Rapid assay for detecting gyrA and parC mutations associated with fluoroquinolone resistance in Enterobacteriaceae

We developed a PCR-RFLP assay to detect mutations in the quinolone-resistance determining regions of gyrA and parC associated with fluoroquinolone resistance in Enterobacteriaceae. The assay detected mutations associated with reduced susceptibility to fluoroquinolones and may therefore serve as a specific, rapid, inexpensive, and simple testing alternative.     

Effects of porcine pre-ovulatory oviductal fluid on boar sperm function

Sperm storage within the oviductal isthmus prior to ovulation typically involves binding to oviductal epithelial cells, which are thought to modulate sperm functions including internal calcium concentration, membrane fluidity, and motility. Around the time of ovulation the spermatozoa are gradually released so that they eventually encounter the oocytes within the oviductal ampulla. Previous studies have shown that the oviductal epithelial cells selectively sequester high quality spermatozoa, but the role of oviductal fluid as a selective modulator of sperm function has been investigated to a lesser extent. Here we address the hypothesis that oviductal fluid is also likely to modulate sperm function. Using samples of porcine oviductal fluid collected in the follicular phase of the estrus cycle, we show that short exposure (20 min to 50 μg/mL of oviductal fluid proteins) to either of two separate proteins fractions (> or < 100 kDa) promotes boar sperm viability and acrosomal integrity, decreases sperm plasma membrane fluidity (measured using merocyanine S540), and increases zona binding and polyspermy during in vitro fertilization. Exposure to the lower molecular fraction significantly inhibited, but did not abolish, the bicarbonate-induced stimulation of motility. The results show that subpopulations of spermatozoa respond differentially to oviductal fluid, and suggest that exposure to oviductal fluid in vivo could exert a further level of functional sperm selection.     

Cholesterol and the biosynthesis of glycosphingolipids are required for sperm activation in Caenorhabditis elegans

Ejaculated mammalian sperm must acquire fertilization capacity after residing into the female reproductive tract, a process collectively known as capacitation. Cholesterol efflux was required for sperm maturation. Different from flagellated sperm, C. elegans sperm are crawling cells. C. elegans sperm are highly enriched with cholesterol though this animal species lacks biosynthetic pathway for cholesterol and its survival requires an exogenous cholesterol supply. The low abundance of cholesterol in C. elegans lipid extract is thought insufficient to form lipid microdomains ubiquitously in this organism. We present evidence that cholesterol is enriched in the plasma membrane of C. elegans spermatids and that cholesterol- and glycosphingolipids (GSLs)-enriched membrane microdomains (lipid microdomains) mediate sperm activation. Disruption of sperm lipid microdomains by acute manipulation of cholesterol in vitro blocks the sperm activation. Restriction of cholesterol uptake also results in the abnormal sperm activation in both males and hermaphrodites. Manipulation of the integrity of lipid microdomains by targeting the biosynthesis of GSLs inhibits sperm activation and the inhibition can be rescued by the addition of exogenous GSLs. The cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, which are exclusively found in lipid microdomains, also affects sperm activation. We conclude that localized signaling mediated by lipid microdomains is critical for worm sperm activation. Lipid microdomains composed of cholesterol and GSLs have been observed in flagellated sperm of several animal species, thus cholesterol, before its efflux from the plasma membrane, might be needed to assemble into a platform for some more important upstream signal sorting during spermatogenesis than was previously thought.     

Protective effects of propolis on cryopreservation of common carp (Cyprinus carpio) sperm

Cryopreservation of sperm is common procedures in aquaculture, particularly used for routine in artificial insemination. However, these application cause damages and adversely affected sperm motility, viability and consequently lower hatching rates. The objective of this study is to determine whether propolis has an effect on cryopreservation and fertilization ability and to investigate the potential protective effect of propolis on spermatozoa of Cyprinus carpio. Many studies have been done in cryopreservation offish spermatozoa, but none of them contain propolis in extender composition. The extenders were prepared by using modified Kurokura Solution to which 10% Me₂SO added with different levels of propolis (0.2, 0.4, 0.6, 0.8 and 1 mg ml⁻¹) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis exhibited higher percentage motility and motility duration than control group (P < 0.05). Especially the group IV (0.8 mg ml⁻¹ propolis) and the group V (1 mg ml⁻¹ propolis) showed significant positive effects on both post thaw motility and hatching ability. The propolis maintained the integrity of the spermatozoa during the cryopreservation process. Evaluating with its contents, it has been shown that propolis is an appropriate cryoprotective agent in fish semen.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

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.     

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 μm s⁻¹ swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 × 10⁶/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.     

Description of Brenneria roseae sp. nov. and two subspecies, Brenneria roseae subspecies roseae ssp. nov and Brenneria roseae subspecies americana ssp. nov. isolated from symptomatic oak

Gram-negative, facultatively anaerobic bacteria were isolated from symptomatic oak tissue in the UK and USA. Partial gyrB sequencing placed ten strains in the genus Brenneria, with B. goodwinii as the closest phylogenetic relative. The strains were investigated further using a polyphasic approach including MLSA (based on partial gyrB, rpoB, infB and atpD gene sequences), 16S rRNA gene sequencing, DNA–DNA relatedness studies and both phenotypic and chemotaxonomic assays. The MLSA and 16S rRNA gene analyses separated the strains into two groups based on origin, suggesting that they belong to Brenneria as two novel species. However, the DNA–DNA relatedness values revealed a closer relationship between the groups and indicated that they should belong to the same species. As the two groups of strains from the UK and USA can be differentiated from each other phenotypically and by ERIC PCR fingerprints, it is proposed to classify them as novel subspecies of a novel Brenneria species. The name Brenneria roseae sp. nov. (FRB 222^T = LMG 27714^T = NCPPB 4581^T) is proposed, with Brenneria roseae subsp. roseae ssp. nov. (FRB 222^T = LMG 27714^T = NCPPB 4581^T) for the strains from the UK and Brenneria roseae subsp. americana ssp. nov. (FRB 223^T = LMG 27715^T = NCPPB 4582^T) for the strains from the USA.     

SLC6 family transporter SNF-10 is required for protease-mediated activation of sperm motility in C. elegans

Motility of sperm is crucial for their directed migration to the egg. The acquisition and modulation of motility are regulated to ensure that sperm move when and where needed, thereby promoting reproductive success. One specific example of this phenomenon occurs during differentiation of the ameboid sperm of Caenorhabditis elegans as they activate from a round spermatid to a mature, crawling spermatozoon. Sperm activation is regulated by redundant pathways to occur at a specific time and place for each sex. Here, we report the identification of the solute carrier 6 (SLC6) transporter protein SNF-10 as a key regulator of C. elegans sperm activation in response to male protease activation signals. We find that SNF-10 is present in sperm and is required for activation by the male but not by the hermaphrodite. Loss of both snf-10 and a hermaphrodite activation factor render sperm completely insensitive to activation. Using in vitro assays, we find that snf-10 mutant sperm show a specific deficit in response to protease treatment but not to other activators. Prior to activation, SNF-10 is present in the plasma membrane, where it represents a strong candidate to receive signals that lead to subcellular morphogenesis. After activation, it shows polarized localization to the cell body region that is dependent on membrane fusions mediated by the dysferlin FER-1. Our discovery of snf-10 offers insight into the mechanisms differentially employed by the two sexes to accomplish the common goal of producing functional sperm, as well as how the physiology of nematode sperm may be regulated to control motility as it is in mammals.     

Cryopreservation of sperm in farmed Australian greenlip abalone Haliotis laevigata

This study investigated factors important to the development of the liquid nitrogen (LN) vapor sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata, including (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature (height above LN surface); (3) thawing temperature; (4) sperm to egg ratio; and (5) sugar supplementation, using sperm motility, fertilization rate or integrity/potential of sperm components and organelles as quality assessment indicators. Results suggested that among the single CPAs evaluated 6% dimethyl sulfoxide (Me2SO) would be the most suitable for sperm cryopreservation in this species. The highest post-thaw sperm motility was achieved with the sperm that had been exposed to LN vapor for 10 min at 5.2 cm above the LN surface, thawed and recovered in 60 and 18 °C seawater bathes, respectively after at least 2 h storage in LN. The highest fertilization rates were achieved at a sperm to egg ratio of 10,000:1 or 15,000:1. Addition of 1% glucose or 2% sucrose produced significantly higher post-thaw sperm motility than 6% Me2SO alone. Among the three cryoprotectant solutions further trialled, 6% Me2SO + 1% glucose produced the highest fertilization rate of 83.6 ± 3.7%. Evaluation of sperm has shown that the addition of glucose could significantly improve the sperm plasma membrane integrity and mitochondrial membrane potential. These results demonstrated a positive role of glucose in the improvement of sperm cryopreservation in farmed greenlip abalone.     

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

Introduction

Acquired AmpC enzymes, classified as miscellaneous extended-spectrum β-lactamase (ESBL_M) enzymes according to a recently proposed β-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL_M are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla_AmpC detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes.

Material and methods

Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/− cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla_AmpC real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing.

Results and discussion

The real-time PCR assay was able to detect and sub-classify all acquired bla_AmpC genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla_AmpC real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.     

Sperm structure and sperm transfer in Pseudopythina subsinuata (Bivalvia; Galeommatoidea)

Males and females of the commensal protandric bivalve Pseudopythina subsinuata have paired seminal receptacles, the interior of which contains many slender elongate cells. The testis produces small euspermatozoa and comparatively few and much larger paraspermatozoa. The ?16-μm-long and 3-5-μm-thick paraspermatozoa have a terminally placed irregularly cork-screw-shaped acrosome and a bundle of ca. 16 flagella emerging from behind the nucleus. The role of the paraspermatozoa is obscure. Euspermatozoa are transferred to the seminal receptacles of the females and attach with the tip of the acrosome to the elongate cells. Most females contain one to three “sperm trees”, structures consisting of a short stem and numerous branches. They are firmly implanted in the abfrontal part of the gill filament and protrude into the posterior part of the suprabranchial (brooding) chamber. Implantation of the trees causes the gill tissue to swell around the stem and some of the nearest filaments to coalesce. All branches are densely coated with euspermatozoa that are attached by means of their acrosomes. It is conjectured that the syncytial and multinucleate trees arise from seminal receptacle cells that detach from the receptacle and thereupon fuse. A similar process is known in the allied P. tsurumaru, but the resulting structure (“sperm-carrying body”) is not attached to the gills.     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.     

Evidence for the existence of two new members of the family Chlamydiaceae and proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov.

The family Chlamydiaceae with the recombined single genus Chlamydia currently comprises nine species, all of which are obligate intracellular organisms distinguished by a unique biphasic developmental cycle. Anecdotal evidence from epidemiological surveys in flocks of poultry, pigeons and psittacine birds have indicated the presence of non-classified chlamydial strains, some of which may act as pathogens. In the present study, phylogenetic analysis of ribosomal RNA and ompA genes, as well as multi-locus sequence analysis of 11 field isolates were conducted. All independent analyses assigned the strains into two different clades of monophyletic origin corresponding to pigeon and psittacine strains or poultry isolates, respectively. Comparative genome analysis involving the type strains of currently accepted Chlamydiaceae species and the designated type strains representing the two new clades confirmed that the latter could be classified into two different species as their average nucleotide identity (ANI) values were always below 94%, both with the closest relative species and between themselves.     

Cryopreservation of sperm of an indigenous endangered fish species Nandus nandus (Hamilton, 1822) for ex-situ conservation

This study dealt with the development of cryopreservation protocol for Nandus nandus, which entailed a number of experiments. Sperm was collected by sacrificing males. The collected sperm was suspended in extenders. Activation of sperm motility was evaluated in different osmolalities of NaCl. Motility of sperm decreased as the osmolality of the extender increased and was completely inhibited at almost 319 mOsmol/kg. To evaluate the toxicity of cryoprotectant, sperm was incubated with DMSO, methanol and ethanol at 5%, 10% and 15% concentrations, respectively, for 5–35 min. Five and ten percent of cryoprotectants produced better motility during 5 and 10 min incubation. Sperm incubated with 15% cryoprotectant seemed to be toxic and this concentration was excluded in the subsequent trials. Three extenders, namely, Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were employed to preserve the sperm. Alsever’s solution with 10% DMSO showed best performance producing 90.0 ± 1.8% and 75.0 ± 2.5% equilibration and post-thaw motility followed by that of 82.5 ± 4.2% and 62.5 ± 5.5% with Alsever’s solution plus methanol, respectively. Between two diluents, sperm preserved with Alsever’s solution plus DMSO produced highest fertilization (76.7 ± 3.3%) and hatching (43.8 ± 7.9%) while fresh sperm yielded 83.3 ± 6.7% and 64.0 ± 10.4% fertilization and hatching, respectively. The protocol developed through the study can be applied for long-term conservation of genetic materials of the endangered fish N. nandus and the cryopreserved sperm can be used in artificial breeding for generating new individuals.     

Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria

Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.     

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.