Total Hatching Rate Research Articles

Maximizing the hatchability of Locusta migratoria (Orthoptera: Acrididae) eggs under artificial hatching conditions

AbstractLocusta migratoria is a potential alternative food and animal feed additive in South Korea and has already been registered in the European Union. We investigated the optimal conditions to improve hatchability of L. migratoria eggs under varying hatching conditions for improved artificial rearing. The parameters such as temperature, photoperiod, illuminance, or relative humidity were altered, while the other factors were kept constant. Among the four different temperature treatments (25, 27, 30, and 33°C), the 33°C temperature showed the highest total hatching rate of 85.6% at days 8–12 after oviposition. For photoperiod, hatchability was the highest (81.2%) at 9 L/15D, followed by 80.0% at 12 L/12D, and 72.6% at 15 L/9D. Under illuminance conditions of 600, 1,800, and 3,500 lux, 3,500 lux displayed the highest total hatching rate of 76.1% at days 7–11 after oviposition. Among the relative humidity (RH) levels of 50%, 65%, and 80%, 65% RH provided the highest hatching rate of 86.4% at days 8–13 post‐oviposition. In short, the most effective conditions for hatchability were 33°C, 9 L/15D, 65% RH, and 3,500 lux. Under these conditions, the hatching rate increased significantly to 98.8%. Application of these conditions will contribute to improve hatchability, which will be necessary for industrialization of L. migratoria.

Incubation temperature effects on some hatching parameters of silver rasbora (Rasbora argyrotaenia) egg

ABSTRACT This study aims to observe the effects of incubation temperature on some hatching parameters of silver rasbora (Rasbora argyrotaenia) egg and determine the optimum temperature to incubate it. A total of 2,000 fertilized eggs were assigned to 20 incubation cylinder plastic tanks (10 L capacity; 30 cm diameter). Every five tanks were placed in four different containers (120 × 80 × 46 cm3) with incubation temperature treatment including 25.0°C–30.0°C (room temperature), 28.0°C, 30.0°C, and 32.0°C. The increase of temperature caused accelerated hatching time, yolk absorption time, and larvae development; it also increased the yolk absorption rate and larvae abnormality rate. A decrease in hatching and survival rate occurred at the highest temperature treatment, so the optimal incubation temperature for silver rasbora egg is 30°C based on the best results of the total hatching rate and survival rate of larvae.

Level of in vitro storage of the European catfish (Silurus glanis L.) eggs at different temperatures.

European catfish (Silurus glanis) is a commercially important freshwater fish originating from Eastern Europe. The objective of this study was to examine the short-term storage of its eggs especially in relation to maintaining a low level of malformation in newly hatched fry. The eggs from freshly spawned individuals were stored separately in cell incubators at 17 and 22°C under aerobic conditions. Changes in fertilization, hatching, and malformation were examined in eggs stored at 1, 3, 5, and 7h post-stripping. The sperm used for fertilization showed very good motility rates (84-90%) and curvilinear velocity (110-125μm/s), and straight-line velocity did not drop below 77μm/s. For all females, a temperature of 17°C was better than 22°C for egg storage in vitro. Egg fertilization and total hatching decreased rapidly after 7h storage at 17°C. The storage time of eggs in vitro to fertilization should therefore not exceed 5h at 17°C. In all females, there was no difference in the total number of eggs hatching between 1 and 3h of egg storage at 17°C. The storage time of eggs did not correlate with the level of malformations of the fry. However, the level of hatching and malformations was clearly affected by the storage temperature of eggs when it was > 17°C. Analysis showed that the storage time of eggs, temperature of storage, and individual females had a significant influence on fertilization and total hatching rates. Regression analysis confirmed a low correlation of fertilization and hatching rates with storage time of eggs.

Optimization of cold-shock conditions for the induction of triploidy in the Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel)

Pacific bluefin tuna (Thunnus orientalis) is one of the most important species for aquaculture in Japan. In recent years, the reduction in muscle fat content during sexual maturation in farmed T. orientalis has become a serious problem. Herein, as a first step toward sterilization to avoid the deterioration of flesh quality associated with sexual maturation, we examined cold-shock conditions for inducing triploidy in T. orientalis. In this study, three treatment variables (initiation timing, duration, and water temperature of the cold-shock treatment) were considered, using the fertilized eggs collected immediately after spontaneous spawning in a land-based tank. We found that the optimal cold-shock conditions for T. orientalis were 5 °C for 15 min starting from 5 min after fertilization. Under these optimal conditions, we obtained high triploidization (96.6 ± 3.3%) and total hatching (64.5 ± 8.7%) rates. Therefore, using eggs obtained by spontaneous spawning in a land-based tank and the triploidization method established in this study, it is possible to repeatedly and efficiently produce triploid T. orientalis. Statement of relevanceIn recent years, deterioration of flesh quality associated with sexual maturation in aquaculture-produced Thunnus orientalis has become a serious problem. To avoid this problem by sterilization technique, in this study, we have developed an efficient method for the induction of triploidy in T. orientalis, using the fertilized eggs collected immediately after the spontaneous spawning in a land-based tank. We believed that the results of present study are a valuable first step toward the sterilization of T. orientalis.

AQUA_2020_1080: Sperm management of European catfish (Silurus glanis L.) for effective reproduction and genetic conservation

The European catfish (Silurus glanis; also called wels or sheatfish) is one of the largest commercially important freshwater fish species. The objective of this study was to examine the storage time of its semen in an immobilization solution (IS) to enhance conservation and aquaculture programs. Changes in spermatozoan motility was examined in semen stored on ice in 20 mL tubes in IS for 0, 1, 7, 14 and 28 days post-stripping (DPS). Stored sperm samples were pooled and used for fertility testing at 1 and 7 DPS. The IS maintained sperm motility at >90% during days 1–7 of semen storage, and curvilinear velocity (VCL) and straight-line velocity (VSL) were up to the levels of 163 and 90 μm s−1, respectively. Spermatozoa stored for 14 days had 60% motility and VCL and VSL values were similar to those of 7 DPS. Sperm motility was only 1% at 28 DPS with VCL and VSL of 79 and 24 μm s−1, respectively. The fertilization and total hatching rates of eggs activated with sperm stored for 7 days were reduced, but the slight decrease was compensated by the increase in supply of spermatozoa which resulted in overall 80% level of fertility and hatching. The number of larval malformations was not correlated with storage time of sperm. Based on the results of this study, sperm from the European catfish can be collected and stored in IS for as long as 1 week, retaining quality sufficient for fertilization to yield normal fry.

Acute exposure of zebrafish (Danio rerio) larvae to environmental concentrations of selected antidepressants: Bioaccumulation, physiological and histological changes

Antidepressants have been detected in surface waters worldwide at ng–μg/L concentration. These compounds can exert adverse effects on fish even at low levels. But, all previous analyses have concentrated on adult fish. The aim of the study was to assess the effect of environmental concentrations of sertraline, paroxetine, fluoxetine and mianserin, and their mixtures on such unusual endpoints as physiological and histological changes of zebrafish (Danio rerio) larvae. We also determined the bioconcentration of the pharmaceuticals.Fish Embryo Toxicity test was used to analyze the influence on developmental progression. Histological sections were stained with hematoxylin and eosin. Proliferating cells in liver were determined immunohistochemically by detection of Proliferating Cell Nuclear Antigens. The bioconcentration factor was measured by liquid chromatography coupled to mass spectrometry. Pharmaceuticals were used at low, medium and high concentrations in mixtures and at medium concentration as single compound.Exposure to the analyzed pharmaceuticals increased the rate of abnormal embryo and larvae development, accelerated the hatching time and affected the total hatching rate. Three-times lower proliferation of hepatocytes was observed in larvae exposed to paroxetine, mianserin, sertraline and the mixture of the pharmaceuticals at the highest concentrations. The highest bioaccumulation factor (BCF) was obtained for sertraline. The BCF of the analyzed compounds was higher if the organisms were exposed to the mixtures than to single pharmaceuticals. To conclude, the exposure of zebrafish larvae to selected antidepressants and their mixtures may cause disturbances in the organogenesis of fish even at environmental concentrations.

Morphometric characteristics and time to hatch as efficacious indicators for potential nanotoxicity assay in zebrafish.

Although the effects of nano-sized titania (nTiO2 ) on hatching events (change in hatching time and total hatching) in zebrafish have been reported, additional consequences of nTiO2 exposure (i.e., the effects of nTiO2 -induced changes in hatching events and morphometric parameters on embryo-larvae development and survivability) have not been reported. To address this knowledge gap, embryos 4 h postfertilization were exposed to nTiO2 (0, 0.01, 10, and 1000 μg/mL) for 220 h. Hatching rate (58, 82, and 106 h postexposure [hpe]), survival rate (8 times from 34 to 202 hpe), and 21 morphometric characteristics (8 times from 34 to 202 hpe) were recorded. Total hatching (rate at 106 hpe) was significantly and positively correlated to survival rate, but there was no direct association between nTiO2 -induced change in hatching time (hatching rate at 58 and 82 hpe) and survival rate. At 58, 82, and 106 hpe, morphometric characteristics were significantly correlated to hatching rate, suggesting that the nTiO2 -induced change in hatching time can affect larval development. The morphometric characteristics that were associated with change in hatching time were also significantly correlated to survival rate, suggesting an indirect significant influence of the nTiO2 -induced change in hatching time on survivability. These results show a significant influence of nTiO2 -induced change in hatching events on zebrafish embryo-larvae development and survivability. They also show that morphometric maldevelopments can predict later-in-life consequences (survivability) of an embryonic exposure to nTiO2 . This suggests that zebrafish can be sensitive biological predictors of nTiO2 acute toxicity. Environ Toxicol Chem 2018;37:3063-3076. © 2018 SETAC.

Unusual incubation behavior and embryonic tolerance of hypothermia in the Sichuan Partridge (Arborophila rufipectus)

Incubation is an energetically demanding phase of avian reproduction. Single-sex incubators are often faced with a direct conflict between incubation and foraging. We report an unusual incubation behavior of the Sichuan Partridge (Arborophila rufipectus), with long recesses of females and embryonic hypothermia, at Laojunshan National Nature Reserve, southwest China. The Sichuan Partridge is a globally endangered species that inhabits subtropical evergreen and deciduous broadleaf forests at 1100–2250 m a.s.l. Female Sichuan Partridges incubated eggs alone, and typically took one long incubation recess of 4.5 ± 1.2 h (mean ± SD) day−1. They left the nests at 7:36 a.m. (±50 min) and returned at 12:06 p.m. (±70 min). Nest attentiveness was 81.2 ± 5.2% of the entire incubation period. Females prolonged the recess duration and decreased nest attentiveness significantly in response to high disturbance risk and bad weather. Although eggs experienced ~4.2 h below 26 °C (the physiological zero temperature) for each recess, the total hatching rate was 88.4%, suggesting that embryonic hypothermia had minimal negative effect on the hatching rate. For Sichuan Partridge, the ability to withstand embryonic hypothermia may be an adaptation to cold montane environments.

Effect of Cadmium on the Embryonic Development and Hatching Rhythm of Big Fin Squid Sepioteuthis lessoniana, Lesson (1830) from Palk Strait, East Coast of India

In the present study, the impact of cadmium on embryonic development, morphology of eggs and newly hatched larvae of the Sepioteuthis lessoniana were described using the laboratory-reared specimens originating from Thondi, South East coast of India. The eggs were exposed to a concentration of 25 μg Cd/L for up to hatching. The average incubation period of the eggs varied between 18-23 days and the estimated mean temperatures were 18.6 ± 0.7°C for control, and 19.2 ± 1.1°C for Cd exposed groups. The results revealed that a total hatching rate of 99.2 ± 1.6% was achieved in control group and only 65.2 ± 8.8% in treated group. The deformities of the paralarvae were illustrated with light and scanning electron microscopy. In conclusion, the present concentration exhibited toxic effects to the eggs and paralarvae of S. lessoniana. Therefore, Cd level as high as we used in this study could give good information on dangerous Cd levels on squid.

Efficacy of the hatching event in assessing the embryo toxicity of the nano-sized TiO2 particles in zebrafish: A comparison between two different classes of hatching-derived variables

The aim of the present study was to evaluate the nano-TiO2 toxicity to zebrafish embryos through evaluating the success in hatching in relationship with hours post-exposure instead of considering just the total hatching rate. Zebrafish embryos 4h post-fertilization were exposed to nTiO2 (0, 0.01, 10, and 1000µgmL−1) for 130h. The hatching rate (HR) was calculated for each concentration (treatment). The HR magnitude was significantly (p<0.001) correlated (using simple regression) to hours post-exposure time interval (hpe; 34, 58, 82, 106, and 130), noted as HR.hpe. The HR descriptive statistics (HRds) and the parameters of the regression models (i.e., constant, x, F, and r2) were recruited to define 15 HRds- and 4 h.hpe-derived variables, respectively. The efficacy of the variables was evaluated. Exposure to nTiO2 led to a significant: premature hatching and general decrease in time required for normal hatching; and change in HR and hpe interrelations in a dose-dependent manner. The major change in hatchability between the treatment and control occurred at 58 hpe (62 hpf), when the treatment with nTiO2 induced significant premature hatching compared to only 6% of the hatched embryos in the control at the same time point. EC10 and EC50 values that cause premature hatching at 58 hpe for nTiO2 are 0.073µgmL−1 and 107.2µgmL−1 respectively. In general, this study shows multivariate differences among exposure concentrations of nTiO2 recruiting hatching-derived endpoints.

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation

Summary The objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus-oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8-16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

Effect of salinity during resting egg formation and hatching on descendent reproduction in the rotifer Brachionus rotundiformis Tschugunoff

This study investigated the effect of salinity experienced during resting egg formation and hatching on the descendents' reproduction and on resting egg hatching in the monogonont rotifer Brachioraus rotundiformis Tschugunoff. The study was divided into two parts. First, the resting eggs formed at 17 psu were incubated at four salinities (8, 11, 22 and 33 psu) and then the hatchlings were cultured at the same salinities as during incubation. Second, the resting eggs formed at the four salinities were incubated at the same salinities as during their formation, and the hatchlings were cultured at 33 psu. The resting eggs formed at a salinity of 17 psu and incubated at four different salinities showed a higher hatching percentage at lower salinities (8 and 11 psu). When resting eggs were incubated at the same salinities as at formation (8, 11, 22 and 33 psu), the eggs showed no significant differences in a total hatching rate among treatments (83.3-86.7%). There was no significant difference in population growth when hatchlings did not experience a change in salinity. However, the population growth of hatchlings at 8 psu was comparatively arrested and at 33 psu caused by the acute increase in salinity. With regard to sexual reproduction, rotifers showed different patterns that were associated with the salinity experienced during resting egg hatching. This study shows that the salinity experienced during resting egg formation is the optimal one for egg hatching. Moreover, the salinity experienced during resting egg hatching strongly affects the descendents' sexual reproduction more than the salinity experienced during egg formation.

Desiccation tolerance in fully developed embryos of two cicadas, Cryptotympana facialis and Graptopsaltria nigrofuscata

AbstractIn two Japanese cicadas, Cryptotympana facialis and Graptopsaltria nigrofuscata, with different habitat distributions, fully developed embryos hatch in response to high humidity due to rainfall. Despite the advantage of hatching on rainy days, this trait burdens embryos with an extra period of desiccation until the unpredictable advent of rain. We compared the ability of the fully developed embryos of these cicadas to endure periods of low humidity. Eggs were exposed to a combination of different humidities (43% and 75% relative humidity, RH) and durations (0–15 days), and then transferred to an environment with 100% RH to stimulate hatching. In both species, total hatching rates decreased as duration increased, although there was no significant effect of humidity. In C. facialis, a considerable proportion of the eggs hatched during the desiccation period, and the hatching rate was higher at 75% RH than at 43% RH. After transfer to 100% RH, most hatching occurred within a day regardless of the desiccation level. In G. nigrofuscata, no nymphs hatched during the desiccation periods. However, more eggs required more than a day after transfer to 100% RH to hatch after desiccation at 43% RH than at 75% RH. Consequently, the overall proportion of timely hatching of eggs (eggs hatching within a day of moisture supply) was higher after desiccation at 43% RH in C. facialis, but it was higher after desiccation at 75% RH in G. nigrofuscata. These different physiological responses of the two species may reflect adaptation to habitat dryness.

Viability, growth and external morphology of meiotic- and mitotic-gynogenetic diploids in red sea bream, Pagrus major

Two types of gynogenetic diploids were artificially induced in the red sea bream (Pagrus major Temminck et Schlegel), either by suppressing the first cell cleavage (mitotic-G2N) or by retaining the second polar body (meiotic-G2N). The eggs of red sea bream were inseminated with UV-irradiated (3000 erg mm−2) sperm of Japanese parrot fish (Oplegnathus fasciatus Temminck et Schlegel), and hydrostatic pressure shock of 700 kg cm−2 for 5.5 min at 46 min after insemination (mitotic-G2N) and cold shock of 1 °C for 30 min at 3 min after insemination (meiotic-G2N) were applied to the eggs, sequentially. The total hatching rate and hatching rate of normal larvae of the normal diploid, meiotic-G2N and mitotic-G2N were 86.5 and 94.9%, 38.1 and 45.8%, and 12.8 and 35.0%, respectively. The induction of mitotic-G2N was confirmed by isozyme marker analysis. The standard deviations, variances and coefficients of variation of the body weight, standard length and body depth in 91-day-old juveniles were always large in mitotic-G2N, small in normal-2N and intermediate in meiotic-G2N. The variances in the number of pectoral fin rays and caudal fin rays of mitotic-G2N were significantly higher than those of normal-2N. The incidences of deformities were highest in the mitotic-G2N group. The survival rates and growth performance of the meiotic- and mitotic-G2N were significantly lower than those of normal-2N. Both G2N survived for 3 years to the adult stage.

Effects of Cadmium (CdCl2) on development and hatching of eggs in European squid (Loligo vulgaris Lamarck, 1798) (Cephalopoda: Loliginidae)

This is the first study related to the effects of cadmium (Cd) on the development and hatching of eggs of Loligo vulgaris. The eggs were exposed to concentrations varying from 10 to 100,000 microg Cd/l for up to 1 month. During the study period natural sea water salinity (37 PSU), ambient temperature (ranging 16.5-21.5 degrees C) and light conditions of 12-h light: 12-h dark for 744-h were used prior to the hatching. For 96-h, mortality rate of 100% of the eggs was found at cadmium concentration of 100,000 microg Cd/l, and the same mortality rate of the eggs were found at 1,000 and 10,000 microg Cd/l for 744-h and 696-h, respectively. Hatching took place between hours 456 and 576 in the control group and between hours 480 and 576 in 10 and 100 microg Cd/l groups. Total hatching rates were calculated to be 98.2 +/- 1.6% for control group, 99.4 +/- 0.6% for 10 microg Cd/l group and 98.6 +/- 1.2% for 100 microg Cd/l group. Hatching successes were estimated as 69.5 +/- 13.8% for control group, 84.5 +/- 8.2% for 10 microg Cd/l group and 76.9 +/- 5.9% for 100 microg Cd/l group. In the 100,000 microg Cd/l treatment, the egg degeneration began from animal pole of the eggs in 16 h, and 50% and 100% of them died in the initial stage at 72-h and 96-h, respectively. In the 10,000 microg Cd/l group, the eggs which reached at stage 19 (IX) were deformed and 50% and 100% of them died at 312-h and 696-h, respectively. In 1,000 microg Cd/l group, however, the eggs developed abnormally from stage 21 (X-XI) and reached at stage 29 (XIX), and 50% and 100% of them died in 624-h and in 744-h, respectively. There were significant differences in survival rates of the eggs among the concentrations (p < 0.05). The current study demonstrated that Cd adversely affects the development, survival and hatching of L. vulgaris eggs; however, no mortality was observed in 10 microg Cd/l and 100 microg Cd/l groups.

161 LIPID CONTENT, RESISTANCE TO CRYOPRESERVATION, AND SEX RATIO OF IN VITRO BOVINE BLASTOCYSTS PRODUCED IN A SERUM-FREE SYSTEM

In vitro-produced (IVP) bovine blastocysts are known to be more sensitive to cryopreservation than their in vivo counterparts. Removing serum from the culture medium decreases sanitary risk and could improve embryo resistance to cryopreservation by preventing the accumulation of intracellular lipids. Our objectives were to evaluate the lipid content, resistance to cryopreservation, and sex ratio of IVP embryos cultured in a serum-free system. Oocytes from slaughterhouse ovaries were matured in a serum-free enriched medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) and cultured in 5% O2 in modified SOF supplemented with 5% FCS (FCS) or with insulin-transferrin-selenium (ITS) and 0.1 mg/mL polyvinylpyrrolidone (PVP) (ITS-PVP) or 4 mg/mL BSA (ITS-BSA) (Daniaux et al. 2005 Reprod. Fertil. Dev. 17, 217). Day 5 morulae were stained with the fluorescent dye Nile Red in order to evaluate their lipid content (Genicot et al. 2005 Theriogenology 63, 1181). Day 7 blastocysts (diameter ≥160 µm) were selected, classified according to their size, and frozen in HEPES-SOF containing 1.5 M ethylene glycol, 0.1 M sucrose, and 1.8 mg/mL wheat peptones (George et al. 2002 Reproduction 29, 51). The lipid content was significantly lower in morulae cultured in ITS-BSA compared with the two other media (320 ± 10 arbitrary fluorescence units vs. 383 ± 12 in FCS and 406 ± 10 in ITS-PVP; n = 271; ANOVA2: P &lt; 0.01). After cryopreservation, a higher total hatching rate was found 24 h post-thawing in blastocysts cultured in ITS-BSA and for both serum-free conditions at 48 h (Table 1). In particular, embryos ≤180 µm cultured in FCS were less resistant to cryopreservation than embryos of the same size produced without serum. Expanded blastocysts cultured in ITS-BSA were sexed by PCR (Grisart et al. 1995 Theriogenology 43, 1097) and a higher proportion of male embryos was found (62.7%; n = 51). In conclusion, a complete serum-free system was set up from oocyte maturation to embryo cryopreservation that gave high quality embryos resistant to cryo-preservation. Embryos produced in ITS-BSA presented a lower lipid content, but a shift of the expanded blastocyst sex ratio toward males was observed. Table 1. Hatching rates post-thawing as a function of the blastocyst size and the culture medium

155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P &gt; 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P &lt; 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P &gt; 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P &lt; 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching. This project was supported by a grant from the National Institutes of Health (U42 RR 018877).

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF+5% FCS for 9 days; (2) KSOM+0.1% BSA for 4 days and then KSOM+1% BSA to Day 9; (3) SOF+5% FCS for 4 days and then KSOM+1% BSA to Day 9; and (4) KSOM+0.1% BSA for 4 days and then SOF+5% FCS to Day 9. Treatment 4 (sequential KSOM–SOF culture system) improved (P>0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM+BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM–SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5M glycerol); or (2) slow freezing (1.36M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P<0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.

Enhanced hatching rate of bovine IVM/IVF/IVC blastocysts using a 1.48-micron diode laser beam.

Our purpose was to test whether zona pellucida (ZP) drilling using a 1.48-micron diode laser beam on bovine IVM/IVF/IVC blastocysts is effective for embryo hatching. Blastocysts produced in vitro at day 7 after IVF were divided into control and laser-drilled groups, respectively. When the rates of in vitro development of bovine embryos were examined, the average cleavage rate (> or = two-cell) was 82.3% and the blastocyst rate at day 7 after IVF was 32.5%. Using these blastocysts, when the laser drilling effect was investigated at 48 hr after treatment, the total hatching rate in the laser-drilled group (98.0%) was significantly higher than that in the control group (60.0%) (P < 0.001). Especially, the hatched rate of the laser-drilled group (68.0%) was significantly enhanced compared with that of the control group (30.0%) (P < 0.001). These results demonstrated that laser ZP drilling on bovine IVM/IVF/IVC blastocysts can significantly increase the hatching rate.

Optimum incubation conditions of the domestic guinea fowl egg.

1. During artificial incubation of 8000 guinea fowl eggs, the effects of temperature, relative humidity, rate of inflowing air and age of the laying flocks were determined. 2. Total duration of incubation was divided into setter (0 to 24 days) and hatcher (25 to 28 days) periods. Eggs transferred at the end of the setter period, (ET, % of fertile eggs) and hatching rate (HR, % of transferred eggs) were calculated, as well as the total hatching rate expressed as percentage of fertile eggs. 3. In the range 36 to 39 degrees C, temperature affected significantly (P < 0.001) ET and HR, with optima at 37.2 and 37.0 degrees C +/- 0.1 degrees C respectively. 4. During incubating period, relative humidity in the range 40 to 64% (water vapour partial pressure, PH2O = 17 to 34 Torr at 36 to 39 degrees C), significantly (P < 0.001) affected the total diffusive water loss and hatchling mass, both expressed as percentage of fresh egg mass. ET was significantly affected by water loss, the highest ET being for water loss of 13.3 +/- 0.5%. Optimal relative humidity was calculated to be 48 to 52% (PH2O = 23 to 25 Torr at 37.2 degrees C). 5. The rate of inflowing air significantly (P < 0.001) affected HR, with an optimum at 3.1 LSTPD/(h.egg). 6. The age of the laying flock significantly (P < 0.001) increased water loss; this was explained by a parallel increase of the mass specific shell conductance to water vapour. 7. Finally optimum incubation conditions were deduced, giving total hatching rates of 78 to 81% of fertile eggs, improving by 5 to 8% the best results obtained routinely in commercial practice.