Peronella japonica, a direct developer, exhibits certain peculiar features during development, particularly heterochrony, a change in the relative timing of expression among tissues and organs. One of the important heterochronical changes in the species was found in the development of the amniotic cavity, a component of an adult rudiment. In indirect developers the amniotic cavity is formed on the left side of the larval body in the late pluteus stage. In P. japonica the organ is formed at the gastrula stage in the region located on the midline of the larval body.

In the present study, the ability of partial embryos isolated from 8- or 16-cell stage embryos of P. japonica to differentiate an amniotic cavity was investigated to assess the regulative potential of a direct developer.

The embryos were dissected at 8-cell stage with a glass needle to obtain half embryos. Some of the half embryos were further divided into four blastomeres to obtain mesomere pairs. Each half embryo and blastomere that did not form micromeres but divided equally during the next cleavage was identified as an animal cap and presumptive mesomere pair. Isolated animal caps and mesomere pairs were cultured, and differentiation of the amniotic cavity was examined at 24 and 48 h after fertilisation, when the organ in the normal embryos had already completed differentiation.

Protein phosphorylation is highly coupled with sperm motility activation in several animal species. The micro-tubule based flagellar motor protein, dynein, is a candidate for a phosphoprotein related to sperm activation in many animal species (Morisawa & Hayashi, 1985; Hayashi et al., 1987; Dey & Brokaw, 1991; Stephens & Prior, 1992; Inaba et al., 1998, 1999). Sperm motility of the ascidians Ciona intestinalis and C. savignyi is activated by a factor derived from unfertilised eggs named sperm activating and attracting factor (SAAF). SAAF elevates the intracellular cyclic AMP (cAMP) level by a mechanism dependent on membrane hyperpolarisation and extracellular Ca2+ (Yoshida et al., 1994; Izumi et al., 1999). Experiments using demembranated Ciona sperm showed that cAMP is required prior to ATP for the activation of axonemal movement (Opreska & Brokaw, 1983; Morisawa et al., 1984; Brokaw, 1985; Dey & Brokaw, 1991; Chaudhry et al., 1995) and that many sperm flagellar proteins including dynein light chain are phosphorylated during incubation of demembranated sperm with ATP and cAMP (Dey & Brokaw, 1991). However, there is no evidence of which proteins are phosphorylated during the SAAF-dependent activation of Ciona sperm motility.

Our study aimed to establish the response of Salminus franciscanus to hypophysation and describe the main morphological events of its embryonic process. Wild fish were captured in São Francisco River and selected broodstock (females: 66.4 ± 11.1 cm and 4.04 ± 2.32 kg; males: 58.3 ± 10.2 cm and 3.62 ± 1.12 kg) were kept at 26.1 ± 0.6°C for induction of final maturation/gamete release via the hypophysation technique. In females, two doses (0.8 and 5.6 mg/kg body weight) of crude pituitary extract of common carp (Cyprinus carpio) were administered with a 14 h interval. For males, a single dose (2.7 mg/kg body weight) of crude pituitary extract was applied at the same time as the females’ second dose. Oocytes and sperm were manually stripped 8 h after a females’ second hormonal dose. Fertilization was carried out using the dry method. Eggs were kept in funnel-type 60 L incubators at 24.3 ± 0.3°C and were analyzed and photographed every 10 min. After hormonal induction, 60% of females and 100% of males reacted positively and no broodstock mortality was recorded. The females released an average of 385.2 ± 78.4 g of oocytes and the fertilization rate observed was 50.4 ± 12.3%. The blastopore closure occurred at 7.5 h, somite formation at 12 h and hatching at 20 h post-fertilization. In general, the results of this study improve the understanding of the reproductive biology of dourado and confirm its potential for fish farming in the neotropical region.

Commercial application of embryo transfer in pig breeding is dependent on the storage of embryos. The aim of this study was to assess the embryo quality of in vitro-produced blastocysts after 3 h liquid storage at 37°C in CO2-free medium by evaluating morphology, in vitro developmental capacity and apoptosis. Blastocysts at days 5 and 6 post-fertilization were randomly allocated to the storage group (HEPES-buffered NCSU-23 medium including bovine serum albumin in a portable embryo transport incubator at 37°C) or a control group (porcine blastocyst medium in a conventional culture incubator). Thereafter, blastocysts were evaluated for morphology and stained to assess apoptosis straight after the 3 h storage period or after a further 24 h conventional incubation. There was no significant difference between the storage and control group after 3 h storage and the further 24 h conventional incubation for any of the parameters, nor for apoptosis straight after the 3 h storage. Embryos that reached the blastocyst stage at day 5 showed less apoptosis (6.6% vs 10.9%, P = 0.01) and a trend for a higher rate of developmental capacity (70.6% vs 51.5%, P = 0.089) than embryos reaching the blastocyst stage on day 6. In conclusion, in vitro-produced porcine blastocysts can be stored for 3 h at physiological temperature in transportable incubators using a CO2-independent medium without compromising quality.