Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring a-fetoprotein, alpha-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-alpha-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-alpha-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

Chorioallantoic and yolk sac placentation in Thrichomys laurentinus (Echimyidae) and the evolution of hystricognath rodents

The evolutionary history of Hystricognathi is associated with major transformations in their placental system. Data so far indicate that key characters are independent from size dimensions in medium to very large species. To better understand the situation in smaller species, we analyzed placental development in a spiny rat, Thrichomys laurentinus. Fourteen individuals ranging from early implantation to near term were investigated by histology, immunohistochemistry, proliferation activity and electron microscopy. Placentation in Thrichomys revealed major parallels to the guinea pig and other hystricognath rodents with respect to the early and invasive implantation, the process of trophoblast invasion, the internal organization of the labyrinth and the trophospongium as well as the establishment of the complete inverted yolk sac placenta. In contrast to systematically related small-sized species, the placental regionalization in Thrichomys was characterized by a remarkable lobulated structure and associated growing processes. Reverse to former perspectives, these conditions represented ancient character states of hystricognaths. The subplacenta was temporarily supplied by both the maternal and fetal blood systems, a rare condition among hystricognaths. The extraplacental trophoblast originating from the subplacenta was partly proliferative in mid gestation. In conclusion, the presented results indicated that only minor variations occurred in small-sized hystricognath species, independent of their systematic interrelationships. Previous views were supported that placentation in hystricognaths followed an extraordinary stable pattern, although the group had distinct habitats in South America and Africa that were separated 30-40 million years ago. J. Exp. Zool. (Mol. Dev. Evol.) 318:13-25, 2012. (C) 2011 Wiley Periodicals, Inc.

Morphology and Involution of the Yolk Sac during Early Gestation Bovine (Bos indicus)

Background: The bovine yolk sac derives from visceral endoderm and its development occurs between days 18-23 of gestation. The study of this membrane is important for comparative data and has already been performed in rodents, sheep and in cattle, especially Bos taunts. In species Bos indicus the yolk sac has not quite been studied and is believed that there are morphological differences between these species. The yolk sac undergoes a process of involution and degeneration during embryonic development and none vestige of it is found in late gestation. The period in which occurs the involution of the yolk sac coincides with the period of increased pregnancy loss in cattle, and changes in the morphology of this membrane may indicate the reasons for such high loss rates. Thus, considering that the yolk sac is important for embryonic circulation and metabolic transmission, besides participating actively in the process of cattle placentation, this study aimed characterize morphologically the involution of the bovine yolk sac. Materials, Methods & Results: The early gestational period was determined between days 20 and 70 post-insemination (p.i), according to the exterior characteristics of embryo/fetus. For macroscopic analyzes the uterus was dissected to expose the fetal membranes and subsequently the embryo/fetus was photographed. The samples were fixed for light microscopy and transmission electron microscopy. The yolk sac that emerges from the ventral part of the embryo was prominent and composed by a central part with two thin peripheral projections of different lengths. The bovine yolk sac with about 9 cm on day 25 p. i. of pregnancy permanently decreased its total length during this study. Histologically, the yolk sac is composed of three cell layers: the mesothelium, the mesenchyme and the endoderm. In mesenchyme are found blood islets. In the endoderm are formed cells invaginations toward the mesenchyme originating small canaliculi. The ultrastructure of yolk cells presented many mitochondria, rough endoplasmic reticulum, vesicles, euchromatin and the presence of two nucleoli, Discussion: The real first blood circulation in the bovine is attached with the development of yolk sac, differently from other membranes, such as the corium, that does not present evidence of vascularization by the age of 20-30 days. The erythroblasts found in the yolk sac are related to vasculogenesis and the process of differentiation of blood cells during the erythropoiesis. It could be observed on the histology of the yolk sac, in embryos of 30-50 days old, the presence of canaliculi and small folds of the epithelium. The canaliculi collapse is associated with the degeneration of the endoderm wall of the yolk sac. The organelles present in the endoderm cells of the yolk sac are associated with the function of protein metabolism and in the exchange of substances between the mesenchyme and the mesothelium, For these findings, could be observed that the yolk sac epithelium is found active until the 50th day of gestation, and thereafter regresses. However, remnants of this membrane may be present until the 70th day, These features may represent a presence of an active chorionvitelline placenta in this period responsible for the maintenance of pregnancy whereas the chorioallantoic placenta is not definitively established.

Development of of yolk sac inversion in Galea spixii and Cavia porcellus (Rodentia, Caviidae)

Caviomorph development includes an inverted yolk sac. Since principle processes are not understood, we investigated its differentiation in Galen and re-examined material from the guinea pig. Galea showed the typical caviomorph conditions in blastocyst development and the nature of the definitive yolk sac, formed of the visceral layer that became villous, proliferative, vascularized and attached to the uterus and placenta. In contrast to what was known before, in both species parts of the parietal yolk sac and a yolk sac cavity were temporarily present. Data suggest that early yolk sac development in caviomorphs is more complex than thought before. (C) 2012 Elsevier Ltd. All rights reserved.

An unusual feature of yolk sac placentation in Necromys lasiurus (Rodentia, Cricetidae, Sigmodontinae)

We studied the development of the inverted yolk sac in a New World rodent, Necromys lasiurus during early placentation. Ten implantation sites were investigated by means of histology, immunohistochemistry and electron microscopy. The yolk sac was villous near its attachment to the placenta. Elsewhere it was non-villous and closely attached to the uterus. The uterine glands were shallow and wide mouthed. They were associated with vessels and filled with secretion, suggesting the release of histotroph. This feature was absent at later stages. The intimate association of the yolk sac with specialized glandular regions of the uterus may represent a derived character condition of Necromys and/or sigmodont rodents. (C) 2012 Elsevier Ltd. All rights reserved.

Modulation of plasma levels and percentages of incorporation of ω-3 PUFAs in egg yolk under the influence of supplementation sources rich in omega 3 to diet of laying

Hundred forty-four Shaver White laying hens were used over a 4 week experimental period to investigate the effect of 3% of soybean oil, corn oil (MIL), canola oil, flaxseed oil (LIN), salmon oil (SAL) or tuna and sardine oil (SR/AT) added to the diets, upon the fatty acid egg yolk composition, blood plasma levels and incorporation time of each fatty acid into the egg yolk. Hens were allocated into 72 cages and the experimental design was a 6 x 6 randomized factorial model. Hens fed 3% of different oils, responded with increased polyunsaturated fatty acids omega 3 (ω-3 PUFAs), except for corn oil. The addition of flaxseed, soybean or corn oil into the diet increased the PUFAs levels into the egg yolk and in the blood plasma. Adding tuna and sardine oil into the diet increased the concentration of yolk saturated fatty acids. The levels of ω-3 PUFAs were increased in the tuna and sardine oil treatment, while the flaxseed oil increased the plasma fatty acids. The deposition of 349.28 mg/yolk of a-linolenic fatty acids (ALA) was higher in the group fed LIN, while the higher equal to 157.13 mg DHA/yolk was observed in group SR/AT. In the plasma, deposition increased from 0.33% (MIL) for 6.29% ALA (LIN), while that of DHA increase of 0.47% (MIL) for 4.24% (SAL) and 4.48% (SR/AT) and of 0.98% (MIL) for 6.14% (SR/AT) and 8.44% (LIN) of ω-3 PUFAs. The percentage of EPA into the yolk and plasma was higher for the hens fed 3% tuna and sardine oil diet, as well as the levels of yolk DHA. The concentration of DHA into the plasma was higher for the salmon and tuna/sardine oil treatments. The PUFAs yolk decreased during the first eight days of experiment, while the ω-3 PUFAs increased during the same period. The concentration of ALA increased until ten days of experiment, while the percentage of EPA and DHA increased up to the eighth experimental day

Lipid profile of the yolk under the influence of supplementaries sources rich in Ω-3 PUFAs in the diet of laying hens in the time

Two hundred eighty-eight 32-wk-old Hisex White laying hens were used in this research during a 10 weeks period, arranged in a 2 x 5 completely randomized factorial design, with three replicates of eight birds per treatment. Two groups: fish oil (OP) and Marine Algae (AM) with five DHA levels (120, 180, 240, 300 and 360 mg/100 g diet) were assigned including two control groups birds fed corn and soybean basal diet (CON) and a diet supplemented with AM (AM420) to study the effect of time 0, 2, 4, 6 and 8 weeks (wk) on the efficiency of egg yolk fatty acid enrichment. The means varied (p<0.01) of 17.63% (OP360) to 22.08% (AM420) is the total Polyunsaturated Fatty Acids (PUFAs) and 45.8 mg/g (OP360), 40.37 mg/g (OP360, 4 wk) to 65.82 mg/g (AM420) and 68.79 mg/g/yolk (AM120, 8 wk) for n-6 PUFAs. On the influence of sources and levels in the times, the means of n-3 PUFAs increased by 5.58 mg/g (AM120, 2 wk) to 14.16 mg/g (OP360, 6 wk) when compared to average of 3.34 mg PUFAs Ω/g/yolk (CON). Usually, the means DHA also increased from 22.34 (CON) to 176.53 mg (μ, OP360), 187.91 mg (OP360, 8 wk) and 192.96 mg (OP360, 6 wk) and 134.18 mg (μ, OP360), 135.79 mg (AM420, 6 wk), 149.75 mg DHA (AM420, 8 wk) per yolk. The opposite was observed for the means AA, so the effect of the sources, levels and times, decreased (P <0.01) of 99.83 mg (CON) to 31.99 mg (OP360, 4 wk), 40.43 mg (μ, OP360) to 61.21 mg (AM420) and 71.51 mg AA / yolk (μ, AM420). Variations of the average weight of 15.75g (OP360) to 17.08g (AM420) yolks of eggs de 32.55% (AM420) to 34.08% (OP360) of total lipids and 5.28 g (AM240) to 5.84 g (AM120) of fat in the yolk were not affected (p>0.05) by treatments, sources, levels and times studied. Starting of 2 week, the hens increased the level of n-3 PUFAs in the egg yolks, being expressively increased (p<0.01) until 4 weeks, which after the increased levels of n-3 PUFAs tended to if stabilize around of time of 8 experimental weeks, when it was more effective saturation of the tissues and yolk.

Vitellogenin content in fat body and ovary homogenates of workers and queens of Melipona quadrifasciata anthidioides during vitellogenesis

Vitellogenin (Vg) is an egg yolk protein that is produced primarily in the fat body of most female insects. In the advanced social structure of eusocial honeybees, the presence of the queen inhibits egg maturation in the workers ovaries. However in the stingless bee Melipona quadrifasciata, the workers always develop ovaries and lay a certain amount of eggs while provisioning the brood cells with larval food during what is known as the worker nurse phase. The present work is a comparative study of the presence of Vg in homogenates of the fat bodies and ovaries of the nurse workers, and the virgin and physogastric queens of M. quadrifasciata. The presence of Vg was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using Apis mellifera anti-egg antibody. Vg was not detected in the fat bodies or ovaries of the workers, but it was found in the ovaries of virgin and physogastric queens and in the fat body of physogastric queens. The results are discussed, taking into account the reproductive state of the individuals and the other possible roles of Vg, such as a storage protein for metoabolism of other organs.

Embryonic development and larval stages of Steindachneridion parahybae (Siluriformes: Pimelodidae) - implications for the conservation and rearing of this endangered Neotropical species

Steindachneridion parahybae is a freshwater catfish endemic to the Paraiba do Sul River and is classified as an endangered Neotropical species. An increasing number of conservation biologists are incorporating morphological and physiological research data to help conservation managers in rescue these endangered species. This study investigated the embryonic and larval development of S. parahybae in captivity, with emphasis in major events during the ontogeny of S. parahybae. Broodstocks were artificially induced to reproduce, and the extrusion occurred 200-255 degree-hours after hormonal induction at 24 degrees C. Larval ontogeny was evaluated every 10 minutes under microscopic/stereomicroscopic using fresh eggs samples. The main embryogenic development stages were identified: zygote, cleavage, including the morula, blastula, gastrula phase, organogenesis, and hatching. The extruded oocytes showed an average diameter of 1.10 +/- 0.10 mm, and after fertilization and hydration of eggs, the average diameter of eggs increased to about 1.90 +/- 0.60 mm, characterized by a large perivitelline space that persisted up to embryo development, the double chorion, and the poles (animal and vegetative). Cell division started about 2 minutes after fertilization (AF), resulting in 2, 4, 8 (4 x 2 arrangement of cells), 16 (4 x 4), 32 (4 x 8) and 64 (2 x 4 x 8) cells. Furthermore, the blastula and gastrula stages followed after these cells divisions. The closed blastopore occurred at 11 h 20 min AF; following the development, the organogenetic stages were identified and subdivided respectively in: early segmentation phase and late segmentation phase. In the early segmentation phase, there was the establishment of the embryonic axis, and it was possible to distinguish between the cephalic and caudal regions; somites, and the optic vesicles developed about 20 h AF. Total hatching occurred at 54 h AF, and the larvae average length was 4.30 +/- 0.70 mm. Gradual yolk sac reduction was observed during the first two days of larval development. The first feeding occurred at the end of the second day. During the larval phase, cannibalism, heterogeneous larval growth and photophobia were also observed. This information will be important in improving the artificial reproduction protocols of S. parahybae in controlled breeding programs.

Gonadal steroids levels and vitellogenesis in the formation of oocytes in Prochilodus lineatus (Valenciennes) (Teleostei: Characiformes)

The objective of this study was to obtain information about the possible mechanisms related to poor reproductive performance in tropical rheophilic fish. To that effect, cages (Cs) and earthen ponds (EPs) were used as experimental systems to provide unsuitable and suitable conditions, respectively, for curimbata (Prochilodus lineatus) breeders. Fish were maintained under experimental conditions for 18 months, and during this period females were randomly sampled every two months for biometric analysis (n=30), blood (n=5/sampling) and ovary (n=5/sampling). After this period EPs females (EPFs) and Cs females (CFs) were submitted to the induced breeding experiments. The results showed that rearing curimbata for such long time in a cage at this stocking density, reduces its growth, plasma E2 levels and vitellogenesis. During vitellogenesis, the mean plasma estradiol levels of CFs were three times lower than those of EPFs (P<0.01). CFs presented poorer results than EPFs for all the examined parameters of reproductive performance. Taken together these data showed that the reduced estradiol levels during vitellogenesis (and the consequently less intense transition from the previtellogenic to vitellogenic phase) and reduced amounts of yolk are mechanisms associated with the formation of low quality oocytes and shortened and delayed breeding season in this species. Moreover, our data showed that the onset of vitellogenesis (six months before the spawning season) must be considered as a key period related to the formation of oocytes of good quality, and adequate management should be provided throughout the year.

Morphological records of oocyte maturation in the parthenogenetic tick Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae)

Oocyte maturation in the thelytokous parthenogenetic tick Amblyomma rotundatum was examined for the first time using light and scanning electron microscopy. The panoistic ovary lacks nurse and follicular cells and is a single continuous tubular structure forming a lumen delimited by the ovarian wall. Oocytes of tick species are usually classified according to cytoplasm appearance, the presence of germinal vesicle, the presence of yolk granules, and the chorion. However, for this species, we also use oocyte size as an auxiliary tool since most oocytes were in stages I-Ill and were histologically very similar. Oocytes were classified into five development stages, and specific characteristics were observed: mature oocytes with thin chorion, pedicel cells arranged forming an epithelium with two Or more oocytes attached by the same structure, and a large number of oocytes in the process of reabsorption. (C) 2011 Elsevier GmbH. All rights reserved.

Fatty acids as a tool to compare cachara (Pseudoplatystoma reticulatum) (Siluriformes: Pimelodidae) and hybrid (Pseudoplatystoma corruscans x Pseudoplatystoma reticulatum) larvae during early development

In this study, we investigated the physiological alterations during ontogeny for cachara (Pseudoplatystoma reticulatum) and their hybrid larvae (Pseudoplatystoma corruscans x P. reticulatum) using lipids and fatty acids as physiological tools to elucidate the basis for differences in these groups' productivity in an industrial setting. Eggs and larvae samples were collected during January and February of 2008 in the city of Bandeirantes, MS, and were divided into three primary phases: phase I (0-16 h after fertilization); phase II (24 h after fertilization to 6 days after fertilization); and phase III (7-25 days after fertilization). The larvae of both groups showed a high degree of similarity, suggesting that the hybrid larvae showed a high level of heritability from the cachara broodstock. Analysis of the total lipid content provided evidence that there is no alteration in lipid concentration during ontogeny for both groups (i.e., the cachara and hybrids). However, the fatty acid profile showed that during the endogenous feeding period (phase II), when the larvae must use the energy reserves from the mother, the cachara larvae used mainly monounsaturated fatty acids for development. This is typical for most fish species, though notably, the hybrids preferentially used saturated fatty acids. Furthermore, certain specific changes demonstrate unique patterns of energy utilization and structural substrates, which may aid in elucidating the empirical differences reported by fish farmers (i.e., that the hybrids perform better than cacharas in captivity).

The influence of captive breeding on the fatty acid profiles of Salminus hilarii (Characiformes: Characidae) eggs and larvae

In addition to the strong influence of the broodstock diet on the development and survival of offspring, domestication may also interfere with the larval life success. We obtained eggs from wild and domesticated Salminus hilarii females and domesticated males. Wild females were caught in the Tiete River and tributaries, and the domesticated females were born three years before the beginning of the experiment in the Ponte Nova Fish Farm. Animals from both groups were fed with the same feed to exclude feed variables. The eggs and larvae were sampled at 0, 8, 16, and 28 h after spawning (HAS), with the last sampling (28 HAS) coinciding with hatching time. After hatching, samplings proceeded at 32, 48, 66, and 96 HAS, with the last sampling (96 HAS) corresponding to the end of yolk sac consumption. Finally, the last experimental period was during the larvae exogenous feeding phase, at 102, 118, 166, and 214 HAS. Our data revealed that domestication of S. hilarii females influenced fatty acid (FA) metabolism during embryo and larva development. However, the structure of membrane phospholipid FA remained mostly stable, with changes principally in the neutral fraction. When the external conditions, mainly water and feed quality, remained constant, domestication of S. hilarii females did not significantly affect the structural FA composition but influenced the selectivity of consumption and/or storage of specific FA.

Enterobacteria Isolation in Ostrich Eggs (Struthio camelus)

This study was conducted to determine the presence of enterobacteria in the eggs of ostriches reared on a farm with a history of reproductive failure. Ninety samples from twenty eggs were submitted to bacteriological tests. The results showed Enterobacteria growth in 100% of the eggs. The microorganisms isolated were Hafnia alvei in 50% (10/20), Serratia spp. in 20% (4/20), Escherichia coli in 15% (3/20), and Citrobacter freundii in 15% (3/20). All eggs presented poor eggshell quality, which favored enterobacteria contamination. Hafnia alvei was present only in the internal egg structures (albumen and yolk sac), suggesting the possibility of vertical infection.

Vitrification of primordial germ cells using whole embryos for gene-banking in loach, Misgurnus anguillicaudatus

In gene-banking, primordial germ cells (PGCs), which are embryonic precursor cells of germ cells, are useful for cryopreservation because PGCs have a potential to differentiate into both eggs and sperm via germ-line chimera. Here, we have established vitrification methods for PGCs cryopreservation using 12- to 17-somite stage embryos in loach, Misgurnus anguillicaudatus, which were dechorionated, removed their yolk and injected with green fluorescent protein (GFP) -nos1 3'UTR mRNA to visualize their PGCs. In order to optimize cryopreservation medium for vitrification, the toxicity of cryoprotectants was analyzed. Different concentrations (2, 3, 4, 5 m) of dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and propylene glycol (PG) as cryoprotectants were tested. Then, 5 m DMSO showed significantly-high toxicity. Based on this information, combinations called DMP (2 m (14.2% [v/v]) DMSO, 2 m (8.1% [v/v]) MeOH and 2 m (14.4% [v/v]) PG), DP (2 m (14.2% [v/v]) DMSO and 4 m (28.7% [v/v]) PG) and DE (2.1 m (15% [v/v]) DMSO and 2.7 m (15% [v/v]) EG) were evaluated for their toxicities and efficacy of PGCs cryopreservation using two types of equilibration step: direct immersion of cryopreservation media (one-step) and serial exposure to half and full concentration of cryopreservation media (two-step). Viable PGCs were obtained from post-thaw embryos which were cryopreserved by DP and DE with both 1- and 2-step equilibrations. Despite DP showing the highest toxicity, it gave the highest survival rate of embryonic cells after cryopreservation. When PGCs recovered from vitrified embryos were transplanted into host embryos at the blastula stage, the transplanted PGCs were able to migrate to a host genital ridge similarly as endogenous PGCs. It suggests that our methods could be useful to create a germ-line chimera for the production of gametes from PGCs of cryopreserved embryos.