Brian Harvey

Cryopreservation of Sarotherodon mossambicus spermatozoa

Abstract A method for the cryopreservation of spermatozoa from Sarotherodon mossambicus is described in which 5% methanol and 15% milk powder served as cryoprotectants. Postthaw motility is roughly double that obtained by substituting 5% or 10% DMSO for methanol. Data detailing cryoprotectant concentration optima, cooling and warming rate optima, the effect of dilution and of equilibration time are presented. In fertility tests, cryopreserved sperm yielded 64.3 ± 34.2% of embryos developing to the stage of blastopore closure, compared with 57.5 ± 10.5% for control, unfrozen milt.

Permeability of intact and dechorionated zebra fish embryos to glycerol and dimethyl sulfoxide

Intact developing embryos of the zebra fish Brachydanio rerio were exposed to [14C]DMSO and [3H]glycerol (1 M in Fish Ringer) to assess the degree of permeation of these cryoprotectants. Glycerol enters the embryo more easily, although reaching only about 8% of the expected equilibrium level after 2 hr at room temperature; DMSO reaches only about 2.5% of this level. In an attempt to identify the barrier to permeation, dechorionated embryos were similarly exposed to isotopic DMSO. Permeation increased severalfold, indicating that the chorion retards the free exchange of solute. Embryos are unaffected by exposure to 1 M DMSO in Fish Ringer at 23 degrees C for up to 1 hr. The number of embryos hatching after 1-hr exposure to DMSO at varying concentrations was significantly reduced at 1.5 and 2 M. Embryos exposed to 1 M glycerol for 1 hr at 23 degrees C showed disruption of periblast cells and separation of the blastoderm; it was impossible to remove glycerol either by abrupt or very slow dilution.

Cryoprotectant penetration and supercooling in the eggs of salmonid fishes

Abstract 1. 1. The freezing point of isolated, unfertilized eggs of rainbow trout ( Salmo gairdneri ) is −1.7 °C. 2. 2. Experiments using radiolabeled cryoprotectants show that the degree of permeation of methanol, dimethyl sulfoxide, and glycerol is inversely proportional to the molecular weights of the compounds; glycerol does not penetrate the egg, while methanol, which penetrates with the greatest rapidity, achieves no more than 23% of the expected equilibrium concentration after 2 hr exposure at 0 °C. 3. 3. Eggs treated with 10% DMSO and 10% sucrose in Fish Ringer resist intracellular freezing longer when cooled at 0.01 °C/min than do those in Fish Ringer alone; intracellular freezing is, however, inevitable with both cryoprotectants, although eggs that have remained unfrozen for several hours appear viable upon slow rewarming. These results make it likely that earlier studies reporting survival of “cryopreserved eggs” have in fact been dealing with supercooled eggs. 4. 4. Scanning electron micrographs of cooled and frozen DMSO-treated eggs show a progressive deterioration of the outermost layer of the zona radiata following cooling and freezing, and suggest that intracellular freezing is occurring following nucleation by way of pore canals in the zona that are exposed as cooling progresses.

Induced spawning of sea bass, Lates calcarifer, and rabbitfish, Siganus guttatus, after implantation of pelleted LHRH analogue

Abstract Captive Lates calcarifer broodstock at Tigbauan, Iloilo (Philippines) were implanted with cholesterol-based pellets of the LHRH analogue D-Trp6-desGly10-LHRH ethylamide or D-hArg(Et2)6,Pro9-NHet-LHRH at doses between 9.0 and 23.5 μg/kg body weight. In May, one of ten LHRH-treated females released partially hydrated ova into the tank 4 days after implantation. In July, at least one (and probably four) of five LHRH-treated females spawned in the tank 2 days after implantation; 2.6 million hatchlings were collected. In August, both LHRH-treated females spawned in the tank 2 days after implantation; 978 000 hatchlings were collected. None of the sham-operated control fish spawned in any of the experiments. Captive Siganus guttatus broodstock implanted with silastic-based pellets of the LHRH analogue D-Nal (2)6 LHRH spawned 1–2 days earlier than sham-operated controls.

Cooling of embryonic cells, isolated blastoderms, and intact embryos of the zebra fish Brachydanio rerio to −196°C

Single cells from the developing embryo of the zebra fish survive freezing when protected with 1 M DMSO and cooled to −196 °C in two steps. Cell survival drops from 85 to 26% when clumps of 5–10 cells are similarly frozen, and to 2% when isolated blastoderms are treated in the same way. This drastic decrease in survival is interpreted as an example of the “scale-up problem,” in which diffusional barriers prevent cryoprotectant equilibration and osmotic dehydration in large cell assemblanges. Isolated blastoderms develop considerably in culture, and retain some of this ability following cooling to −25 °C after protection with DMSO or glycerol. Intact embryos protected with high concentrations of glycerol (2.8 M) tolerate slow cooling to −196 °C surprisingly well, with most of the embryonic cells morphologically intact and actively extruding lobopodia. Glycerol could, however, only be removed from cells by disrupting the embryo so that diffusional barriers were removed. DMSO (2.8 M) was ineffective in preserving embryos or cells cooled to −196 °C.

Topical absorption of gonadotropin-releasing hormone (GnRH) in goldfish.

The studies reported here show that exogenous GnRH can be absorbed by goldfish from the intraperitoneal (ip) cavity, gill surface, or surrounding water. Mammalian rather than teleost GnRH was applied in order to ensure that GnRH measurement in plasma did not reflect the native form. A radioimmunoassay (RIA) specific to mammalian GnRH was used to measure the concentration of absorbed GnRH; validation for this approach was provided by HPLC and cross-reactivity studies in which mammalian GnRH was shown not to be present in control goldfish brain or pituitary extracts. Plasma concentration of GnRH was highest at the first sampling time, 4 min after administration, for all three routes. For intraperitoneal injection, plasma concentration was halved in 12 min, a period comparable with the half-life in rats. The pituitary content of GnRH also increased rapidly during the first 4 min after ip injection and remained high for 60 min. Absorption of GnRH from the gill was equally effective with water or dimethyl sulfoxide (DMSO) as vehicle.

Chilled storage of Sarotherodon mossambicus milt

Abstract Post-activation motility of undiluted milt of Sarotherodon mossambicus stored at 5°C declined to zero within 60–120 h. Dilution (1:1) of milt with a simple egg yolkcitrate diluent prolonged post-activation motility for up to 18 days. Fertility of milt stored in this diluent was 16% after 18 days; addition of glucose, glycine and sucrose to the medium raised fertility to 54%. At the longest storage period tested (21 days) milt was 21% fertile. Determination of the duration of spermatozoan motility in mannitol solutions of varying osmolalites indicates that little movement occurs at osmotic pressures > 300 mOs kg −1 . Data obtained with milt stored at varying osmolalities suggest that raising the osmolality to 373 mOs kg −1 by adding sucrose to the diluent has a beneficial effect on milt storage. Milt stored at 415 mOs kg −1 was 71% fertile after 18 days at 5° C. The effects of oxygenation, container configuration and antibiotics on spermatozoan storage are also described.

Induced spawning of maturing milkfish (Chanos chanos Forsskal) with gonadotropin-releasing hormone (GnRH) analogues administered in various ways☆

Abstract The response of mature female captive milkfish to mammalian and salmon gonadotropin-releasing hormone analogues (mGnRH-A and sGnRH-A) was investigated. Prior to spawning, six groups of three females received (1) 10–16 μg mGnRH-A from an osmotic pump implanted intraperitoneally (IP); (2) 100 μg mGnRH-A from a cholesterol/cellulose pellet implanted IP; (3) 10 μg/kg mGnRH-A as an intramuscular (IM) injection; (4) 10–16 μg sGnRH-A from an osmotic pump implanted IP; (5) 100 μg sGnRH-A from a cholesterol/cellulose pellet implanted IP, and (6) a cholesterol/cellulose pellet without analogue implanted IP. The most effective treatment was 100 μg sGnRH-A/fish given in a cholesterol/cellulose pellet; all ( 3 3 ) of the fish spawned. However, mGnRH-A was more effective ( 2 3 ) compared with sGnRH-A ( 1 3 ) if osmotic pumps were used to administer GnRH-A. If the dose and method of administration were not considered, then the salmon and mammalian GnRH analogues were equally effective (62–67%) for induction of ovulation and natural spawning in milkfish. Gonads of control fish regressed. At the doses tested, injections or pellet implantations were more effective compared with osmotic pumps. All pellet-implanted and injected females responded to treatment and 75% ( 6 8 ) spawned; half ( 3 6 ) of the pump-implanted females spawned. Spawning occurred from 18 to 36 h after treatment.

Short-term storage of Sarotherodon mossambicus ova

Abstract Unfertilized ova stripped from Sarotherodon mossambicus were stored in coelomic fluid in closed humidified containers. Gram-negative bacteria proliferating after 19 h were inhibited by 200 μg ml −1 kanamycin sulfate. Post-storage fertility declined after 1 1 2 h exposure to temperatures below 18–20°C; the optimal temperature for storage for 19 h was 20°C and produced 35% fertility. Post-storage fertility was increased to 55% by oxygenation of sample containers.