Improving postcryopreservation survival capacity: an embryo-focused approach

The major challenge for a greater dissemination of in vitro produced (IVP) bovine embryos is to improve embryonic survival after cryopreservation. The involvement of embryonic lipids on this issue is well documented. However, it has been recognized that not only the amount of lipids that affects embryo cryotolerance, but the embryo survival capacity after cryopreservation is a rather multifactorial event. In this review, some strategies to improve embryonic lipid composition and postcryopreservation survival by modifying the embryos themselves to make them more cryopreservable are overviewed. The use of semi-defined and defined serum-free culture media, the addition of some chemicals in the culture media to modify embryo lipid composition, and the modulation of embryo cell membrane fluidity by cholesterol or unsaturated fatty acids added to the culture media and oocyte/embryo donor nutritional management with a diet enriched in polyunsaturated fatty acids, were described as alternatives for the improvement of IVP embryo survival after cryopreservation.

Reproductive responses of rabbit does to a supplemental lighting program

The objective was to investigate whether the productivity of rabbit does can be improved, when natural photoperiod is decreasing, by adopting a supplemental lighting program. Three experiments were conducted involving two groups: control, submitted to the natural decreasing photoperiod, and supplemented with a lighting program which provided 14 h light/24 h beginning at 10 weeks of age. In the first experiment, 20 nulliparous does, 10 from each group, were euthanized 8 h after being presented to a buck; the overall number of follicles, whose diameter exceeded I mm, was determined macroscopically. The right ovaries were collected, histologically analyzed, and electronically measured. In the second experiment, 30 nulliparous does, 15 from each group, were presented to a buck (day 1). Receptive does were euthanized on day 8 to evaluate embryonic survival (number of normal embryos/ovulation rate). In the third experiment, 48 nulliparous does, 24 from each group, were followed from the first presentation to the buck until the weaning of the first litter. The effect of treatment on reproductive and body weight traits of does, and litter performance traits, at birth and weaning, was evaluated. The average number of follicles whose diameter exceeded 1 mm was higher in the treatment group (12.05 +/- 1.07 vs. 8.63 +/- 1.00, P=0.03 7). Receptive does of the treatment group had heavier ovaries relative to those of the control group (790 +/- 59 vs. 470 +/- 64 mg, P=0.004), whereas no treatment difference regarding this trait was found for non-receptive ones. Treatment had a favorable effect on pregnancy rate of total exposed and of receptive does (80.0% vs. 33.3%, P=0.01, and 92.3% vs. 50.0%, P=0.02, respectively). The number of underdeveloped embryos was lower (0.067 +/- 0.380 vs. 2.500 +/- 0.455, P=0.004), embryonic survival up to day 8, and uterus weight was higher in the treatment group (0.839 +/- 0.075 vs. 0.534 +/- 0.087, P=0.033 and 13.83 +/- 0.72 vs. 10.99 +/- 0.84, P=0.037, respectively). Number of presentations tended to be lower (1.32 +/- 0.17 vs. 1.75 +/- 0.16, P=0.077) and adjusted litter size in the first reproductive cycle tended to be higher (7.09 +/- 0.89 vs. 5.22 +/- 0.68, P=0.091) in the treatment group relative to the control.

Phosphatidylcholine and Sphingomyelin Profiles Vary in Bos taurus indicus and Bos taurus taurus In Vitro- and In Vivo-Produced Blastocysts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of heat stress on development, quality and survival of Bos indicus and Bos taurus embryos produced in vitro

Heat stress is an important cause of poor development and low survival rates in bovine embryos. Experiments were conducted to test the hypothesis that Bos indicus embryos are more resistant to heat stress than are Bos taurus embryos. In experiment 1, Nelore and Jersey embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 6 hours), developmental ratios were assessed at Day 7 (Day 0 = day of fertilization), and blastocysts were frozen for RNA extraction. Experiment 2 evaluated expression of COX2, CDX2, HSF1, and PLAC8 in previously frozen blastocysts. In experiment 3, Nellore and Angus embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 12 hours) and transferred to recipients on Day 7. In experiment 4, embryos developed as in experiment 3 were fixed for Terminal deoxynucleotidyl transferase dUTP nick end labeling labeling and total cell counting. In experiment 1, heat stress decreased the percentage of Jersey oocytes that became blastocysts, but had no effect on Nellore embryos (34.6%, 25.0%, 39.5%, and 33.0% for Jersey control, Jersey heat-stressed, Nellore control, and Nellore heat-stressed oocytes, respectively; P < 0.05). In experiment 2, heat stress decreased (P < 0.05) expression of CDX2 and PLAC8, with higher expression of these genes in Nellore embryos than in Jersey embryos. Heat stress also decreased (P < 0.05) expression of COX2 in Jersey embryos, but had no effect on Nellore embryos. Expression of HSF1 was decreased (P < 0.05) by heat stress in both breeds, with a greater effect in Nellore embryos. In experiment 3, heat stress tended (P = 0.1) to decrease the percentage of pregnancies among cows (Day 30 to 35) that received Angus embryos. In experiment 4, heat stress increased (P < 0.05) the percentage of apoptotic blastomeres, but had no breed-specific effects. In addition, Nellore embryos had fewer (P < 0.05) Terminal deoxynucleotidyl transferase dUTP nick end labeling- positive blastomeres than did Angus embryos. We concluded that the detrimental effects of heat stress were dependent upon embryo breed and were more evident in Bos taurus embryos than in Bos indicus embryos. © 2013 Elsevier Inc.

Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effects of different dietary lipid sources on the survival, growth, and fatty acid composition of South American catfish, Pseudoplatystoma fasciatum, Surubim, juveniles

The present study examines the effect of four semi-purified diets (casein-gelatin based) where the source of fatty acids was free (esterified) oleic acid and linoleic acid (LA) (LOA diet), linseed and olive oil (predominantly LA and linolenic acid) (LO diet), cod liver oil (rich in highly unsaturated fatty acids) (CLO diet), and soybean lecithin (phospholipids; mostly LA) (LE diet) on the growth of juvenile South American catfish (surubim, Pseudoplatystoma fasciatum, Pimelodidae) (0.98 +/- 0.04 g individual weight). Fish were fed at a restricted-readjusted feeding rate for 8 wk. At the end of the experiment, LE-diet-fed fish grew significantly larger than those of the other three groups (P < 0.05). Considerable cannibalism was observed in all the treatments. It is suggested that the quantitative growth performance may possibly change under other conditions, with less or no cannibalism. Survival did not differ significantly among the fish fed four different diets. Muscle and liver lipid contents did not vary among dietary treatments (P > 0.05), but whole-body lipid concentrations were affected by dietary treatments. Fish fed LE diet contained significantly lower lipid level than those fed three other diets (P < 0.05). Muscle and liver fatty acid profiles reflected dietary fatty acid composition. Arachidonic acid level was significantly higher in muscle and liver of fish fed LOA and LE diets than in those fed LO and CLO diets. The results suggest that the efficiency of elongation and desaturation of 18C fatty acids depends on the dietary lipid source, and South American catfish has considerable capacity to transform linoleate to arachidonate.

Efeito da escassez de alimento no desenvolvimento pós-embrionário e no potencial reprodutivo de Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae) em laboratório

As conseqüências da escassez alimentar no período pós-embrionário e potencial reprodutivo de Chrysoperla externa (Hagen) em laboratório foram avaliadas. Larvas de 1.°, 2.º e 3.° ínstares receberam alimento apenas no 1.°, 2.º ou 3.° dia após a ecdise. Nos períodos em que antecederam ou sucederam os testes com os respectivos ínstares, as larvas receberam uma, duas e três unidades de alimentação (UA) a cada dois dias para o 1.°, 2.º e 3.° ínstares, respectivamente. Cada UA constou de um disco de cartolina contendo ovos de Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae). Diariamente, avaliou-se a viabilidade nos diferentes ínstares, da fase larval e pupal, assim como os períodos de pré-oviposição e oviposição, o número e a viabilidade de ovos. A escassez de alimento durante o 1.° ínstar pode ser suprida no decorrer do desenvolvimento larval. Entretanto, as larvas precisam encontrar alimento nas primeiras 48 horas de vida, pois a viabilidade nesse ínstar foi decrescendo com a ausência de alimento, podendo ocorrer 100% de mortalidade quando as larvas não se alimentam em até 48 horas após a eclosão. Os períodos de pré-oviposição e oviposição, assim como a fecundidade, não foram influenciados pela escassez de alimento durante o 1.° ínstar larval. Durante os 2.° e 3.° ínstares larvais, C. externa necessita de alimentação rica em proteína, pois a ingestão de apenas água e açúcares nesses estádios ocasionou alta mortalidade. Entretanto, a fecundidade e viabilidade dos ovos não foram afetadas pela escassez de alimento nesses ínstares.

Aspectos biológicos de Chrysoperla raimundoi Freitas & Penny (Neuroptera, Chrysopidae)

Os crisopídeos são importantes predadores e são encontrados em diversos agroecossistemas. A temperatura é um dos fatores determinantes para o desenvolvimento dos insetos, sendo que existe um gradiente de variação aceitável para cada espécie. Portanto, objetivou-se com este estudo verificar a influência de diferentes temperaturas sobre o desenvolvimento embrionário e pós-embrionário de C. raimundoi assim como verificar as conseqüências nas diferentes gerações. As fases jovens foram criadas em três ambientes com temperaturas constantes de 19,0, 25,0 e 31,0ºC e em um ambiente externo sem controle dos fatores abióticos. Foram observados a duração do período embrionário, do primeiro, segundo e terceiro ínstares, a duração do período larval, pupal e ovo-adulto, assim como, a sobrevivência em cada estágio, estádio e a porcentagem de emergência. O desenvolvimento embrionário e pós-embrionário diferiu entre as gerações F2 e F4. Quanto à influência de diferentes temperaturas para a geração F2, os indivíduos criados a 31,0ºC demonstraram redução na taxa de desenvolvimento com relação aos outros dois ambientes. Para a geração F4, observou-se que as diferentes temperaturas influenciaram no desenvolvimento, sendo que a 19,0ºC houve prolongamento do mesmo.

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere- (NT-B) and fibroblast- (NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p < 0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p < 0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

In vitro Survival of Follicles Collected from Domestic Cats' Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF-1

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of moderate and severe heat stress on avian embryonic Hsp70 gene expression

Stress response is a universal mechanism developed by all organisms to deal with adverse changes in the environment, which lead to the synthesis of heat shock proteins (Hsps). In this study, the effect of moderate (41degreesC) and severe (44degreesC) heat stress on Hsp70 transcript expression pattern was investigated during chicken embryogenesis. Acute exposure to severe heat stress for one hour resulted in a fifteen-fold increase in Hsp70 mRNA levels. The return of stressed embryos to normal incubation temperature resulted in Hsp70 mRNA levels five-fold higher than control after three hours and normal levels after six hours. Moderate heat stress did not induce enhancements on Hsp70 mRNA levels. The spatial expression of Hsp70 transcripts was detected in embryos under normal incubation conditions. Whole-mount in situ hybridization analysis showed that Hsp70 transcripts were constitutively present in somite and in distinct encephalic domains (predominantly in prosencephalon and mesencephalon areas) of the chicken embryo. These results showed that Hsp70 induction is dependent on incubation temperature conditions, suggesting that early chicken embryos may induce a quick emergence response to cope with severe heat stress by increasing Hsp70 mRNA levels.

Effect of temperature on incubation period, embryonic mortality, hatch rate, egg water loss and partridge chick weight (Rhynchotus rufescens)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Live and formulated diet evaluation through initial growth and survival of jundiá larvae, Rhamdia quelen

A larvicultura da maioria das espécies de peixes enfrenta o desafio da dependência do alimento vivo (AL) e da falta de dietas formuladas (DF) que atendam plenamente às necessidades das larvas. A baixa digestibilidade e a qualidade nutricional das DFs são alguns dos fatores que explicam o insucesso quando as larvas recebem apenas FD. Para avaliar o efeito da combinação da DF com o AL no crescimento e na sobrevivência de larvas de jundiá (Rhamdia quelen), comparando com o uso separado da DF ou do AL, larvas recém eclodidas (5,57 mm; 1,41 mg) foram estocadas inicialmente em 12 aquários de 10 L (100 larvas por aquário). Quatro réplicas foram alimentadas ad libitum com uma das três dietas por 20 (para DF) ou 48 dias (para AL ou a combinação DF + AL). As larvas alimentadas com apenas DF apresentaram crescimento e sobrevivência reduzidos quando comparadas àquelas alimentadas com AL ou a combinação DF + AL. Adicionalmente, as larvas do tratamento DF + AL apresentaram maior crescimento em peso (170 mg) que aquelas alimentadas apenas com AL (110 mg). O melhor desempenho das larvas alimentadas com DF + AL mostra que a maioria dos nutrientes exigidos pelas larvas é fornecida mais adequadamente quando ambas as dietas são fornecidas juntamente. Contudo, trabalhos sobre nutrição larval poderão contribuir ainda mais sobre a elucidação deste tema quando feitas comparações com o uso combinado de DF + AL, do que apenas testando isoladamente novos ingredientes e fontes protéicas normalmente utilizadas na elaboração de dietas para juvenis e adultos.

Brycon gouldingi (Teleostei, Characidae): aspects of the embryonic development in a new fish species with aquaculture potential

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Observation of the embryonic development in Pseudoplatystoma coruscans (Siluriformes: Pimelodidae) under light and scanning electron microscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)