Effects of induction current and other factors on large-scale electrofusion for pronuclear transplantation of mouse eggs

In this paper, we describe the procedure of large-scale and efficient electrofusion for pronuclear transplantation in mouse eggs and the tolerance of the eggs for electric stimulus, assessed in vitro and in vivo development. The fusion chamber was arranged in parallel by dielectrodes (30-mm length, 1-mm width, and 2-mm height), and 0.3 M mannitol in distilled water was used as a fusion solution. The agglutination cleavage of enucleated eggs with karyoplast was easily orientated in parallel with electrodes by alternating current between 100 and 500 kHz at 2 and 10 V/mm. Immediately after the orientation, a direct current of 150 V/mm was given for 200 μsec twice and repeated three times to induce fusion of the enucleated eggs with karyoplast. More than five eggs, at least, can be submitted to electrofusion at the same time. The eggs that were not fused were treated again in the same manner. The proportion of eggs fused with karyoplast was increased by preincubation in M16 medium prior to submitting them to the electrofusion. When the eggs were incubated for 60 min, 80% of them were fused with karyoplast by the first electric treatment; in contrast, only 19% of the eggs were fused if they were submitted to electrofusion directly. It was found that between the CD-1 and F1 strains there was a difference in tolerance of the eggs to electric stimulus and that this was depend on the nuclei but not on cytoplasm. The proportion of development to blastocyst in the eggs fused with the pronuclear karyoplast derived from F1 (75 and 71%) was twice that of the eggs fused with the pronuclei derived from CD-1 strain (25 and 37%). After transfer to recipients, live young were obtained from both the eggs fused with karyoplast following one or two electrofusion exposures.     

Effect of storage temperature and duration on viability of eggs of Helicoverpa armigera (Lepidoptera: Noctuidae)

The ability to store different insect stadia for prolonged periods provides considerable flexibility and ability to conduct experiments properly. Therefore, studies were undertaken to determine the effect of storage temperature and duration on viability of eggs of Helicoverpa armigera (Hübner). The percentage egg hatch and incubation period were significantly (P=0.01) influenced by egg age, storage temperature, and storage duration. Egg hatch ranged from 0.0 to 96.8% across temperatures and storage durations. None of the eggs hatched when stored at -20 and 0 degrees C. The regression model with the optimum Mallow Cp statistic for any of the identified linear and quadratic terms did not improve the precision of prediction in egg hatch beyond 67.0%. Forecasting of incubation period based on egg age, storage duration, and durationxtemperature was quite effective (R2=84.2%). Day degrees required for egg hatching decreased with an increase in temperature from 10 to 27 degrees C, and egg age from 0 to 3 days. The day degree requirements were highest for 0-day-old eggs at 10 degrees C, and lowest at 27 degrees C. Although the incubation period was higher, the hatchability was lower for 0- and 1-day-old eggs stored at constant 10 degrees C, these eggs can be stored for 10 days at 10 degrees C, with a hatchability of >75.0%. It was safer to store the H. armigera eggs for 10 days at 10 degrees C, which will hatch within 1.6 to 2.0 days after restoration at 27 degrees C with a hatchability of >75.0%. This information will be useful in planning and execution of experiments involving H. armigera on various aspects of research in entomology.     

The storage of cow eggs at room temperature and at low temperatures.

The survival and development of cow eggs in the rabbit oviduct after storage at room temperature and after cooling and storage at 0-7-5 degrees C was examined. In PBS medium at room temperature 88% of Day-5 and 85% of Day-3 eggs showed normal development, but in TCM 199, 71% of Day-5 and only 49% of Day-3 eggs showed normal development. Duration of storage (1 1/2-2 hr or 6 1/2-7 1/2 hr) and cleavage stage before storage had no appreciable effect on development. Some retardation of development occurred in Day-3 eggs after 96 hr in the rabbit oviduct when compared to Day-5 eggs after 48 hr. Cooling of Day-5 and Day-6 eggs to 0-7-5 degrees C resulted in degeneration of a large proportion of eggs. Of the factors examined, storage medium (PBS or PBS+20%FCS), storage time (2 min, 24 hr) and storage temperature (0, 2, 5 or 7-5 degrees C) had little effect, but slower cooling rates tended to improve survival of eggs although the differences were not significant. More morulae (greater than 32 cells) than 8-to 24-celled eggs developed normally.     

Preservation of rainbow trout (Oncorhynchus mykiss) eyed eggs using a perfluorochemical as an oxygen carrier

The objective of this study was to maintain the viability of chilled rainbow trout (Oncorhynchus mykiss) eyed eggs during storage using oxygenated perfluorochemical (PFC). Three trials were conducted using eggs at 161, 180 or 217 degree days (days from fertilization x incubation temperature in degrees C). A separate trial was conducted for 147 degree day eggs that were not at the eyed stage. For each trial, eggs were stored in a moisture-saturated atmosphere at 1 degrees C in PFC, water, and 1:1 combinations of PFC and PBS, PFC and 0.3 M glucose, PFC and mineral oil, or PFC and water. The PFC was oxygenated before each trial and all media were oxygenated at weekly intervals during the storage period. Eggs from each trial were also incubated without storage to provide Day 0 results. After 3 and 5 weeks of storage, eggs from each medium were incubated at 10 degrees C until hatch. Hatching percentage was expressed as a percentage of Day 0 results. The percentage of normal alevins that hatched was also determined. There were interactions (P < 0.01) between stage of development and treatment for hatching percentage after 3 and 5 weeks of storage. After 3 weeks of storage, eggs stored at 161, 180, or 217 degree days without PFC had hatching rates of 0-14.3% but eggs stored in any medium with PFC had hatching percentages from 75.1 to 106.4% of Day 0 values. After 5 weeks of storage, eggs stored at 161 degree days in PFC plus PBS or PFC plus water, and eggs stored at 217 degree days in PFC or PFC plus water, had higher (P < 0.05) hatching percentages than eggs stored in any of the other media. Eggs stored at 161 degree days for 5 weeks in PFC and water had a higher (P < 0.05) percentage of normal alevins hatching than eggs stored in PFC and PBS. Because of their early developmental stage, eggs stored at 147 degree days had low hatching percentages, except eggs stored for 3 weeks in PFC or PFC plus PBS. Chilling eyed eggs of rainbow trout to 1 degrees C and storing them in water with PFC as an oxygen carrier can preserve their viability for 5 weeks.     

Survival of parasite eggs upon storage in sludge

Destruction rates of parasite eggs in stored sludge were examined to help understand the fate of these agents of enteric diseases in sludge lagoons. Eggs from the roundworms, Ascaris spp., Toxocara spp., Trichuris spp., and the tapeworm, Hymenolepis spp., were treated with domestic sludges by aerobic or anaerobic processes. Sludge samples seeded with eggs were stored at 4 or 25 degrees C or in a container inserted into the ground to simulate lagoon conditions. The number of eggs recovered from the samples decreased with storage time. The viability and infectivity of eggs recovered were related to the storage temperature; i.e., the eggs stored at 4 degrees C remained viable longer than those stored at 25 degrees C. After 25 months at 4 degrees C, the Toxocara eggs and some Ascaris eggs remained both viable and infective, whereas most of these eggs stored at 25 degrees C were rendered nonviable after 10 to 16 months of storage in sludge. Although storage temperature was found to be the most important factor affecting the destruction and viability of these eggs, other factors, such as the type of sludge digestion, whether or not the eggs were digested along with the sludge or added later, storage in the soil versus sludge, pH, and egg species also exhibited some minor effects. These controlled laboratory studies suggest that lagooning of sludge can be an effective method for the elimination of parasite eggs, particularly in warmer geographical locations.     

Survival of parasite eggs upon storage in sludge.

Destruction rates of parasite eggs in stored sludge were examined to help understand the fate of these agents of enteric diseases in sludge lagoons. Eggs from the roundworms, Ascaris spp., Toxocara spp., Trichuris spp., and the tapeworm, Hymenolepis spp., were treated with domestic sludges by aerobic or anaerobic processes. Sludge samples seeded with eggs were stored at 4 or 25 degrees C or in a container inserted into the ground to simulate lagoon conditions. The number of eggs recovered from the samples decreased with storage time. The viability and infectivity of eggs recovered were related to the storage temperature; i.e., the eggs stored at 4 degrees C remained viable longer than those stored at 25 degrees C. After 25 months at 4 degrees C, the Toxocara eggs and some Ascaris eggs remained both viable and infective, whereas most of these eggs stored at 25 degrees C were rendered nonviable after 10 to 16 months of storage in sludge. Although storage temperature was found to be the most important factor affecting the destruction and viability of these eggs, other factors, such as the type of sludge digestion, whether or not the eggs were digested along with the sludge or added later, storage in the soil versus sludge, pH, and egg species also exhibited some minor effects. These controlled laboratory studies suggest that lagooning of sludge can be an effective method for the elimination of parasite eggs, particularly in warmer geographical locations.     

In vitro storage of unfertilized eggs of the Eurasian perch and its effect on egg viability rates and the occurrence of larval malformations

Ova ageing is the most important factor affecting fish egg quality after ovulation. Long-term storage of fish ova, using cryopreservation and vitrification techniques, has been unsuccessful to date. Instead, short-term in vitro ova storage has been used successfully and optimized in some cultured fish species. In vitro ova storage can drastically improve mass production of larvae and juveniles in the hatcheries by providing the possibility of the synchronous artificial fertilization for different females. To study how long unfertilized eggs of Eurasian perch (Perca fluviatilis L.) can retain their fertilizing ability after stripping, eggs were stored at temperatures of 4°C, 8°C and 12°C for 72 h post-stripping (HPS). The stored eggs of four female perch were separately fertilized at 0 h (i.e. control eggs fertilized before storage) and at 6-hour intervals during the experimental period of 72 h. The embryos reaching the eyed-egg and hatched-larvae stages, eyed-egg mortality and larval malformation rates were recorded as indices of egg quality. The results indicated that the maximum eyed eggs and hatched larvae (86% and 63%, respectively) were observed for eggs fertilized immediately after stripping, whereas the storage of the eggs at 4°C for 48 HPS decreased the eyed-egg and hatched-larvae rates to 46% and 17%, respectively. The use of a higher storage temperature resulted in a more rapid decrease in egg viability: eyed-egg and hatched-larvae rates of 23% and 9%, respectively, were obtained after 48 HPS storage at 8°C and 2% and 1% for eggs stored at 12°C. Eyed-egg mortality and larval malformation rates were not significantly affected by post-stripping ova ageing for at least up to 36 h. Thereafter, both values increased significantly and were measured to be the highest in the most aged ova. The present study demonstrated that stripped Eurasian perch eggs can be stored for at least 12 h at 4°C to 12°C without a significant reduction in their quality.     

Viability of nuclei of two-cell mouse embryos stored at 4 degrees C and fused with blastomeres of fresh two-cell embryos.

This study compares the resistance of the nuclei and the cytoplasm of two-cell mouse embryos to short-term storage at low temperature above 0 degrees C. Two-cell embryos were stored at 4 degrees C for 24-96 h in PB1 containing 0.25, 0.5, 0.75, and 1.0 M sucrose. The development to blastocysts in culture was highest in the presence of 0.5 M sucrose. However, only 3% of the embryos developed into blastocysts after 96 h of storage. On the other hand, the viability of the nuclei of two-cell embryos stored at 4 degrees C was significantly prolonged when they were transplanted into a blastomere of enucleated fresh F1 (C57BL/6JXCBA) two-cell embryos. The proportions of chimeric embryos that developed to blastocysts were 88, 67, 76, 71, 64, 45, 32, and 20% following storage for 0, 48, 72, 96, 120, 144, 168, and 192 h, respectively. In addition, there was no difference in the coat color of the young derived from nuclei stored at 4 degrees C or fresh nuclei, although the proportions of chimeric embryos that developed into live young after transfer tended to decrease with increased storage time. Moreover, the viability of nuclei stored at 4 degrees C for 192 h was confirmed in the germ cell population of chimeric mice mated with albino mice. These results demonstrated that the nuclei in the two-cell mouse embryos were more resistant to storage at low temperature than the cytoplasm.     

Electrofusion for the pronuclear transplantation of mouse eggs

The present study was undertaken to determine the efficiency of HVJ treatment and electrofusion for pronuclear transplantation in the mouse. The output voltage and duration of the pulses were fixed to 200 μsec at 10 V or to 150 μsec at 15 V for electrofusion, because the maximum rates of blastomere fusion of 2-cell embryos and development of fused embryos in vitro were obtained under these conditions. Although the proportion of eggs with fused karyoplast (78%) and the fused eggs developed to morulae or blastocysts (67%) was significantly lower than those obtained after HVJ treatment (94% and 94%), the proportion of pregnant recipients and young obtained after treatment of fused eggs was not significantly different between these two procedures. It is advised that electrofusion can be used as a fusogenic procedure for pronuclear transplantation in the mouse in some cases where HVJ cannot be applied.     

Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.     

Progeny quality of Gonatocerus ashmeadi (Hymenoptera: Mymaridae) reared on stored eggs of Homalodisca coagulata (Hemiptera: Cicadellidae)

This study assessed the effects of refrigerated storage on the suitability of eggs of the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae), as hosts for propagation of the parasitoid Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae). Development of the host eggs was terminated by chilling at 2 degrees C for 5 d before storage was initiated at 10 degresC for up to 70 d. Parasitism, adult emergence rate, developmental time, and sex ratio were used to gauge the suitability of the eggs as hosts after storage. In addition to these measures, demographic growth parameters also were used to assess the quality of the wasp progeny through the F2 generation. Host eggs stored 20 d remained fully acceptable to the wasps for attack. Although the parasitism rate decreased with storage time, > 80% adult parasitoid emergence was realized from eggs stored 30 d. After 70 d storage, adult emergence rate was decreased by 48%, fecundity decreased by 53%, female production by 19%, developmental time was extended 3 d, and female longevity was shortened 5 d. The emergence pattern of F1 but not F2 adults varied with storage time of the parental and grandparental hosts, respectively. For the F1 generation, emergence rate, development, and sex ratio did not vary with storage time when the F1 parents parasitized fresh host eggs. Demographic parameters for the F, population showed that net reproductive rate was > 20 although it decreased significantly after their parental host eggs were stored for > 30 d. The intrinsic and finite rates of increase, population doubling time, and mean generation time decreased only after storage for 60 d. Our results show that short-term cold storage could be used for maintaining wasp populations in a mass-rearing program and that the detrimental effects of chilling host eggs in storage for over 30 d do not extend to F2 generation.     

Effect of different cold storage periods on parasitization performance of Trichogramma cacoeciae (Hymenoptera,Trichogrammatidae) on eggs of Ephestia kuehniella (Lepidoptera,Pyralidae)

The parasitism rates by Trichogramma cacoeciae Marchal (Hymenoptera, Trichogrammatidae) using Ephestia kuehniella Zell. (Lepidoptera, Pyralidae) eggs held at 0, 4 and 8°C and for up to 31 days was measured. Parasitism was lowest on eggs held at 8°C and highest on eggs held at 0°C. The highest parasitism, 97.8%, was measured for parasitoids attacking eggs held for 3 days and stored at 0°C. Parasitism of eggs stored at all three temperatures decreased with increasing duration of storage. The number of T. cacoeciae successfully developing and emerging as adults after storage in E. kuehniella eggs held at 0, 4 and 8°C was measured. Parasitoid emergence was >83% from E. kuehniella eggs stored at 8°C for 3 weeks. Storage at 0°C caused a significant decline in parasitoid emergence after 2 weeks (P<0.05). Storage at 0°C for more than 4 weeks reduced fecundity by 50%. T. cacoeciae parasitized the highest number of E. kuehniella eggs 1 day after adult emergence. The oviposition period lasted 6–7 days, although the parasitoids lived up to 13–14 days. Impact of storage time and temperature on parasitism rates by T. cacoeciae stored while in E. kuehniella eggs was measured. As storage time and temperature increased, subsequent parasitism rates of resulting adult T. cacoeciae decreased. Eggs of E. kuehniella can be stored at 0°C for up to 31 days. Trichogramma cacoeciae developing in eggs of E. kuehniella can be stored at 4°C for up to 5 weeks prior to release.     

The effects of low temperature on fertilized rabbit ova in vitro,and the normal development of ova kept at low temperature for several days

Fertilized rabbit ova at the 2-blastomere stage kept in rabbit serum were stored at low temperatures for various lengths of time. They were then cultured at 38 degrees C. for about 24 hours to determine their viability. A number of the viable ova were finally transplanted into recipient does. It was found that rapid cooling of ova to 5 degrees or to 0 degrees C. was more harmful to the subsequent viability of ova than slow cooling. Rapid cooling was not more lethal to the ova than slow cooling, but did prevent their future normal cleavage. There was no difference between those ova cooled rapidly or slowly to 10 degrees C. It was concluded that temperature shock has an adverse effect on ova, especially at the lower temperatures, though temperature shock can be remedied by acclimatization (slow cooling). Thus, the physiological significance of temperature shock would seem to be broadened. The optimal temperature for the storage of ova was investigated. It was found that 10 degrees C. was the best temperature; at this temperature viable ova were obtained after storage for 144 to 168 hours. At 0 degrees , 5 degrees , or 15 degrees C. the ova were viable for 96 to 120 hours, while at 22-24 degrees C., only for 24 to 48 hours. The percentage of dead ova was low at a favorable temperature, increasing only at the end of the storage period. At an unfavorable temperature, however, the rate of death increased steadily from beginning to end of storage. The percentage of abnormally cleaved ova (arrested cleavage and fragmentation) remained at a low level at first at a favorable temperature, but then increased just before or during death of the ova. A critical time for the viability, the abnormal cleavage, and the death of ova was characteristic of each temperature. About 24 to 28 per cent of the viable ova remaining after being stored at 0-15 degrees C. for 2 to 4 days and cultured at 38 degrees C. for 24 hours were capable of development into normal young. The compatibility of serum and ova, the absence of a correlation between the viability of the ova and the source of the fertilizing spermatozoa, and the fertilization of superovulated ova (i.e., the percentage of fertile does in follicular phase and in luteal phase, the percentage of unfertilized ova and of fertilized ova at different stages, the percentage of does that had produced a normal number of ova or had produced a large number of ova, etc.), are reported. The possibility of a more efficient utilization of the germ cells of valuable animals by means of the present techniques, and the possibility of a new approach to the experimental investigation of mammalian genetics and development, have been mentioned.     

Storage and incubation of Echinostoma revolutum eggs recovered from wild Branta canadensis, and their infectivity to Lymnaea tomentosa snails

Echinostoma revolutum eggs recovered from naturally infected wild Canada geese (Branta canadensis) were cold stored (4-6 degrees C) for up to 72 weeks. Successful hatching followed incubation for from 6 to 8 days at an optimum temperature of between 25 and 30 degrees C. A partial life cycle from adult worm to metacercarial encystment in Lymnaea tomentosa snails was completed in the laboratory. Snails were infected both by free miracidia and by ingestment of unhatched embryonated eggs. Infection was equally successful in environmental temperature ranges from 10 to 25 degrees C, and at challenge levels of 2, 5 or 10 embryonated eggs per snail. Exposure to 10 eggs was lethal. Ingestion by snails of embryonated eggs with successful infection at 10 degrees C suggests that embryonated eggs may be used to infect wild snails when the environmental water temperature has reached 10 degrees C.     

Quality assessment of Riptortus pedestris (Hemiptera: Alydidae) eggs cold-stored at different temperature and relative humidity regime

Supplementation of host resource can be more economical method for the biological control of insect pest compared to direct release of adult parasitoids. Periodical release of non-viable cold-stored eggs of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) has been found to enhance parasitism of this pest in soybean fields. To find the optimum environmental conditions for cold storage of these host eggs, we evaluated nine different combinations of temperature (2, 6, and 10 °C) and relative humidity (high 90–95%, medium 70–75%, and low 30–35%). After 30 d of cold-storage, eggs were weighed and held at 26.6 °C and 75% relative humidity for 8 d before testing. To test the eggs’ suitability as hosts following cold storage, females of Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) were released individually onto batches of eggs, and parasitization rates and the development, emergence, sex ratio, adult longevity, and size of parasitoid progeny were examined. Eggs stored at high relative humidity showed less weight loss than those stored at low relative humidity. The number of eggs parasitized was highest (5.9/15) on eggs stored at 6 °C and high relative humidity. Developmental times and adult emergence were optimal on host eggs stored at 2 °C and high relative humidity. A significantly lower proportion of eggs produced male parasitoids when eggs were stored at 2 or 6 °C. Adult longevity was not affected by egg storage conditions, but adult size of progeny decreased in eggs stored at 10 °C. In conclusion, eggs of R. pedestris stored below 6 °C and with a high relative humidity maintained the best quality for parasitization by O. nezarae.     

From oocyte to embryo: a model, deduced from in vitro fertilization, for natural selection against chromosome abnormalities

A cytogenetical analysis was performed on 151 unfertilized oocytes, 22 fertilized eggs at the pronuclear stage, and 108 cleaved embryos obtained in the course of in vitro fertilization (IVF). Thirty-two per cent of unfertilized oocytes were abnormal, carrying nullisomies or disomies, mainly of D and G chromosomes, and a structural anomaly (Gq-) in one case. Fertilized eggs showed frequent asynchronism in the development of pronuclei and only 2 out of 8 karyotyped pronuclei were normal. Cleaved embryos were classified according to the number of pronuclei observed 17 hours after insemination. One per cent displayed a single pronucleus, and haploid chromosome complements were found in the corresponding cleaved embryos which were considered to be parthenotes. The rate of chromosome abnormalities of diploid eggs depended on their morphological aspect. Healthy cleaved embryos carried 12.5% of anomalies while this rate reached 37% in fragmented embryos (p less than 0.05). Lastly, 6% of fertilized eggs displayed three pronuclei or more. Only 41% of the corresponding embryos were triploid. Diploidy or diploidtriploid mosaicism were often encountered. This leads to a 21% rate of abnormalities in the preimplantation embryos. Parental karyotyping and HLA typing were carried out in a series of eight couples with in vitro idiopathic infertility or recurrent embryo degeneration in vitro. No abnormality was noted. According to these results, a model of natural selection of normal conceptuses is proposed.     

Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa

Experiments were conducted to determine temperatures between 24 and 4 degrees C at which stallion spermatozoa are most susceptible to cold shock damage. Semen was diluted to 25 x 10(6) spermatozoa/ml in a milk-based extender. Aliquots of extended semen were then cooled in programmable semen coolers. Semen was evaluated by computerized semen analysis initially and after 6, 12, 24, 36 and 48 hours of cooling. In Experiment 1A, semen was cooled rapidly (-0.7 degrees C/minute) from 24 degrees C to either 22, 20, 18 or 16 degrees C; then it was cooled slowly (-0.05 degrees C/minute) to a storage temperature of 4 degrees C. In Experiment 1B, rapid cooling proceeded from 24 degrees C to either 22, 19, 16, or 13 degrees C, and then slow cooling occurred to 4 degrees C. Initiating slow cooling at 22 or 20 degrees C resulted in higher (P<0.05) total and progressive motility over the first 24 hours of cooling than initiating slow cooling at 16 degrees C. Initiation of slow cooling at 22 or 19 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of cooled storage than initiation of slow cooling at 16 or 13 degrees C. In Experiment 2A, semen was cooled rapidly from 24 to 19 degrees C, and then cooled slowly to either 13, 10, 7 or 4 degrees C, at which point rapid cooling was resumed to 4 degrees C. Resuming the fast rate of cooling at 7 degrees C resulted in higher (P<0.05) total and progressive motility at 36 and 48 hours of cooled storage than resuming fast cooling at 10 or 13 degrees C. In Experiment 2B, slow cooling proceeded to either 10, 8, 6 or 4 degrees C before fast cooling resumed to 4 degrees C. There was no significant difference (P>0.05) at most storage times in total or progressive motility for spermatozoa when fast cooling was resumed at 8, 6 or 4 degrees C. In Experiment 3, cooling units were programmed to cool rapidly from 24 to 19 degrees C, then cool slowly from 19 to 8 degrees C, and then resume rapid cooling to storage temperatures of either 6, 4, 2 or 0 degrees C. Storage at 6 or 4 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of storage than 0 or 2 degrees C.     

Cold storage of Trichogramma ostriniae reared on Sitotroga cerealella eggs

Efficient storage of the biological controlagent Trichogramma ostriniae couldimprove current parasitoid production methodsby making the system more flexible andefficient. Initial studies compared emergencerates of T. ostriniae reared onSitotroga cerealella eggs held at 6 °C, 9 °C, 12 °C,15 °C, and 24 °C for up to 8 weeks after parasitism.At 15 °C, emergence occurred in <2 weeks.Emeregence was >80% for parasitized eggsstored at 9 °C and 12 °C for 4 and 6 weeksrespectively. Storage at 6 °C caused asignificant decline in emergence after 2 weeks. Subsequent trials focused on fitness of storedT. ostriniae. Percentage of emergedfemales parasitizing eggs, female longevity,and fecundity were quantified after storage.The percentage of females successfullyparasitizing O. nubilalis eggs wasgreatest for the 9 °C four-week treatment(100%). Compared to 24 °C controls, storage at12 °C for 6 weeks or at 9 °C for 8 weeks reduced thepercentage of females parasitizing. Longevityof females held in cold storage was generallyless than that of controls. Rates of parasitismby stored Trichogramma was generallysimilar to controls after 2 to 4 weeks' storageat 9 °C and 12 °C but declined with storage longerthan 4 weeks. Emergence of progeny of cold-storedfemales was lower than controls for alltreatments. The percentage of female progenyfrom cold stored females was comparable tocontrols up to 4 weeks of storage.     

Effect of cold storage on survival, reproduction and development of adults and eggs of Franklinothrips vespiformis (Crawford)

Cold storage effects on both female adults and eggs of the predatory thrips Franklinothrips vespiformis (Crawford) (Thysanoptera: Aeolothripidae) were investigated. The effect of low temperatures (5.5, 7.0, 8.5, 10.0 and 12.5 °C) on survival of F. vespiformis adults was firstly recorded. Survival times were significantly reduced at the lower temperatures tested, whereas storage at 10.0 and 12.5 °C provided the longest survival. Life-history consequences of exposing adults to moderately low temperatures were examined in terms of pre-oviposition period, oviposition rate, egg viability and survival after storage. Adults stored at 7.0 °C showed longer pre-oviposition period and shorter longevity than unstored females but other reproductive attributes were not significantly affected by storage regime. Low temperature and storage period affected egg viability and subsequent development of pre-imaginal stages. No eggs hatched after a 20-day period of storage at 5.5 and 7.0 °C, whereas eggs stored at 12.5 °C hatched significantly faster than ones stored at 10.0 °C and unstored eggs. Increasing the egg storage period from 10 to 20 and 30 days decreased the oviposition rate of adults and egg viability. An essential component in the successful mass rearing and distribution of these predators is the development of a reliable storage schedule of eggs and adults. Long-term storage was unsatisfactory, however their short-term storage (3.5 weeks at 10.0 and 12.5 °C for adults and 4–5 weeks at 12.5 °C for eggs) gave satisfactory results, which suggest the efficacy of such storage during the mass production of the biocontrol agent.     

Morphological criteria of egg quality in marine fishes: Activation and cleavage of eggs of <Emphasis Type="Italic">Zebrasoma scopas</Emphasis> (Acanthuridae)

Morphological changes of the eggs of Zebrasoma scopas obtained just after ovulation and after the storage of ovulated oocytes in the ovarian cavity of the female (in vivo) or in the external medium (in vitro) are described. The dynamics of cortical reaction is followed in noninseminated oocytes, as well as in inseminated oocytes of various quality. The average proportions of eggs exhibiting normal cleavage in the control series (comprised the eggs inseminated just after ovulation) and in the series stored in vivo for 2 and 4 h at 27°C are 83, 30, and 35%, respectively. The average proportions of eggs with normal cleavage in the control series and in the series stored in vitro for 2 and 4 h at 25°C are 88, 79, and 34%, respectively. The storage of oocytes in vitro at 7°C for 2 h leads to the abrupt decrease of their quality with the proportion of normally cleaved eggs reaching only 15%. The types of abnormal cleavage leading to subsequent degradation of cells of the blastodisc and total mortality of eggs are as follows: (1) mitotic cleavage of nuclei accompanied by incomplete cytotomy, (2) mitotic cleavage of nuclei without cytotomy, (3) a weak adhesion between cells, and (4) desynchronization of cell divisions. The methods for the assessment of egg quality just after ovulation and at initial developmental stages are suggested for marine teleost fishes. Published in Russian in Voprosy Ikhtiologii, 2008, Vol. 48, No. 4, pp. 537–552. The article was translated by the authors.