Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton))

The aim of the present study was to investigate the spontaneous motility of spermatozoa and to optimize sperm collection, short-term sperm storage, and fertilization in zebrafish Danio rerio. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation then damped from the end of the flagellum for 35 s and fully disappeared at 61 s after activation. For artificial fertilization, milt must be added to an immobilizing solution, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 and Kurokura as isotonic solutions were shown to be suitable extenders to store sperm for fertilization for 6 h. E400 stored sperm for 12 h at 0–2 °C. Sperm motility decreased only to 36% at 12 h post stripping for the E400 extender and to 19% for the Kurokura extender. To achieve an optimal level of fertilization and swim-up larvae rates, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µL of activation solution has proven to be more successful than using a Petri dish. The highest fertilization and swim-up larvae rates reached 80% and 40–60%, respectively, with milt stored for 1.5 h in the E400 extender at 0–2 °C.

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242 EFFECTS OF TWO COMMERCIAL BOVINE SEMEN EXTENDERS FOR SHORT-TERM STORAGE ON MOTILITY PATTERN OF CHILLED BISON SEMEN

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab242 ◽

2011 ◽

Vol 23 (1) ◽

pp. 219

Author(s):

B. M. Toosi ◽

G. Gratton ◽

C. Lessard ◽

G. P. Adams

Keyword(s):

Sperm Motility ◽

Repeated Measures ◽

Test Tube ◽

Computer Assisted ◽

San Jose ◽

Short Term ◽

Development Fund ◽

Straight Line ◽

Wood Bison

Difficulties of adequate cryopreservation of bison semen has limited the success of artificial insemination and in-vitro embryo production in bison. We evaluated the effects of short-term cooling on motility of bison sperm using two commercial semen extenders (Triladyl® and Andromed®; Minitube, Ingersoll, ON, Canada). Semen was collected by electroejaculation of mature wood bison (n = 3) and plains bison (n = 3) twice a week for 2 wk. Upon collection, the ejaculate was divided into 3 equal aliquots, which were then diluted 1:2 (vol/vol) in Triladyl or Andromed, or were not extended (n = 24 samples per treatment). Samples were maintained at 37°C until transfer to the laboratory (≤2 h). One millilitre of each sample was then placed into a test tube (15 mL, BD Falcon, BD Biosciences, San Jose, CA, USA) and were kept in water bath set at 5°C inside a walk-in refrigerator (4°C). Characteristics of sperm motility were evaluated before cooling (Day 0) and every 24 h after cooling for 5 days using a computer-assisted semen analyzer. Total motility (TM), progressive motility (PM), velocity curved line (VCL), velocity average path (VAP), and velocity straight line (VSL) were compared among treatments by ANOVA for repeated-measures. Values are expressed as mean ± SEM. After collection, the PM of the raw semen and semen extended in Triladyl or Andromed were not significantly different (63.1 ± 4.4%, 63.3 ± 3.1%, and 56.9 ± 4.5%, respectively). Cooling semen for a period of 24 h resulted in a decrease (P < 0.05) in PM in all 3 groups (4.4 ± 2.0%, 22.7 ± 2.9%, and 28.7 ± 4.3%, respectively). The PM of semen extended in Tryladyl or Andromed was greater than that of raw semen on Day 1 (P < 0.05). Semen extended in Triladyl and Andromed maintained PM on Day 2 (24.7 ± 3.3% and 21.8 ± 3.8%, respectively), but PM declined progressively to 1.1 ± 0.6% and 6.3 ± 2.1% by Day 5. A similar pattern was observed for the TM. The VCL, VAP, and VSL parameters for semen extended with Triladyl and Andromed decreased gradually between Day 0 and Day 5 (P < 0.05). From Day 1 to 4 after cooling, these velocity parameters were not significantly different between semen extended with Triladyl or Andromed; however, they were greater than those of raw semen (P < 0.05). All sperm velocity parameters for the raw semen declined by more than 60% between Days 0 and 2 (P < 0.05). On Day 0, VCL for semen extended with Andromed (152.2 ± 4.3) was greater than that of semen extended with Triladyl and raw semen (P < 0.05; 122.5 ± 7.0 and 122.4 ± 6.6, respectively). The VCL then decreased to 98.9 ± 12.9, 100.5 ± 10.8, and 18.6 ± 6.8 in Andromed, Triladyl and raw groups respectively on Day 2 (P < 0.05), followed by a further decline to 51.8 ± 14.3, 19.9 ± 10.0, and 9.0 ± 5.0, respectively, observed on Day 5. In conclusion, both Triladyl and Andromed improved characteristics of sperm motility of chilled bison semen. Despite an initial decrease within the first 24 h, bison sperm extended with Triladyl or Andromed maintained an acceptable degree of motility for up to 2 days after chilling to 5°C. Supported by Agriculture and Agri-Food Canada, Agriculture and Development Fund, and Canadian Animal Genetic Resources program.

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The impact of the short-term human sperm storage in the cryoprotectant-free medium on sperm motility and vitality

Fertility and Sterility ◽

10.1016/j.fertnstert.2019.07.432 ◽

2019 ◽

Vol 112 (3) ◽

pp. e119

Author(s):

Nabil Sayme ◽

Marija Kljajic ◽

Thomas Krebs ◽

Dieter Maas

Keyword(s):

Sperm Motility ◽

Human Sperm ◽

Sperm Storage ◽

Free Medium ◽

Short Term ◽

The Impact

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Artificial fertilization of oocytes and sperm activation in pacu: effects of the spermatozoa:oocyte ratio, water volume, and in natura semen preservation

Revista Brasileira de Zootecnia ◽

10.1590/s1516-35982011000100001 ◽

2011 ◽

Vol 40 (1) ◽

pp. 1-6 ◽

Cited By ~ 19

Author(s):

Eduardo Antônio Sanches ◽

Diego Mendes Baggio ◽

Pitágoras Augusto Piana ◽

Bruno Estevão de Souza ◽

Robie Allan Bombardelli

Keyword(s):

Sperm Motility ◽

Water Volume ◽

Survival Ratio ◽

Sperm Activation ◽

Fertilization Rates ◽

Effect Of Water ◽

Semen Preservation ◽

Artificial Fertilization ◽

Sperm Survival

The objective of this work was to investigate artificial fertilization and the duration of sperm motility in pacu with different insemination doses, water volume, and in natura semen preservation. It was carried out four experimentsfor evaluation of insemination doses (7x10³, 7x10(4), 7x10(5), 7x10(6), and 7x10(7) spermatozoa oocytes-1) on the artificial fertilization of oocytes; the effect of water volume (0.5, 15.0, 30.0, 45.0, and 60.0 mL water mL-1 of oocyte) with insemination doses of 105,481 and 210,963 spermatozoa oocytes-1; the effect of semen dilutions (0.005, 0.05, 0.5, and 5.0 µL semen mL-1 of water) on sperm motility duration; and the effect of storage at 15ºC for 9h on sperm motility duration and sperm survival ratio. The highest results obtained were: insemination doses from 7x10³ to 7x10(7) spermatozoa oocytes-1; from 15 to 60mL water mL-1 of oocytes; semen dilution of 0.005 µL semen/mL water and 98.65% sperm survival until 2h45min 36s preservation time. Preservation at 15ºC for 9h does not influence sperm motility duration. The highest fertilization rates can be observed by using 0.27 to 270 µL semen mL-1 of oocytes with 15 at 60 mL water for activation.

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Morphological and Immunohistochemical Modifications in Zebrafish (Danio rerio) Gills After Short-Term Exposure to the Fungicide Tebuconazole

Zebrafish ◽

10.1089/zeb.2018.1638 ◽

2019 ◽

Vol 16 (1) ◽

pp. 65-76 ◽

Cited By ~ 4

Author(s):

Rachele Macirella ◽

Manuela Tripepi ◽

Elvira Brunelli

Keyword(s):

Danio Rerio ◽

Short Term ◽

Short Term Exposure

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Landlocked Fall Chinook Salmon Sperm Motility after Short Term Milt Storage

International Journal of Innovative Studies in Aquatic Biology and Fisheries ◽

10.20431/2454-7670.0301002 ◽

2017 ◽

Vol 3 (1) ◽

Keyword(s):

Sperm Motility ◽

Chinook Salmon ◽

Short Term

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Phosphofructokinase activity and acidosis during short-term tetanic contractions

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y91-046 ◽

1991 ◽

Vol 69 (2) ◽

pp. 298-304 ◽

Cited By ~ 16

Author(s):

Lawrence L. Spriet

Keyword(s):

Glycolytic Enzyme ◽

Test Tube ◽

Mammalian Skeletal Muscle ◽

Short Term ◽

Short Supply ◽

In Vivo Experiments ◽

Anaerobic Energy

Anaerobic energy production is essential for the production of muscular tension when the demand for energy is greater than can be provided aerobically and when oxygen is in short supply. The largest source of anaerobic energy is from the glycolytic pathway. With sustained tetanic contractions, muscle glycolytic activity is high and hydrogen ions (H+) accumulate while tension production decreases. The increasing [H+] and decreasing tension led to the suggestion that H+ inhibits the activity of the regulatory glycolytic enzyme phosphofructokinase (PFK). Early in vitro work confirmed the H+ sensitivity of PFK in the test tube, indicating that little PFK activity should persist at a pH of 6.9–7.0. However, in situ and in vivo experiments suggested that significant PFK activity was maintained during intense contractions when muscle pH decreased to 6.4–6.6. There are several concerns associated with the application of in vitro findings to in vivo exercise situations: (i) there is little in vitro work in mammalian skeletal muscle with substrate and modulator concentrations representative of exercise, (ii) most in vitro analyses of PFK activity are performed following the dilution of the enzyme in mediums with low protein concentration, and (iii) do the modulators identified in vitro exist in high enough in vivo concentrations at rest and during exercise to contribute to the regulation of PFK? More recent in vitro and in situ PFK experiments have overcome some of these concerns. They confirm that during intense, short-term tetanic contractions, PFK activity is well matched to the ATP demand despite decreases in pH to ~6.4–6.5. A combination of decreased inhibitor (ATP) and increased substrate (fructose 6-phosphate) contents coupled with increases in the contents of several positive modulators may be responsible for the maintained PFK activity. This combination reduces the pH-dependent ATP inhibition of PFK and extends the physiological pH range of the enzyme to the range normally measured during this type of muscular activity.Key words: glycolysis, phosphofructokinase, anaerobic metabolism, acidosis.

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Seminal and spermatic characteristics of fresh semen and the effects of sperm cooling in Steindachneridion melanodermatum (Garavello, 2005)

Semina Ciências Agrárias ◽

10.5433/1679-0359.2015v36n6supl2p4493 ◽

2015 ◽

Vol 36 (6Supl2) ◽

pp. 4493

Author(s):

Ronan Maciel Marcos ◽

Giovano Neumann ◽

Cesar Pereira Rebechi de Toledo ◽

João Marcos Sena ◽

Gilmar Baumgartner ◽

...

Keyword(s):

Sperm Motility ◽

Storage Temperature ◽

Sperm Concentration ◽

Plasma Osmolality ◽

Storage Period ◽

Sperm Velocity ◽

Powdered Milk ◽

Sperm Activation ◽

And Storage ◽

Fresh Semen

<p>This study describes the seminal and spermatic characteristics of fresh semen of <em>Steindachneridion melanodermatum </em>and investigates the effects of dilution, temperature, and storage period on its spermatic parameters. Sperm samples were collected from nine hormonally-induced males. The following parameters in fresh sperm were analyzed: seminal plasma osmolality (OSM), seminal pH, sperm motility (MOT), sperm velocity (SV) (including sperm curvilinear velocity (CVV), sperm straight-line velocity (SLV), and sperm average path velocity (APV)), total time of sperm motility (TEMP), sperm concentration (CONC), and index of sperm normality (NORM). Sperm samples from each male were diluted in a solution containing 5% fructose and 5% powdered milk, and stored at 10°C and 25°C. The same was carried out for sperm samples not subjected to dilution. From these samples, MOT, CVV, SLV, APV, SV, and TEMP were measured after 0 h, 5 h, 9 h, 18 h, 27 h, 36 h, 45 h, and 54 h. Males released 11.74 ± 5.38 mL of sperm, with an osmolality of 258.78 ± 29.36 mOsm.kg-1 and pH of 7.11 ± 0.31. The sperm presented a MOT of 99.86 ± 0.31% at a concentration of 1.03 × 1010 ± 3.65 × 109 spermatozoa.mL-1 with CVV of 185.58 ± 14.11 ?m.s-1, SLV of 49.15 ± 4.66 ?m.s-1, APV of 87.02 ± 4.13 ?m.s-1, SV of 106.52 ± 4.45 ?m.s-1, TEMP of 79.31 ± 5.62 s, NORM of 75.81 ± 5.71%. The results indicate that sperm motility, sperm velocity, and total time of sperm activation were affected by dilution, storage temperature, and storage period (p < 0.05). Procedures for semen storage should be performed with undiluted sperm cooled at 10°C, or kept undiluted at 25°C for up to 27 h.</p>

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77 ADDITION OF CHOLESTEROL IN FRESH GOAT SPERM IMPROVES CRYOSURVIVAL RATES

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab77 ◽

2013 ◽

Vol 25 (1) ◽

pp. 186

Author(s):

B. G. Silva ◽

E. A. Moraes ◽

C. S. Oliveira ◽

W. D. Ferrari Junior ◽

W. C. G. Matos ◽

...

Keyword(s):

Liquid Nitrogen ◽

Sperm Motility ◽

Phase Contrast ◽

Room Temperature ◽

Petri Dish ◽

Egg Yolk ◽

Irreversible Damage ◽

Hypoosmotic Swelling Test ◽

Contrast Microscopy ◽

Working Solution

Cryopreservation causes irreversible damage to goat sperm membranes, measured by a loss of motile and functional normal cells, compared with fresh sperm. The objective of this study was to determine if the addition of cholesterol-loaded cyclodextrin (CLC) to goat semen improved sperm cryosurvival. The CLC was prepared as described by Purdy and Graham (2004 Cryobiology 48, 36–45) with some modifications: 200 mg of cholesterol were dissolved in 1 mL of chloroform and 1 g of methyl-beta-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents allowed to evaporate on a warm plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C. Thirty ejaculates from 5 bucks were collected, diluted 1 : 1 in Tris diluent, divided into 7 equal aliquots, and centrifuged at 800g for 10 min. The sperm pellets were resuspended in Tris diluent, to which 0, 0.75, 1.5, 3.0, 4.5, 6.0, or 7.5 mg of CLC/120 million sperm were added. All treatments were incubated for 15 min at room temperature and then cooled to 4°C over 2 h. The samples were then diluted with Tris-egg-yolk diluent containing 2% glycerol, and the sperm were packaged into 0.5-mL straws, frozen in static liquid-nitrogen vapour for 20 min, and plunged into liquid nitrogen. Straws were thawed in 37°C water for 30 s, extended in Tris, and analyzed using optic microscopy. To test thermal resistance, after thawing, 0.5 mL of semen from each treatment were placed in 1.5-mL Eppendorf tubes in a water bath at 37°C for 3 h. At 0, 60, 120, and 180 min, subsamples were evaluated for sperm progressive motility. A hyposmotic test was also conducted by adding 10 µL of sperm to 2 mL of each solution and incubating them for 1 h/37°C. Sequentially, 20 µL of sperm was diluted in hypoosmotic solution (150 mOsm), and the samples were evaluated using phase-contrast microscopy. A total of 100 spermatozoa were counted in at least 5 different fields, and sperm tails were classified as either noncoiled or coiled. Data were analyzed using ANOVA, and treatment means were separated, using the SNK test at 5% probability. The sperm motility (50.4, 33.8, and 22.5%) was significantly higher for sperm treated with 0.75 mg of cholesterol after 0, 60, and 120 min of incubation after thawing, when compared with other treatments. No treatment differences in the hypoosmotic swelling test were observed. The addition of 0.75 mg of cholesterol to fresh goat semen improved sperm motility after cryopreservation for up to 3 h. Supported by FACEPE and CAPES.

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