cryopreservation of E. lanceolatus by comparing different extenders produced by mixing various cryoprotectants (dimethyl sulfoxide, methanol and glycerol) and diluents (MPRS, TS‐2, TS‐19, Cortland and Hank's). Using computer‐assisted sperm analysis (CASA) and morphological analysis, the sperm motility and fertilization rates from post‐thaw sperm were comparable to untreated controls. The results revealed that MPRS (containing 12% DMSO) or TS‐19 (containing 12% DMSO), were the optimum extenders for protecting the sperm from cryo‐damage in liquid nitrogen. The post‐thaw sperm maintained high motility (90.61 ± 3.03%) and a fertilization rate (92.27 ± 2.43%) similar (P > 0.05) to fresh sperm (94.34 ± 4% and 94.10 ± 1.87%). This study is the first to report on the successful sperm cryopreservation of E. lanceolatus and provides an important tool for repopulating this species through aquaculture.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Sperm motility parameters for Steindachneridion parahybae based on open‐source software

The objective of this work was to evaluate the sperm motility of 13 Steindachneridion parahybae males using open‐source software (ImageJ/CASA plugin). The sperm activation procedure and image capture were initiated after semen collection. Four experimental phases were defined from the videos captured of each male as follows: (i) standardization of a dialogue box generated by the CASA plugin within ImageJ; (ii) frame numbers used to perform the analysis; (iii) post‐activation motility between 10 and 20 s with analysis at each 1 s; and (iv) post‐activation motility between 10 and 50 s with analysis at each 10 s. The settings used in the CASA dialogue box were satisfactory, and the results were consistent. These analyses should be performed using 50 frames immediately after sperm activation because spermatozoa quickly lose their vigor. At 10 s post‐activation, 89.1% motile sperm was observed with 107.2 μm s^?1 curvilinear velocity, 83.6 μm s^?1 average path velocity, 77.1 μm s^?1 straight line velocity; 91.6% were of straightness and 77.1% of wobble. The CASA plugin within ImageJ can be applied in sperm analysis of the study species by using the established settings.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Sperm quality analysis in XX, XY and YY males of the Nile tilapia (Oreochromis niloticus)

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offspring), but they also constitute major tools to study sex determinism mechanisms. In other species, sexual genotype and sex reversal procedures affect different aspects of biology, such as growth, behavior and reproductive success. The aim of this study was to assess the influence of sexual genotype on sperm quality in Nile tilapia. Milt characteristics were compared in XX (sex-reversed), XY and YY males in terms of gonadosomatic index, sperm count, sperm motility and duration of sperm motility. Sperm motility was measured by computer-assisted sperm analysis (CASA) quantifying several parameters: total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity and linearity. None of the sperm traits measured significantly differed between the three genotypes. Mean values of gonadosomatic index, sperm concentration and sperm motility duration of XX, XY and YY males, respectively ranged from 0.92 to 1.33%, from 1.69 to 2.22 ×10⁹ cells mL⁻¹ and from 18′04″ to 27′32″. Mean values of total motility and curvilinear velocity 1 min after sperm activation, respectively ranged from 53 to 58% and from 71 to 76 μm s⁻¹ for the three genotypes. After 3 min of activity, all the sperm motility and velocity parameters dropped by half and continued to slowly decrease thereafter. Seven min after activation, only 9 to 13% of spermatozoa were still progressive. Our results prove that neither sexual genotype nor hormonal sex reversal treatments affect sperm quality in male Nile tilapias with atypical sexual genotype.     

Dimethylformamide as a cryoprotectant for canine semen diluted and frozen in ACP-106C

The objective was to assess the effect of adding various concentrations of dimethylformamide on characteristics of canine semen diluted in powdered coconut water (ACP-106C; ACP Biotecnologia, Fortaleza, CE, Brazil) and frozen at −196°C. Fifteen ejaculates were collected by manual stimulation from five adult Boxer dogs. The sperm-rich fraction was diluted in ACP-106C (ACP Biotecnologia) containing 10% egg yolk and divided into four aliquots. The cryoprotectants used for each aliquot were 6% glycerol (control group; CG) or 2%, 4%, or 6% dimethylformamide (DF2, DF4, and DF6, respectively). After thawing, total motility (mean ± SEM) for CG (58.4 ± 24.6) was higher (P < 0.05) than that of the other groups (2% dimethylformamide, 24.4 ± 12.3; 4% dimethylformamide, 26.5 ± 16.1; and 6% dimethylformamide, 21.7 ± 17.9). Furthermore, there was a greater percentage of fast, average, and slow moving sperm (assessed with computer-aided semen analysis; CASA) in CG in comparison with the other three groups. Therefore, based on concentrations tested in this study, dimethylformamide, together with ACP-106C (ACP Biotecnologia) and 10% egg yolk as a diluent, yielded unsatisfactory in vitro results for freezing canine semen.     

Effectiveness of the sperm quality analyzer (SQA-Vp) for porcine semen analysis

The Sperm Quality Analyzer (SQA-Vp) was evaluated for assessing concentration and motility of porcine semen. Both fresh and diluted semen from 50 different boars from a commercial artificial insemination (AI) centre were investigated. For the fresh ejaculate, the concentration obtained with SQA-Vp was compared with a photometer and a haemocytometer. For the diluted samples, the concentration and motility were compared with computer assisted semen analysis (CASA) and visual sperm analysis. The agreement between methods was studied with Bland-Altman plots and the repeatability with coefficient of variation (CV) as well as Bland-Altman plots. The sperm concentration (x10⁶/ml) obtained with SQA-Vp (379.3 ± 134.9) for fresh ejaculates agreed well with concentration by the photometer (447.2 ± 154.2; difference= -67.9 x 10⁶/ml; difference + 2SD = 55.3 x 10⁶/ml; difference - 2SD = -191.1 x 10⁶/ml) and with the haemocytometer (332.8 ± 141.11; d = 92.8; d + 2SD = 448.6; d - 2SD = -263). For diluted semen, the agreement between the concentration (x10⁶/ml) assessed with SQA-Vp (20.4 ± 4.3) was good with CASA (23.2 ± 5.8; d = -2.8; d + 2SD = 6.2; d - 2 SD = -11.8) but poor with the haemocytometer (18.8 ± 5.0; d = 1.6; d+ 2SD = 12.2; d - 2SD = -9). The % motile spermatozoa assessed by SQA-Vp (65.8 ± 10.0) in diluted semen agreed well with CASA (72.2 ± 13.7; d = -6.4; d+ 2SD = 20; d - 2SD = -32.8) and with visual assessment (64.1 ± 11.6; d = 1.7; d+ 2SD = 30.9; d - 2SD = -27.5). The SQA-Vp showed a good repeatability (CV; repeatability coefficient) for measuring the concentration of both fresh (3.9%; d = 10.7; d + 2SD = 30.9; d - 2SD = -9.5) and diluted semen (2.6%; d = 1.0; d + 2SD = 2.38; d - 2SD = -0.42) and for motility (3.2%; d = 0.9; d + 2SD = 8.5; d - 2SD = -6.7). The mean SQA-Vp values fell between the other methods′ results for both fresh and diluted semen. Moreover the repeatability was acceptable. Therefore SQA-Vp can be used as a valid device for sperm quality analysis in pigs.     

Effects of extenders, cryoprotectants and freezing methods on sperm quality of the threatened Brazilian freshwater fish pirapitinga-do-sul Brycon opalinus (Characiformes)

The objective was to develop a suitable freezing method to cryopreserve Brycon opalinus (Characiformes) sperm. Extenders (NaCl and glucose at 325 and 365 mOsm/kg), cryoprotectants (dimethyl sulfoxide = dimethyl sulfoxide (DMSO) and methyl glycol = methyl glycol (MG)), equilibration times (15 and 30 min), thawing temperatures (30 and 60 °C), and straw sizes (0.5 and 4.0 mL) were tested. Sperm were frozen in a liquid nitrogen vapor vessel at −170 °C and subsequently stored in liquid nitrogen. Post-thaw sperm quality was always evaluated in terms of motility (expressed as percentage of motile sperm), duration of motility and vitality (eosin-nigrosin staining, expressed as percentage of intact sperm). The best freezing method was also tested for fertility and hatching (expressed as the percentage of fertilized eggs). Post-thaw sperm quality was highest when sperm were cryopreserved in Glucose 365 mOsm/kg and MG, after a 30-min equilibration and thawed at 60 °C for 8 s, of regardless straw size: 74 ± 7% motile sperm, 47 ± 4 s of motility duration, 69 ± 3% intact sperm, 64 ± 4% fertilization and 63 ± 3% hatching. The freezing method developed in the present study was efficient and can be used to maximize larvae production for both aquaculture purposes and for conservational programs, since B. opalinus is a threatened species.     

Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (V_o) of 12.1 ± 0.2 μm³ (SEM), a water permeability (L_p) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) μm/min/atm, and a cryoprotectant permeability (P_s) of 0.10 ± 0.01 (SEM) × 10⁻³ cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 °C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.     

Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Inês cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10⁶ sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST⁺ (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI⁻/PSA⁻ (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.