Morphological development of the swim bladder in hatchery-reared striped trumpeter Latris lineata

This study examined swim bladder morphogenesis in three cohorts of striped trumpeter (Latris lineata), a euphysoclist species with physostomous larvae. The swim bladder was first discernible 1–2 days after hatching as an evagination on the dorsal surface of the incipient digestive tract. It comprised a cluster of mesenchymal cells surrounding an inner primordium of epithelial cells. At mouth opening in larvae of 5.3 mm standard length (SL), the swim bladder was noticeably enlarged. Histologically, the swim bladder lumen was dilated and liquid filled. The pneumatic duct was first seen during the dilation stage and the rete mirabile began forming among the connective tissue surrounding the swim bladder. Initial swim bladder inflation occurred on day 11 post‐hatching in Cohort 1, at 14°C, and day 9 post‐hatching, in Cohorts 2 and 3, at 16°C. Histologically, the lumens of inflated swim bladders were ellipsoid and the epithelium was squamous, except for cuboidal gas gland cells at the anterio‐ventral and anterio‐lateral regions of the swim bladder. During the initial inflation interval the pneumatic duct was dilated in larvae both with and without swim bladder inflation. The pneumatic duct began to regress in some larvae over 7.5 mm SL. The swim bladder of striped trumpeter was similar to larvae of other altricial perciform marine fish in respect to organ derivation, tissue differentiation, luminal dilation and initial gaseous inflation. However, variations, particularly the delay in initial swim bladder inflation until after the start of feeding, were observed that could be fundamental to problems encountered during larval rearing.     

Visual field of cultured striped trumpeter Latris lineata (Teleostei) larvae feeding on rotifer prey

The visual field of striped trumpeter Latris lineata larvae fed rotifer prey was determined from analysis of feeding behaviour in the horizontal plane. The visual field was forward and laterally directed, characterised by maximum reactive distances (distance at which the predator first detects and reacts to the prey) of 5.07 mm and 5.25 mm on days 13 and 17 post-hatching, respectively, 97% of mean larval length. This confirmed the predicted horizontal visual field, forward and laterally directed, derived from higher cone cell densities in the dorso-temporal and medial regions of the retina compared with ventral regions. The visual field of prey detection expanded laterally with ontogeny as a wider range of reactive angles was used by 17 day-old than 13 day-old larvae. Larvae displayed a saltatory searching pattern, periodically stopping to scan for prey throughout the visual field, and exhibited a side-to-side movement of the head as they approached and stopped, prior to striking at a detected prey item. Larvae on day 17 post-hatching terminated 35% of feeding sequences at the pre-strike position, at a mean distance from prey of 0.58 mm.     

Estimating gonadal development using a non-destructive measurement of ovarian length in live Tasmanian striped trumpeter, Latris lineata (Forster, 1801)

Sexually mature female striped trumpeter Latris lineata (Forster, 1801) were sampled monthly for two spawning seasons until the start of gonadal recrudescence, and then fortnightly until ovulations ceased. Oocyte size and ovarian length, measured by inserting a semi-rigid biopsy catheter to the full extent of insertion, were recorded at each sample time. Ovarian length was expressed as proportion of fork length to provide a gonad index (GI). In non-ovulating females, there was little change in GI throughout the year. However, in ovulating females, GI increased from 18.3 five months before the first spawning season to 27.6 at the peak of the season in October, decreasing to 19.1 the following May and then increasing again to a maximum of 31.1 the following October, in concert with annual changes in reproductive condition. There was a positive linear correlation between GI and oocyte size during the period of oocyte growth ( r  = 0.75, n = 302). Based on the range of GI values for each stage in oocyte development (primary, cortical alveoli, vitellogenic, maturing and hydrated), GI was 90% accurate at assessing fish as pre-vitellogenic and 83% accurate at assessing fish as undergoing final oocyte maturation. This study demonstrated that measurement of GI by catheterization provides a rapid and non-destructive method for assessing maturational status of striped trumpeter broodstock.     

Effects of glycine and alanine on short-term storage and cryopreservation of striped bass (Morone saxatilis) spermatozoa

Three experiments were designed to examine the effects of the amino acids glycine and alanine on short-term storage and cryopreservation of striped bass spermatozoa. In the first experiment, the effect of glycine on post-equilibration motility was evaluated. In the presence of 2.5 or 5.0% Me(2)SO, glycine treatments (25, 50, and 75 mM) yielded higher (P<0.05) post-equilibration motility at all equilibration times examined compared to the control. There was no difference (P>0.05) among these three glycine treatments. In the second experiment, glycine and alanine at concentrations of 25, 50, 75, or 100mM were evaluated for post-thaw motility in the presence of 2.5 or 5% Me(2)SO. When compared to the control, both the glycine and alanine treatments showed positive effects on post-thaw motility at all concentrations tested. The highest (P<0.05) post-thaw motility was achieved with 50mM glycine or 75 mM alanine using 5% Me(2)SO. No interaction (P>0.05) between Me(2)SO and glycine or alanine was observed, indicating that the effect of glycine or alanine was independent of the concentrations of Me(2)SO. In the third experiment, glycine was evaluated for sperm motility, after short-term refrigerated storage and after cryopreservation of the same refrigerated semen. Sperm motility decreased after 24h of refrigerated storage in 50mM glycine treatment and the control, when compared to fresh sperm motility. However, 50mM glycine treatment yielded higher (P<0.01) sperm motility after both 24 and 48 h of storage as well as higher (P<0.01) post-thaw motility when compared to the control. An average of 30+/-2.9% and 16+/-2.4% post-thaw motility was achieved with the 50mM glycine treatment after 24 and 48 h of refrigerated semen, respectively.     

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo)

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation.     

Effect of alpha-lipoic acid on oxidative stress,viability and motility in the common carp (Cyprinus carpio) spermatozoa after short-term storage and cryopreservation

As known for different metabolic functions, α-lipoic acid (ALA) has been tested for spermatozoa preservation of animals as well as of human, but not for fish spermatozoa. The present study determined the effects of ALA on short and long-term (cryopreservation) preservation of common carp (Cyprinus carpio) spermatozoa, for the first time. For that, spermatozoa were diluted in extenders containing 0 (control), 0.025, 0.05, 0.1, 0.5, 1, 2, 5, and 10 mM of ALA concentrations in both short-term preservation and cryopreservation. Spermatozoa motility parameters by computer-assisted semen analysis, viability, lipid peroxidation and catalase activity in spermatozoa were conducted in both 2nd and 120th hours of short-term storage and post-thaw samples. Higher percentages of total spermatozoa motility (80 ± 3) and viability (87 ± 3) were observed in 0.5 mM ALA group after 120 h of incubation. In post-thaw samples, higher percentages of these parameters were in 1 mM ALA group (74 ± 3 and 83 ± 2, respectively). Moreover, the results have shown that the addition of ALA until concentrations of 2 mM improved especially spermatozoa curvilinear velocity, maintained viability, and suppressed excessive lipid peroxidation during the preservations. In conclusion, the additions of 0.5 mM ALA for short-term preservation and 1 mM ALA for cryopreservation were the optimal concentrations, and shown the protective effects on common carp spermatozoa, when considering all measured parameters together.     

Sperm chromatin structural integrity in normospermic boars is not related to semen storage and fertility after routine AI

Standard semen parameters are limited in their capacity to distinguish subfertile boars and to assess storage influences on liquid preserved boar semen. The evaluation of sperm chromatin structural integrity could have potential as a diagnostic tool in AI practice. This study assessed whether the determination of sperm DNA integrity adds a useful diagnostic tool for selection of boar ejaculates in routine AI procedure and assessment of storage effects in diluted semen. Special emphasis was laid on the standard spermatological characterization of semen samples in parallel with the determination of the DNA fragmentation index (DFI) through the sperm chromatin structure assay (SCSA). Six hundred ninety two (692) ejaculates from 79 Piétrain boars in an AI center were analyzed for motility, morphology and DFI over a period of 24 weeks. 95.5% of the semen samples had a DFI < 5% with low distribution of variation for DFI due to boar and ejaculate (< 5%). 61.3% of ejaculates with DFI > 5% showed values below thresholds for sperm motility or morphology. Based on field data from 13,239 inseminations, fertility of boars with temporarily elevated DFI was not impaired (P > 0.05). 24 randomly selected diluted semen samples did not show a significant increase of DFI during 168 h storage (P > 0.05). In a further experiment, 42 diluted semen samples from 14 normospermic boars were assessed for motility, membrane integrity (PI, FITC-PNA) and SCSA parameters. Three single ejaculates showed an increase of DFI at 120 and 168 h storage time. This was accompanied by a pronounced loss of both motility and membrane integrity. In conclusion, the evaluation of sperm chromatin structural integrity by the SCSA has only limited value for identifying sperm deficiencies in normospermic fresh or stored boar semen. Temporarily elevated DFIs seem not to be indicative of subfertility in normospermic boars.     

Cell survival after cryopreservation of dissociated testicular cells from feline species

Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 μm and 20.5 ± 4 μm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.     

Effect of Bradykinin on Murrah buffalo (Bubalus bubalis) semen cryopreservation

Keeping in view the poor freezability of bubaline semen in conventionally used extenders, this study was conducted on three Murrah bulls to improve semen cryopreservation with the incorporation of Bradykinin (0.5, 1.0 and 2.0 ng ml(-1)) in routinely used egg yolk tris-glycerol (EYTG) extender. Bradykinin (2.0 ng ml(-1)) had significant (P<0.05) beneficial effect on live sperm % (81.6+/-1.8) and hypo osmotic swelling (HOS) % (63.0+/-1.3) as compared to their respective control values of 73.4+/-2.1 and 56.3+/-2.0 at 0 h post freezing. The post-thaw progressive sperm motility in semen samples diluted with EYTG containing 2.0 ng ml(-1) Bradykinin (65.5+/-1.4) was also significantly (P<0.01) higher than control (60.3+/-1.9) at 0 h post freezing. Thus incorporation of 2 ng ml(-1) Bradykinin in buffalo semen diluted in EYTG extender may be useful in improving the quality of cryopreserved bubaline semen.     

Changes in sperm quality and lipid composition during cryopreservation of boar semen

Egg yolks are commonly used in diluents in order to improve the freezability of semen. Two aspects of the role of lipids in boar semen freezability are reported in this article. The first one concerns the eventual exchanges of lipid components between the spermatozoa and the yolk-based diluent during cryopreservation. Two types of yolk have been considered as ingredients in diluents for cryopreservation: yolks with a standard fatty acid composition and yolks enriched in docosahexaenoic acid (DHA). The relation between lipid exchanges and the quality of fresh semen is considered. The other aspect concerns the possibility to enhance the freezability of boar spermatozoa by altering the plasma membranes under the influence of dietary fatty acids. Spermatozoa were damaged significantly by the cryopreservation cycle in all experiments. Spermatozoa with the best fresh quality had accumulated the largest quantity of lipids upon thawing. A general decrease in the proportion of polyunsaturated fatty acids was observed after thawing. The yolks enriched in n-3 fatty acids failed to improve the quality of sperm following cryopreservation. The proportion of DHA was significantly higher in spermatozoan phospholipids from thawed cells that had been in contact with n-3 yolks. A significant reduction in cholesterol was observed in spermatozoa after the cryopreservation cycle, which correlated with an increased number of acrosome-reacted cells and changes in the parameters of motility. The addition of 3% fish oil to the daily boar ration significantly increased the content of DHA (from 33 to 45% of the total fatty acids) in the spermatozoa. Ejaculate concentrations were significantly increased in the experimental group. DHA-enriched semen did not show improved freezability, at least not as assessed by in vitro parameters.     

Comparative semen cryopreservation in ferrets (Mustela putorius furo) and pregnancies after laparoscopic intrauterine insemination with frozen-thawed spermatozoa

A study was conducted to determine an optimum technique for semen cryopreservation and the biological competence of frozen-thawed ferret spermatozoa. Fifty-two fresh electroejaculates from 4 males were evaluated for sperm percentage motility, forward progressive motility, motility index (SMI) and acrosomal integrity. To determine the optimum temperature for maintaining sperm motility in vitro and the influence of glycerol on sperm motility, seminal aliquants were diluted in TEST diluent (containing either 0 or 4% glycerol) and maintained at 25 degrees or 37 degrees C. For cryopreservation, semen was diluted in each of 3 cryodiluents (TEST, PDV, BF5F), cooled for 30 min at 5 degrees C and pelleted on solid CO2 or frozen in 0.25 ml straws (20 degrees C/min to -100 degrees C). Following thawing, SMI and acrosomal integrity were determined. Ten females with maximum vulval swelling were given 90 i.u. human chorionic gonadotrophin and laparoscopically inseminated in utero with spermatozoa previously frozen using the optimum diluent and freeze-thaw method. The maintenance temperature of 25 degrees C was superior (P less than 0.05) to 37 degrees C for sustaining sperm motility, and glycerol did not influence (P greater than 0.05) motility for up to 11 h of culture. After thawing, motile spermatozoa were recovered in all treatment groups, but sperm motility and normal acrosomal ratings were highest using the PDV diluent, the pelleting method and thawing at 37 degrees C (P less than 0.05). Seven of the 10 ferrets (70%) inseminated with spermatozoa frozen by this approach became pregnant and produced 31 kits (mean litter size 4.4; range 1-9 kits).(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation

The influence of permeating and of non-permeating cryoprotectants on motility, eosin permeability (sperm cells viability test) and leaking lactate dehydrogenase activity of spermatozoa of the rainbow trout ( Oncorhynchus mykiss ) was investigated. Correlations between sperm motility rate, percentage of eosinimpermeable (viable) cells and LDH activities established the three parameters as indicators for damages to rainbow trout spermatozoa. High sperm motility rates, velocities, and numbers of linear swimming sperm cells, high numbers of eosinimpermeable spermatozoa and a low extracellular LDH activity were evaluated as positive quality characteristics of semen. With a buffered physiological saline solution as basic extender we found a mixture of 0.67 mol L (5%) dimethylsulfoxide (DMSO) and 0.13 mol/L (1%) glycerol to be the most effective cryoprotectant, followed by DMSO (0.67 mol/L [5%]-1.85 mol/L [15%]) and glycerol (0.65 mol/L [5%]-1.78 mol/L [15%]) and propanediol (1.24mol L [10%]-1.78 mol/L [15%]). Addition of hen egg yolk (7%, 20%) and of 15 mmol/L (0.5%) sucrose significantly increased the semen quality in comparison to the same extender without these additives. Bovine serum albumin (1.5%, 3%) had no influence on the investigated semen parameters.     

Preliminary study on the cryopreservation of tropical bagrid catfish (Mystus nemurus) spermatozoa; the effect of extender and cryoprotectant on the motility after short-term storage

The effects of different extenders, and cryoprotectants on the motility of tropical bagrid catfish (Mystus nemurus) spermatozoa were evaluated after short-term storage. Three extenders, physiological saline, Ringer or saline at three levels of sperm to extender dilutions (1:20, 1:30, or 1:40) and four cryoprotectants (DMSO, ethanol, glycerol or methanol) at three concentrations (5, 10, or 15%) were examined in two separate experiments. In the first experiment, milt was suspended in the respective extender at the three milt to extender dilution ratios in two sets of tubes. Extended milt in the first set of tubes was stored at -4 degrees C, and motility assessed after 24h, while the second set was kept at 23 degrees C and sperm motility was assessed immediately and at 30-min intervals thereafter. Ringer retained sperm motility better than the other extenders at all dilution levels at temperatures of 23 and -4 degrees C respectively. At 23 degrees C, the sperm motility was almost completely lost after 150 min except for those in Ringer at 1:20 dilution level which still had a motility of 18% (compared to those kept at -4 degrees C for 24, which had motility from 39 to 71%, regardless of extender). In the second experiment, various cryoprotectants were added to solutions of milt (that was diluted in Ringer at 1:20 ratio and cryopreserved in liquid nitrogen for 15 days). Sperm cryopreserved in 10% methanol had the highest motility (58%) compared with those in the other cryoprotectants at all concentrations.     

The in vitro quality of frozen-thawed Asian elephant (Elephas maximus) spermatozoa in semen supplemented with Equex STM paste and oxytocin during and after cryopreservation

The effects of Equex STM paste (Equex) and oxytocin (OT) on the in vitro quality of frozen-thawed Asian elephant sperm were investigated in the study. The viability of frozen-thawed sperm was significantly higher in the Equex-treated (1 and 2%) groups than in the control group. There were no differences in the examined sperm parameters among the control and OT-treated (0.05–5 IU) groups.     

Sperm cryopreservation of the critically endangered olive barb (Sarpunti) Puntiussarana (Hamilton, 1822)

The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntiussarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever’s solution prepared at 296 mOsmol kg⁻¹. Sperm were activated with distilled water (24 mOsmol kg⁻¹) to characterize motility. Maximum motility (90%) was observed within 15 s after activation, and sperm remained motile for 35 s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was ?287 mOsmol kg⁻¹. To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10 min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5 °C to −80 °C at 10 °C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7–15 days at −196 °C. The highest motility in thawed sperm 61 ± 8% (mean ± SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.     

Refrigerated storage and cryopreservation of black drum (Pogonias cromis) spermatozoa

Procedures were developed for the collection, refrigerated storage and cryopreservation of black drum spermatozoa. Sperm samples were collected by removing and slicing the testis, and suspending the spermatozoa in Hanks' balanced salt solution (HBSS) at 200 mOsm/kg. Threshold activation (10%) of black drum spermatozoa occurred at 370 mOsm/kg, and complete activation occurred at 580 mOsm/kg in HBSS. Sperm cells activated in artificial seawater had higher motility than those activated in HBSS at osmolalities from 350 to 500 mOsm/kg. Spermatozoa stored at 4 degrees C in HBSS or artificial seawater at osmolalities from 202 to 290 mOsm/kg retained motility longer than did those stored at other osmolalities Dilution rate had no effect on sperm storage time at 4 degrees C. Four chemicals were evaluated as cryoprotectants: dimethyl sulfoxide (DMSO), n,n-dimethyl acetamide (DMA), methanol, and glycerol. Glycerol and DMA at concentrations of 10% significantly reduced motility within 52 min. Spermatozoa were cryopreserved at 3 freezing rates (-27, -30, or -45 degrees C/min) in a nitrogen vapor shipping dewar or a computer-controlled freezer. Spermatozoa frozen using 10% DMSO had the highest post-thaw motility at a freezing rate of -27 or -30 degrees C/min. Spermatozoa frozen using 5% glycerol, 5% DMSO, or 10% DMSO had the highest post-thaw motility at a freezing rate of -45 degrees C/min.