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.     

Water balance in eggs of striped bass (Morone saxatilis)

Measurements of embryonic volume revealed regulation capability before hatch. Water diffusion permeability, determined as tritiated water efflux, was found to be low, but within the range for fish eggs. The perivitelline fluid is readily interchangeable with the external medium due to a high chorion permeability to both water and salt.     

Effects of hypothermic storage on intracellular calcium, reactive oxygen species formation, mitochondrial function, motility, and plasma membrane integrity in striped bass (Morone saxatilis) sperm

Experiments were conducted to determine the effect of hypothermic 24 h storage on striped bass sperm cell plasma membrane integrity, free intracellular Ca²⁺ ([Ca²⁺]_i), mitochondrial membrane potential (ΔΨ_m), and reactive oxygen species (ROS) formation (oxidation of hydroethidine to ethidium) as determined by flow cytometry; motion activation and ATP concentration as determined by Luciferin-Luciferase bioluminescence assay. Semen was stored for 1 or 24 h at 4 °C in an O₂ atmosphere undiluted or diluted (one volume semen with 3 volumes diluent) with T350 (20 mM TRIS base-NaCl, 350 mOsm/mL, pH 8) or with seminal plasma in the presence of various treatments. Viability (% cells excluding propidium iodide) approached 100% after 1 h storage in undiluted or diluted semen. After 1 h of storage the [Ca²⁺]_i marker, Fluo-3, was detected in only 3% of sperm cells in undiluted or diluted semen. In contrast to storage for 1 h, after 24 h the incidence Fluo-3 fluorescence intensity was increased (P < 0.05) in > 50% of the viable cells in undiluted and diluted semen along with increased cell death; the presence of 1 mM ethylene glycol tetraacetic acid (EGTA) blocked CaCl₂-induced Fluo-3 fluorescence and cell death. Activation of sperm motility was 82% after 1 h in T350 and decreased (P < 0.05) to 30% after 24 h. However, motility activation failed in the presence of EGTA at 1 or 24 h. During storage ΔΨ_m was not affected by storage time or treatment. In contrast, sperm ATP was greater (P < 0.05) at 1 h than at 24 h and was greater in sperm stored in diluted than undiluted form. While ROS formation was induced by menadione treatment, there was no evidence of storage-induced ROS formation in the absence of menadione. The increased [Ca²⁺]_i found after 24 h indicates a storage induced defect in the maintenance of cellular calcium homeostasis which may be detrimental to sperm activation.     

Isolation and Characterization of Microsatellite Loci for Striped Bass (Morone saxatilis)

Genetic variation has been difficult to detect in striped bass (Morone saxatilis). Therefore, we identified and characterized 13 microsatellite loci to provide additional genetic markers for striped bass. Microsatellites were identified by screening a striped bass genomic library or by using primers developed for European sea bass (Dicentrarchus labrax) microsatellite loci. We found that 6 of the 13 microsatellite loci were polymorphic in DNA samples obtained from wild populations of striped bass. The number of alleles per locus varied from 3 to 12, and the observed heterozygosities ranged from 0.55 to 0.78. These results indicate that microsatellite loci provide more alleles and higher heterozygosities than other genetic markers developed for striped bass. Received November 9, 1999; accepted February 11, 2000.     

Estimation of growth and survival in size-structured cohort data: an application to larval striped bass (Morone saxatilis)

We present a method for estimating growth and mortality rates in size-structured population models. The methods are based on least-square fits to data using approximate models (using spline approximations) for the underlying partial differential equation population model. In a series of numerical tests, we compare our approach to an existing method (due to Hackney and Webb). As an example, we apply our techniques to experimental data from larval striped bass field studies.Research supported in part under grants at Brown University from the National Science Foundation: UINT-8521208, NSFDMS-8818530 (H.T.B., F.K. and CW.); from the Air Force Office of Scientific Research: AFOSR F49620-86-C-0111 (H.T.B., C.W.); and at University of California, Davis from the Alford P. Sloan Foundation (L.W.B.)     

Histopathology, blood chemistry, and physiological status of normal and moribund striped bass (Morone saxatilis) involved in summer mortality ('die-off') in the Sacramento-San Joaquin Delta of California

Summer mortality ('die-off') is common in striped bass, Morone saxatilis (Walbaum), in the San Francisco Bay-Delta region. Tissue and blood samples of moribund and healthy striped bass collected during the summers of 1986–1988 were analysed. Sixteen moribund and 25 healthy reference fish from the Carquinez Strait area and eight fish caught in the Pacific Ocean were studied. Moribund fish plasma was invariably yellow-orange; most of the moribund fish had discoloured livers with haemorrhagic regions, and approximately one-third had haemorrhagic intestines. Piasma levels of aspartale aminotransferase, uric acid, alkaline phosphatase and cortisol were significantly higher than in reference fish from Carquinez Strait and the Pacific Ocean, whereas cholesterol, sodium, chloride, triiodothyronine and glucose levels were significantly lower. Hepatic heavy metal concentrations and bacterial content were similar in moribund and reference fish. Gill Na.⁺, K⁺ -ATPase activity was significantly lower in moribund fish. Liver, kidney, intestine, and thyroid follicles of moribund fish displayed various histopathological changes, and corticosteroidogenic (interrenal) tissue could not be identified positively in moribund fish. These findings are discussed in relation to recent work on the chemical burdens (industrial and agricultural hydrocarbons) found in livers from some of the fish examined in this study.     

Dietary oligonucleotides from yeast RNA influence immune responses and resistance of hybrid striped bass (Morone chrysops x Morone saxatilis) to Streptococcus iniae infection

Three feeding trials were conducted to evaluate potential immunomodulatory effects of nucleotides in the diet of hybrid striped bass. A basal diet was formulated from menhaden fish meal to contain 40% crude protein and 10% lipid. An oligonucleotide product (Ascogen P) from brewer's yeast was added to the basal formulation at the manufacturer's recommended rate of 0.5% to produce the experimental diet. Each diet was fed to four replicate groups of juvenile hybrid striped bass for seven or eight weeks in two separate trials. After Trials 1 and 2, a Streptococcus iniae bath challenge was executed to test the effects of diet on disease resistance. No significant difference in growth performance was observed between fish fed the basal and experimental diets. Body composition of whole fish, hematocrit and serum lysozyme levels were observed to be within normal ranges and not influenced by dietary nucleotides. Neutrophil oxidative radical production of fish fed the nucleotide-supplemented diet was significantly (P=0.011) higher than in fish fed the basal diet. Significantly (P<0.05) enhanced survival after exposure to S. iniae also was generally observed in fish fed the nucleotide-supplemented diet. In addition, fish fed the nucleotide-supplemented diet tended to have a higher antibody response based on microtitration agglutination; however, the difference was not statistically significant because of high variation between individual fish. Long-term (16 weeks) administration of oligonucleotides in Trial 3 failed to show enhancement of immune responses between treatments. It is concluded that dietary oligonucleotides positively influenced immune responses and resistance of juvenile hybrid striped bass to S. iniae infection.     

Effects of osmolality on inner mitochondrial transmembrane potential and ATP content in spermatozoa recovered from the testes of striped bass (Morone saxatilis)

The objective was to determine the effects of osmolality on the energy status of testicular spermatozoa of striped bass incubated in a TRIS free base-NaCl medium (pH 8) adjusted to either 300 (T300) or 600 mOsm/kg (T600) with NaCl. High mitochondrial inner transmembrane potential (DeltaPsim) was assessed (flow cytometry) with the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolyl- carbocyanine iodide (JC-1) and ATP was measured with a luciferin-luciferase assay. Spermatozoa maintained on ice were equally viable (>95% for T300 and T600) for up to 80 min, whereas sperm viability in artificial fresh water (FW) at 27 mOsm/kg decreased (P<0.05) to 67% after 5 min, with only 3.5% viability at 25 min. After 20 min of staining, more spermatozoa (P<0.05) maintained a high DeltaPsim in T300 than in T600 (80 and 50%, respectively). Sperm JC-1 aggregate (Jagg) fluorescence intensity was also greater (P<0.05) in T300 than in T600 (10 and 5 channel number). The Jagg fluorescence was a function of oxidative phosphorylation; the percentage of cells containing Jagg fluorescence decreased to 3% in the presence of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), an uncoupler of cell respiration and oxidative phosphorylation. After incubation for 30 min in the absence of CCCP, sperm ATP concentration was greater (P<0.05) in T300 than in T600 (2.0 vs. 0.2 pmol/10(6) cells), but was below detectability in the presence of CCCP in either medium. In conclusion, we developed a unique approach to assess the energetic status of striped bass spermatozoa during storage and after activation, and concluded that the effects of osmolality must be considered in the design of activating and storage extenders to maintain striped bass sperm motility, viability, and fertility in vitro.     

Effects of sublethal concentrations of zinc and benzene on striped bass, Morone saxatilis (Walbaum), infected with larval Anisakis nematodes

Young of the year striped bass, Morone saxatilis , were exposed to sublethal concentrations of zinc and benzene and infected with larval Anisakis nematodes to determine the effects of these stressors on various physiological parameters. Results indicated that both pollutants and parasites significantly decreased haematocrit values. Parasites alone increased the liver somatic index, decreased haematocrits and, in the initial phase of the experiment, lowered antibody titres. Pollutants alone did not appear to significantly affect antibody titres, nor was there any difference in zinc and benzene uptake in the livers of infected and non-infected fish.     

Measurement of larval striped bass (Morone saxatilis) net avoidance using evasion radius estimation to improve estimates of abundance and mortality

Net avoidance rate increases as a function of larval striped bass size. This causes under-estimation of abundance and overestimation of mortality rate. We modeled net avoidance by assuming that fish avoid the net by swimming a radial distance at a right angle to the net axis. This distance, the evasion radius, was estimated by comparing the calculated densities of striped bass larvae from a series of paired tows involving a large and a small net. Iteration and solution models were used to estimate the evasion radius for each millimeter size group of fish in order to estimate the actual density in the environment. Avoidance of the nets increased with fish length. The ratio of actual density in the environment to the measured density in the small net was used to adjust abundances measured in our ichthyoplankton surveys. After adjusting for net avoidance, mortality rates of striped bass larvae from the Sacramento-San Joaquin Estuary were reduced by 10% compared to the unadjusted rates.      

Effect of glycerol and dimethyl sulfoxide on cryopreservation of rhesus monkey (Macaca mulatta) sperm

Glycerol and dimethyl sulfoxide (DMSO) are widely used as penetrating cryoprotectants in the freezing of sperm, and various concentrations are applied in different species and laboratories. The present study aimed to examine the effect of these two cryoprotectants at different concentrations (2%, 5%, 10%, and 15% glycerol or DMSO) on rhesus monkey sperm cryopreservation. The results showed that the highest recovery of post-thaw sperm motility, and plasma membrane and acrosome integrity was achieved when the sperm was frozen with 5% glycerol. Spermatozoa cryopreserved with 15% DMSO showed the lowest post-thaw sperm motility, and spermatozoa cryopreserved with 15% glycerol and 15% DMSO showed the lowest plasma membrane integrity among the eight groups. The results achieved with 5% glycerol were significantly better for all parameters than those obtained with 5% DMSO. The functional cryosurvival of sperm frozen with 5% glycerol was further assessed by in vitro fertilization (IVF). Overall, 85.7% of the oocytes were successfully fertilized, and 51.4% and 5.7% of the resulting zygotes developed into morulae and blastocysts, respectively. The results indicate that the type and concentration of the penetrating cryoprotectant used can greatly affect the survival of rhesus monkey sperm after it is frozen and thawed. The suitable glycerol level for rhesus monkey sperm freezing is 5%, and DMSO is not suitable for rhesus monkey sperm cryopreservation.     

Lipid and fatty acid composition of striped bass (Morone saxatilis) larvae during development

Changes in lipid class, fatty acid composition, protein, and dry and wet weights of fertilized eggs and developing larvae of striped bass (Morone saxatilis) fed with the live food, Artemia, were investigated. A decrease of wet and dry weights and moisture was observed at the beginning of the larval stage. Larvae regained the original moisture level, and wet and dry weights increased steadily after feeding. Total lipids decreased from 190 μg/egg in fertilized eggs to 151 μg/egg during hatching and increased after feeding. When total lipid contents were expressed as a percentage of larval dry weight, a decline of lipid did not occur until after feeding. Total protein, on the other hand, increased right after feeding, but there was some variation between days. Polar lipids increased significantly from 20 μg/egg at the egg stage to 199 μg/larva at 26 days post-hatching (DPH), 2 days before the onset of metamorphosis, while neutral lipids declined from 175 μg/egg to 80 μg/larva during the same time period. Wax/steryl esters decreased from 150 μg/egg in fertilized eggs to 32 μg/larva at 26 DPH. Triacylglycerols dropped from 21 μg/egg to 15 μg/larva before feeding and increased gradually after feeding. In contrast, the level of cholesterol increased 2–3-fold. There was a significant increase of phospholipids, particularly phosphatidylcholine in larvae after feeding. The fatty acid composition of fish larvae was significantly influenced by the diet, Artemia. There was an indication of catabolism of endogenous eicosapentaenoic and docosahexaenoic acids during metamorphosis.     

Immediate and delayed (after cooling) effects of centrifugation on equine sperm

The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25 × 10⁶ cells/mL, and subjected for 10 min to (1) no centrifugation (NC) or (2) centrifugation at 400 × g, (3) 900 × g, or (4) 4500 × g. Before and after centrifugation (Day 0), and after 24 h of cooling (Day 1), sperm motility was assessed by computer-assisted semen analysis, and samples were stained with SYBR-14/propidium iodide (PI) for viability and with PI/fluorescein isothiocyanate (FITC)-Peanut aglutinin (PNA) (Arachis hypogaea) for acrosomal integrity. The effect of treatment and day on motility, viability, and acrosomal integrity was determined using a mixed linear model. Compared with the other treatments, centrifugation at 4500 × g reduced all end points measured (P < 0.05). Both 400 × g and 900 × g yielded lower recovery rates than that of 4500 × g (NC = 100.0 ± 0.0%; 400 × g = 54.4 ± 8.6%; 900 × g = 75.0 ± 7.1%; 4500 × g = 97.9 ± 2.8%; P < 0.05). Centrifugation at 400 × g or 900 × g did not damage equine sperm. Based on these findings, further studies of centrifugal forces between 900 × g and 4500 × g are warranted to determine the optimal force that maximizes recovery rate, minimizes sperm damage, and does not affect fertility.     

Effects of frozen and liquid hypothermic storage and extender type on calcium homeostasis in relation to viability and ATP content in striped bass (Morone saxatilis) sperm

The effect of hypothermic storage on striped bass sperm calcium homeostasis was determined by Fluo-3 flow cytometry. Calcium homeostasis was defined as the ability of cells to maintain a low concentration of intracellular free calcium as measured by Fluo-3 fluorescence. Sperm were stored frozen in striped bass extender (SBE) and Tris–NaCl medium (T350) modified with 50 mM glycine and 7.5% dimethylsulfoxide and in nonfrozen form diluted 1:3 (vol/vol) in SBE and T350 for 1, 24, and 48 hours at 4 °C in an oxygen atmosphere. Fluo-3 fluorescence was detected in less than 5% of fresh viable sperm cells indicating maintenance of calcium homeostasis. In contrast to sperm in fresh semen, frozen-thawed and nonfrozen sperm cells lost to a considerable extent the ability to maintain low intracellular free calcium even in the absence of exogenous calcium; positive Fluo-3 fluorescence was found in 26% and 39% of thawed sperm frozen in SBE- and T350-based freezing diluents, respectively, and increased (P < 0.05) to 67% during nonfrozen storage in SBE and T350 at 24 and 48 hours. Sperm viability measured by exclusion of propidium iodide by flow cytometry was 99% in fresh milt and maintained at 86% (P > 0.05) in SBE after 48 hours of nonfrozen storage but decreased (P < 0.05) to 55.7% after 48 hours in T350. Energy status in terms of ATP content, determined by luciferin–luciferase bioluminescence assay, was higher (P < 0.05) in sperm frozen in SBE than in T350 during the first 5 minutes post-thaw and decreased to essentially zero by 15 minutes post-thaw and did not differ among nonfrozen storage treatments. In conclusion, sperm cells impervious to propidium iodide after frozen or nonfrozen storage were unable to maintain low intracellular calcium content. SBE is a better medium than T350 for frozen or nonfrozen storage of striped bass sperm. The inability to regulate intracellular calcium in striped bass sperm may be associated with poor activation of motility after 4 °C storage and cryopreservation.     

几种冷冻稀释液与单胺类防冻剂对中缅树鼩精子冷冻存活率的影响

该文实验精子采自昆明地区经笼养驯化的树鼩(Tupaia belangeri),检测其冷冻前后运动度、顶体完整率以及检测部分冷冻精子的受精能力。实验一:选用8种已报道的冷冻稀释液TTE、TCG、TCF、TTG、BWW、BTS、DM、SR稀释鲜精,并添加0.4mol/LDMSO,4℃预冷平衡2h后,TTE、DM和SR稀释液的精子的运动度与鲜精无差别(P>0.05),其余处理组均显著下降(P<0.05)。冷冻复苏后,各组的运动度显著低于预冷平衡处理后的运动度(P<0.05);DM组的复苏运动度显著高于其他稀释液组(P<0.05),BWW组最低(P<0.05)。对于顶体完整率,与鲜精相比,4℃平衡2h后,TTE和DM组精子的顶体完整率显著高于BWW、BTS和SR组(P<0.05)。冷冻复苏后,DM组精子的顶体完整率显著高于其它(除了TTE)冷冻组(P<0.05)。实验二:在DM稀释液基础上分别添加4种浓度0.2、0.4、0.8和1.2mol/L的二甲基甲酰胺(DF)、甲酰胺(F)、二甲乙酰胺(DA)和乙酰胺(A)以及0.4mol/LDMSO,经过预冷平衡处理后,与鲜精相比,各防冻剂组的精子运动度没有下降(P>0.05);冷冻解冻后,各冷冻组精子的运动度显著低于预冷平衡处理后的精子运动度(P<0.05);0.8mol/LDF和0.4mol/LDMSO组精子的运动度显著高于其它冷冻组(P<0.05)。对于顶体完整率,预冷平衡处理后各高浓度组的比率显著下降;冷冻复苏后,0.4mol/LF和0.4mol/LDF组精子的顶体完整率相对较高。实验三,人工授精实验中,DM+0.8mol/LDF冷冻精子的受精率为16.7%,DM+0.4mol/LDMSO的受精率为50.0%。以上实验结果提示,含卵黄的非离子冷冻稀释液对树鼩精子冷冻保护效果好,但单胺类防冻剂的防冻效果还需进一步深入研究。     

Evaluation of sperm functional attributes in relation to in vitro sperm-zona pellucida binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality

The objective of this study was to evaluate sperm functional attributes in relation to in vitro sperm-zona binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality. Frozen-thawed forty-eight ejaculates from eight Surti buffalo bulls (six ejaculates/bull) obtained by artificial vagina were used. Frozen semen from each bull was thawed, pooled, and subjected for sperm functional (six replicates) and in vitro fertilization (four replicates) tests. The progressive forward motility, plasmalemma functional integrity assessed by fluorogenic [6-carboxyfluorescein diacetate (CFDA), and propidium iodide (PI)], hypoosmotic swelling (HOS), and hypoosmotic swelling-Giemsa (HOS-G) test, mitochondrial membrane potential, sperm nuclear morphology, the number of sperm bound to zona and cleavage rate differed significantly (P < 0.05) between bulls. When the animals were grouped based on cleavage rate (group I, >40% cleavage rate, n = 5, and group II, <40% cleavage rate, n = 3), in vitro fertility parameters and all the sperm functional attributes except sperm nuclear morphology differed significantly (P < 0.05). The proportions of sperm with functional plasmalemma in the tail and intact acrosome assessed by HOS-G test (25.33, range: 17.48–40.27) were significantly (P < 0.001) lower than the functional plasmalemma in the tail assessed by HOS test (39.80, range: 27.85–54.67). The number of sperm bound to zona had significant correlations with the mitochondrial membrane potential (r = 0.90, P < 0.01) and plasmalemma integrity (fluorogenic, r = 0.74 and HOS, r = 0.79, P < 0.05) and HOS-G, r = 0.87, P < 0.01). The cleavage rate had significant (P < 0.05) correlations with the mitochondrial membrane potential (r = 0.70) and plasmalemma integrity measured by HOS-G test (r = 0.68). The present study indicates that these attributes could represent important determinants of buffalo sperm quality influencing cleavage rate.