Paramphistomosis is a parasitic disease endemic in ruminants nearly worldwide. In the present study, an in vitro screening of the main anthelmintics used in Mexico was carried out to determine the mean lethal dose for rumen fluke eggs from cattle in a humid, warm region. Rumen flukes were obtained from cattle slaughtered in the states of Tabasco and Chiapas in Mexico. Eggs were collected using a 37-μm sieve and quantified. Then, an in vitro incubation study was performed: 100 eggs were placed into the wells of polystyrene microtiter plates. Anthelmintic products were tested on the eggs at concentrations ranging from 0.0015 to 3.0 mg/ml for rafoxanide, 0.0025 to 10.20 mg/ml for nitroxinil and 0.0015 to 3 mg/ml for closantel to determine the median lethal dose (LD50) and maximum lethal dose (LD99). A control group (water) was included in each plate. Three different species of rumen flukes (Calicophoron brothriophoron, Calicophoron clavula and Paramphistomum cervi) belonging to five isolates were identified. Nitroxinil had the highest efficacy against rumen fluke eggs, with an LD50 of 0.11 to 65 μg/ml, whereas rafoxanide showed the lowest efficacy with an LD50 ranging from 500 to 1713 μg/ml. Closantel showed high variability in the LD50 among the different analysed isolates (17 to 122 μg/ml). The evaluated flukicidal drugs presented differential efficacy against the development of rumen fluke eggs. The efficacy of the drugs will vary depending on the geographical area of origin of the animals.

The aim was to identify cell and genetic predictors of human blastocyst hatching success in assisted reproduction programmes via a prospective case–control study. Blastocysts, donated by couples in assisted reproduction programmes were used. Hatching success assessment was performed after 144–146 h post-fertilization. The mRNA expression levels of cathepsin V (CTSV), GATA-binding protein 3 (GATA3) and human chorionic gonadotropin beta subunit 3, 5, 7 and 8 (CGB) genes were detected by quantitative real-time polymerase chain reaction. The odds ratio (OR) of hatching due to zona pellucida (ZP) thickness, oocyte and sperm quality, embryo quality and mRNA expression of CTSV, GATA3 and CGB genes in blastocysts was determined. From 62 blastocysts included in the study, 47 (75.8%) were unable to hatch spontaneously. The ZP thickening, and oocyte and sperm quality did not affect human blastocyst ability to hatch, except the combination of cytoplasmic and extracytoplasmic oocyte dysmorphisms (OR = 1.25; 95% confidence interval = 1.08, 1.45). Hatching-capable blastocysts had higher Gardner scale grade and mRNA expression of CTSV, GATA3 and CGB genes than hatching-incapable blastocysts. The human blastocyst hatching success depends on the blastocyst Gardner grade, but not on ZP and gamete quality. Blastocyst development was regulated by CTSV, GATA3 and CGB gene expression.

The potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis are major pests of potatoes. The G. pallida (and G. rostochiensis) life cycle includes both diapause and quiescent stages. Nematodes in dormancy (diapause or quiescent) are adapted for long-term survival and are more resistant to nematicides. This study analysed the mechanisms underlying diapause and quiescence. The effects of several compounds (8Br-cGMP, oxotremorine and atropine) on the activation of hatching were studied. The measurements of some morphometric parameters in diapaused and quiescent eggs after exposure to PRD revealed differences in dorsal gland length, subventral gland length and dorsal gland nucleolus. In addition, the expression of 2 effectors (IVg9 and cellulase) was not induced in diapaused eggs in water or PRD, while expression was slightly induced in quiescent eggs. Finally, we performed a comparative study to identify orthologues of C. elegans diapause related genes in plant-parasitic nematodes (G. pallida, Meloidogyne incognita, M. hapla and Bursaphelenchus xylophilus). This analysis suggested that it was not possible to identify G. pallida orthologues of the majority of C. elegans genes involved in the control of dauer formation. All these data suggest that G. pallida may use different mechanisms to C. elegans in regulating the survival stage.

This study was designed to investigate whether a non-invasive birefringence parameter, determined using the Oosight™ imaging system, is useful for estimating the hardness of human zona pellucida (ZP). The value for retardance (R) × thickness (T), but not R or T alone, of ZP was positively correlated (r = 0.92, p < 0.0001) with its hardness estimated by the time required for a 0.1% protease solution to solubilize ZP at 37 °C. In a model experiment to induce ZP puncture by Fluorinert™ fluid microinjection (sham-hatching), the R × T value at the punctured site was positively correlated (r = 0.78, p < 0.01) with the hardness of the ZP as estimated by the maximum expansion rate. The R × T values of ZP in in vitro fertilization-derived embryos (21.6 ± 7.5) and intracytoplasmic sperm injection-derived embryos (20.8 ± 6.3) were significantly higher than that in unfertilized metaphase II oocytes (16.6 ± 6.1; p < 0.05). The R × T value after in vitro hatching of viable blastocysts (10.8 ± 6.2) was significantly lower than that of unexpanded morulae and early blastocysts (19.0 ± 4.0; p < 0.05), while the value of expanding blastocysts (15.3 ± 4.1) was intermediate. In conclusion, hardness of human ZP can be estimated non-invasively by birefringence-based microscopic observation.

In a commercial hatchery, chicks (or poults) hatch over a 24-48 hour period. All chicks remain in the incubator until the majority of the chicks have emerged from the shell. Once removed from the incubator, the newly hatched chick has to undergo several hatchery treatments and is then transported before being placed on the broiler farm. This means that, under practical conditions, chicks are deprived of feed and water for up to 72 hours. In addition, the time of hatch within the hatching window and the spread of hatch cause variability in the amount of time that chicks are feed deprived. Literature on feed deprivation after hatch clearly demonstrates the detrimental effects of any delay in feed access on performance of the chicks with respect to growth, immune system activation, digestive enzyme stimulation and organ development. Improved management strategies, such as shortening the hatching window or the time to first feeding by specific management measures, provide an alternative in dealing with the negative effects caused by a delay in feed access. The development of pre-starter diets that better meet the needs of the newly hatched chicks or in ovo feeding to bridge the gap between hatch and first feeding provide other alternatives in overcoming these problems. However, speculation remains regarding the importance of in ovo or early feeding, or whether the in ovo or early feeding itself is responsible for the beneficial effects reported. The aim of the following review is to discuss the current status of research into early feeding and to stimulate future and further research regarding these topics.

Glycogen synthase kinase-3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. It has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. This enzyme has already been described in Rhipicephalus (Boophilus) microplus and the ovaries of females appeared to be the major site of GSK-3 transcription. The treatment with GSK-3 specific inhibitor (alsterpaullone, bromo-indirubin-oxime 6 and indirubin-3-oxime) caused a reduction in oviposition and egg hatching in completely engorged female ticks. The effect was more pronounced in partially engorged females when alsterpaullone was administrated by artificial capillary feeding. Moreover, GSK-3 gene silencing by RNAi in partially engorged females reduced significantly both oviposition and hatching. The study of tick embryogenesis and proteins that participate in this process has been suggested as an important means for the development of novel strategies for parasite control. GSK-3 is an essential protein involved in embryonic processes and for this reason it has already been suggested as a possible antigen candidate for tick control.

Shelled acanthors (=eggs) of two isolates of Moniliformis moniliformis (Acanthocephala) were tested for their capacity to respond to a hatching stimulus in vitro and for their retention of infectivity to a natural intermediate host (Periplaneta americana). The shelled acanthors were stored for more than 120 weeks (Australian isolate) and for 104 weeks (Texan isolate) together with rat faeces in an incubator maintained at 22·2±0·1°C. In both cases, infectivity to P. americana was shown to have been retained. In vitro tests of the hatching response were carried out on many occasions during this period of faecal storage. Shelled acanthors continued to respond and no differences were detected either between isolates or within an isolate through time.

Natural populations of 3rd-stage Nematodirus battus larvae were present on pastures in North Wales throughout the year; highest numbers were present in late spring, with smaller peaks occurring in autumn. Inter-site variation was observed in the timing and magnitude of these peaks. Hatching on experimental plots occurred 2 months to 2 years following deposition of eggs. Intraspecific and inter-site variation occurred in the timing, and inter-site variation occurred in the magnitude, of the mass hatch on upland and lowland experimental plots. Arrested 4th-stage N. battus were recovered from Welsh Mountain lambs. Percentage arrest and number of arrested worms was greatest during winter and early spring. The prevalence and intensity of N. battus infection in 1-, 2- and 3-year-old Welsh Mountain ewes was low. The plasticity exhibited in the parasite's life-history is discussed in relation to potential changes in the epidemiology of nematodiriasis.

Hatching of eggs of Anguillicola crassus was studied at temperatures between 5 and 30°C. Hatching occurred between 10 and 30°C and the incubation time was inversely related to temperature. Survival, spontaneous and excitatory rate of activity of 2nd-stage larvae of A. crassus were also affected by temperature. The percentage of larvae eaten by Paracyclops fimbriatus was not always influenced by the activity of the larvae. Penetration efficiency into the haemocoel of the intermediate host was highest for freshly hatched larvae and declined gradually with age. Completely non-active larvae were still able to penetrate, although penetration efficiency was lower.

No information is available on the embryonic development of Southern Ocean cephalopods. Estimations of developmental times can only be made by extrapolation using data from other geographical areas. Based on known relationships between environmental temperature and embryonic development time, it appears that below 5°C even the smallest squid eggs measuring 0.6–1.0 mm in diameter need one to two months to develop to hatching. At c. 2°C, the embryonic development of these small eggs would probably cover between three and five months. Very large octopod eggs are known to develop over time spans of at least one year. Protection of the developing embryos either by long-lasting capsules laid at appropriate spawning sites, or by active ‘brooding’ (incirrate octopods) is required for embryonic survival and hatching success. The physiological conditions controlling the onset of hatching at very low temperatures are unknown; postponement of hatching appears to be common in cold waters.

Le complexe d'espèces jumelles mariae se développe dans les creux de rocher (rock pools) contenant de l'eau de mer. L'espèceAedes mariae Sergent & Sergent, 1903 se rencontre sur le pourtour du bassin tyrrhénien et notamment sur la côte rocheuse del'ouest algérien. Le suivi durant une année de la dynamique de la population a permis de démontrer que le cycle biologique estsensiblement différent de celui observé en Méditerranée septentrionale. La présence de femelles actives aux mois de janvier etfévrier et d'un stock réduit d'oeufs dans les gîtes, montrent que la population d'Ae. mariae des côtes occidentales algériennesadopte une double stratégie de développement. Les éclosions les plus importantes sont enregistrées lorsque la conductivité estcomprise entre 52.000 et 75.000 µS.m-1 résultant d'un apport d'eau de mer et de la concentration en sels sous l'effet de l'évaporation.Les conductivités supérieures à 100.000 µS.m-1, entravent l'éclosion des oeufs en favorisant la dessiccation biologique.

Embryonic development time has been of long-standing interest to physiologists and ecologists because of its importance in understanding the behaviour and ecology of reproduction, and the evolution of reproductive strategies in animals. Yet, the relationship of embryonic development time to both neonate mass and incubation temperature remains poorly understood for most taxonomic groups. Here we define the relationship of embryonic development time to neonate mass (embryo mass at birth) and incubation temperature for a broad range of animals, ectothermic and endothermic, invertebrate and vertebrate, microscopic and gigantic. We begin by establishing the relationship of embryonic development time to neonate mass for a broad array of zooplankton, fishes and amphibians for temperatures from 5 to 20°C. Next, we compare the relationships of embryonic development time to neonate mass in these aquatic ectotherms to those for terrestrial ectotherms (reptiles) and endotherms (birds and mammals) in terms of degree-days. The similar nature of these relationships allows us to define embryonic development time as a function of neonate mass for many of the species considered here with a single equation in terms of degree-days for incubation temperatures ranging from 5 to 38°C. This relationship establishes a basis by which to compare differences in embryonic development time among species.

Hatching has been suggested to occur as a result of protease-mediated lysis and the blastocoele tension. However, even if rupturing is initiated at multiple sites, interestingly only a single site is used for escape. This implies that there are several mechanisms involved in hatching. In this study, the involvement of actin filaments in mouse embryo hatching was examined. We treated mouse embryos with cytochalasin B for 12 h or 24 h at the morula, middle blastocyst, expanded blastocyst, lobe-formed blastocyst and hatching blastocyst stages, and measured the amount and distribution of actin filaments using a confocal microscope. At morula, middle blastocyst, lobe-formed blastocyst and hatching blastocyst stages embryonic development was completely arrested by cytochalasin B. However, when transferred to cytochalasin-B-free medium, the embryos resumed development and escaped the zona pellucida. In the expanded blastocysts development was almost completely inhibited by cytochalasin B, but rupturing occurred in some embryos. However, development stopped completely at the ruptured stage. Distribution of actin filaments was prominent at rupturing and hatching sites regardless of cytochalasin B treatment. The amount of actin filaments was prominent at hatching embryos compared with other developmental stages of embryos. These actin filaments were distributed intensively between the trophectodermal cells, and formed locomotion patterns. Taken together, these results suggest that not only tension and lytic enzymes are required to rupture, but the activity of actin filaments may have a crucial role in the process of hatching.

The monogenean (platyhelminth) skin parasite Entobdella soleae from the common sole (Solea solea) lays tetrahedral eggs. One of the 4 corners of the tetrahedron is a detachable operculum which is bonded to the rest of the egg-shell by cement. Most of this cement layer, beginning at the inner surface of the shell and running through almost to the outer surface (a distance of about 2 μm), is more or less uniform in thickness (30–38 nm), or tapers slightly. About 345 nm from the outer surface the cement layer narrows abruptly to about 10 nm. The cement is exposed on the inner surface of the shell, but in most eggs a layer of shell about 10 nm thick covers the narrow outer region of the cement layer. When experimentally perforated eggs were incubated with trypsin, the wide inner layer of cement was digested, but the narrow outer region initially remained intact. These observations are discussed in relation to the following (1) survival of the eggs during embryonic development, (2) hatching, (3) the ‘hinge’ often connecting the operculum to the empty egg-shell, (4) the rapid hatching that occurs in some other monogeneans.

Observations were made in Senegal between 1997−1998 on the length of embryonic development in Octopus vulgaris. The data were from eggs which had been laid by two females in the open sea and fourteen females in concrete tanks. The observations in natural surroundings are the first concerning this species. Hatching occurred after 15−42 d at 27 °C, 29−49 d at 22−23 °C, 57−65 d at 21 °C and 80−87 d at 17 °C. These values are higher than the few data previously recorded in European aquariums, particularly for temperatures under 20 °C.

Apoptosis is a form of naturally occurring cell death during development and it is characterised by extensive DNA fragmentation. Apoptosis is easily detected in the gill epithelium of brown trout embryos in ultrathin sections (Rojo et al. 1997). Here we provide the first biochemical evidence for apoptosis in the gill epithelium of brown trout embryos, using in situ end-labelling of DNA breaks (Gavrieli et al. 1992). Embryos at d 57 of development as well as those at hatching, were processed to analyse the distribution of apoptotic cells in the gills. The extent of apoptosis revealed by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling method technique is considerably greater than apoptosis detected by nuclear morphology. This method revealed that apoptosis was frequent at hatching, although it was also present during embryonic development. The presence and distribution of stained nuclei were different depending on the developmental stage. In embryos of 57 d, apoptotic flattened nuclei were dispersed in the gill epithelium, whereas at hatching, they were mainly grouped in the tips of the filaments and in the gill arches. TUNEL also revealed a distinct pattern of nuclear staining: at hatching, the intense staining covered the entire cell, but in embryos it was restricted to the nucleus. These results show the functional relevance of apoptosis at hatching, when apoptosis seems to be the unique process by which cell numbers in the gill epithelium are adjusted, in order to prepare for the new extrinsic conditions affecting the free-living life of alevins.

The survival of turbot eggs and the rearing capacities of larvae stemmed from artificial fertilization practices using frozen-thawed spermatozoa were evaluated. Furthermore, the viability of sperm samples stored during a 9 month period in liquid nitrogen was assessed. No significant difference in the fertilization rate, hatching rate, survival and wet weight of 10-day old larvae were observed using fresh or frozen-thawed spermatozoa. The motility recorded at 10 s and 60 s post-activation and the fertilization capacity of frozen-thawed spermatozoa were not significantly decreased during a 9 month storage period in liquid nitrogen. These results confirm the high quality of the turbot spermatozoa stemmed from the cryopreservation process, allowing their use for routine aquaculture practices.

The total lipid content of the dry weight of whole cysts and 2nd-stage juveniles (J2) of Globodera rostochiensis was 17·1% in dry cysts, 20·9% in cysts soaked in distilled water (DW), 20·3% in cysts that had been in potato root diffusate (PRD) for 7 days, 7·3% in cysts that had been in PRD for 28 days and 29·2% for hatched J2. The fatty acid composition of the total lipid did not differ between dry cysts, cysts in DW and cysts in PRD for 7 days. However, major differences in the fatty acid composition of all lipid classes were found between rehydrated cysts in PRD and freshly hatched J2. After hatching, the degree of saturation and the percentage of monounsaturated fatty acids decreased and the percentage of polyunsaturated fatty acids increased considerably, especially in the free fatty acid fraction, where C20[ratio ]1 showed an 8-fold decrease and C20[ratio ]4 a 33-fold increase. There was a difference in the fatty acid composition of cysts in PRD for 7 days and cysts in PRD for 28 days (after most of the J2 had hatched); with increased time in PRD the percentage of polyunsaturated fatty acids increased and the percentage of monounsaturated fatty acids decreased in all lipid classes. Differences in the fatty acid profiles between cysts in PRD for 28 days and hatched J2 were found mainly in the free fatty acid and the non-acidic phospholipid fractions. The free fatty acid fraction of the cysts was dominated by monounsaturated fatty acids (52%), whereas the same fraction of hatched J2 contained large amounts of polyunsaturated fatty acids (60%). These polyunsaturated fatty acids, especially C20[ratio ]4, might play an important part in nematode–plant interactions during infection.

The potato cyst nematode (PCN) Globodera rostochiensis, like other parasitic nematodes, needs to synchronize its life-cycle with that of its host. This synchrony is achieved by the invasive-stage juvenile remaining dormant within its egg until stimulated to hatch by the presence of root diffusates of its host. Root diffusates may induce changes in gene expression in PCN, some of which may be important in the transition to a parasitic mode of existence. We have used a range of techniques including differential display to examine gene expression during stimulation and hatching of PCN. We find that few changes in gene expression appear to be induced directly by root diffusates. Instead, changes in gene expression seem to occur during or immediately after the hatching process. These results are discussed in the context of the host–parasite relationship.

Survival of Silurus glanis ovulated oocytes (ova) measured by the capacity of normal development i.e. percentage of normal and abnormal hatched larvae after in vivo and in vitro storage and exposure to sperm activating or immobilizing solutions and urine was studied. In the case of ovulated oocytes left in the ovaries, total hatching and abnormal hatched larvae (in % of hatching) were respectively 74 and 8.2% immediately after ovulation, 77 and 8.6% after 2 h, 54 and 18% after 4 h, and 38 and 50% after 6 h of storage. Four and 6 h stay of ovulated oocytes in ovaries resulted in a significant increase of abnormal hatched larvae (p < 0.01). For the ova stored in vitro, the capacity to undertake a full embryonic development after fertilization was not significantly changed either after 8.5 h at 19 °C or 3.5 h at 25 °C; there was no ova survival at all after 3.5 h storage at 8 °C, 12 h at 19 °C and 8.5 h at 25 °C. There was a significant increase of abnormal larvae after 3.5 h at 25 °C (74%, p < 0.001) and after 8.5 h at 19 °C (37%, p < 0.05). Exposure of non inseminated ova to water buffered to pH 7.0 (5 mM Tris-HCl) to sperm activating solution (17 or 41 mM NaCl, 5 mM Tris-HCl, pH 8.0) or to immobilizing solution (200 mM NaCl, 30 mM Tris-HCl, pH 7.0) resulted in a regular and rapid decrease of their capacity of development; this was 10% of normal hatched larvae within 4 min in water, and within 6-8 min in the NaCl solutions. A similar situation was observed after exposure to urine with a loss of embryonic development of 30% within 3 min. These results indicate that all steps of the whole procedure of gamete collection and artificial insemination should be carried out rapidly as soon as possible after ovulation.