Rodolfo Rumpf

Animal transgenesis: state of the art and applications

There is a constant expectation for fast improvement of livestock production and human health care products. The advent of DNA recombinant technology and the possibility of gene transfer between organisms of distinct species, or even distinct phylogenic kingdoms, has opened a wide range of possibilities. Nowadays we can produce human insulin in bacteria or human coagulation factors in cattle milk. The recent advances in gene transfer, animal cloning, and assisted reproductive techniques have partly fulfilled the expectation in the field of livestock transgenesis. This paper reviews the recent advances and applications of transgenesis in livestock and their derivative products. At first, the state of art and the techniques that enhance the efficiency of livestock transgenesis are presented. The consequent reduction in the cost and time necessary to reach a final product has enabled the multiplication of transgenic prototypes around the world. We also analyze here some emerging applications of livestock transgenesis in the field of pharmacology, meat and dairy industry, xenotransplantation, and human disease modeling. Finally, some bioethical and commercial concerns raised by the transgenesis applications are discussed.

Zebu (Bos indicus) ovarian preantral follicles : morphological characterization and development of an efficient isolation method

Preantral follicles are a major source of oocytes, and their utilization as an important tool to store large number of female gametes for future use in reproductive programs has been investigated. The increasing importance of studies in this subject, together with the important role of Zebu cattle in the economy of tropical and subtropical countries as well as their well-known differences from European cattle, led to this research. The present study aims to determine the best size interval for sectioning ovarian tissue to isolate preantral follicles from Zebu cows using a tissue chopper and to evaluate the follicular quality after isolation. Furthermore, it aims to provide information about the Zebu cow preantral follicle population and use this data (as a control) to evaluate the effectiveness of the tested isolation method. Testing eight different tissue sectioning size intervals, it was possible to conclude that the 125-microm-section interval is shown to be better than the intervals of 25, 50, 175 and 200 microm to isolate preantral follicles from Zebu cow ovaries. The 125-microm interval allowed the recovery of 26,050+/-1611 (mean +/- S.E.M.) preantral follicles per one-half ovary, while the number of preantral follicles in situ estimated by evaluation of histological sections was 35,288+/-2342 per one-half ovary. Thus, the mean (+/-S.E.M.) recovery rate (=[number of preantral follicles isolated/number of preantral follicles in situ in the same ovary] x 100) was 74.3+/-4.3%. The morphometrical analysis showed that Bos indicus preantral follicles are similar to B. taurus preantral follicles based on previous reports. In conclusion, this study showed that a simple, mechanical method can be used effectively to isolate a large number of intact preantral follicles from Zebu cow ovaries, with a high recovery rate.

Recovery, cryopreservation and fertilization potential of bovine spermatozoa obtained from epididymides stored at 5°C by different periods of time.

The objetive of the present study was to evaluate the effect of the interval between animals death and sperm recovery on the freezability and fertilizing ability of spermatozoa from bull epididymides stored for different periods of time. Testis from 25 bulls were collected at the abattoir 2h after the slaughter. In the laboratory spermatozoa from one epididymis were recovered and analysed for motility. The remaining epididymis was stored for 24h (G24), 48h (G48) and 72h (G72) at 5 degrees C. At the end of each time period, spermatozoa were recuperated and cryopreserved in Tris-egg yolk and glycerol. Pre-freeze and post-thaw sperm samples were taken to assess total and progressive motility, concentration, membrane integrity and acrosome integrity. For evaluation of fertilizing ability, in each time period five straws of each bull were thawed, pooled and used for in vitro embryo production. The results showed that after 48h of storage there was a decline in total motility, which did not change until 72h. Progressive motility, plasma membrane and acrosome integrity were not affected by any of the storage periods. Conversely, all sperm parameters, except progressive motility, were reduced after cryopreservation. Embryo production was less (P<0.05) in the treatments than in the reference group. However, there was no differences (P>0.05) in blastoycst rate among experimental groups. Considering all the embryos produced by epididymal spermatozoa a greater proportion of female embryos was observed, which was similar to the reference embryos. The shift observed on sex ratio toward female for those two groups was also observed when they were compared with the expected 1:1 ratio (P<0.05). The results showed the possibility to produced in vitro embryos using cryopreseved spermatozoa from epididymides and stored for long period of time at 5 degrees C. These procedures became an important tool for animal preservation when the sperm cells cannot be cryopreserved immediately after the animals death.

Changes in gene expression profiles of bovine embryos produced in vitro, by natural ovulation, or hormonal superstimulation.

Embryos produced by hormonal superstimulation have been used as an in vivo control in most published research on embryo gene expression. However, it is not known if this is the most appropriate control for gene expression profile studies. We compared the expression of GRB-10, IGF-II, IGF-IIR, MnSOD, GPX-4, catalase, BAX, and interferon-tau genes, in embryos produced in vivo by hormonal superovulation (SOV), by in vitro fertilization (IVF) or in vivo without any hormonal stimulus (NOV). GRB-10 was less expressed in NOV than IVF embryos, whereas no differences were found for the other genes. The genes related to stress response were then grouped and compared; the sum of expression of MnSOD, GPX-4, and catalase genes tended to be greater in IVF than NOV embryos. A correlation analysis was performed; we found a distinct behavior for NOV embryos when compared with SOV and IVF in the expression of GRB-10, IGF-II and IGF-IIR genes. However, the behavior of these genes was similar in SOV and IVF embryos. We conclude that ovarian hormonal stimulation can affect embryos by altering gene expression. Although this conclusion was based on investigation of only a few genes, we suggest that SOV embryos should be used with caution as a control in gene expression studies.

Effects of lowered temperatures and media on short-term preservation of zebu (Bos indicus) preantral ovarian follicles

The maintenance of follicle quality during the transportation of ovaries is essential for the successful cryopreservation and in vitro development of preantral follicles. The objective of this study was to evaluate the effect of cooling ovarian tissue on the conservation of zebu cow preantral follicles. Ovarian pieces were immersed in saline or coconut water (CW) solutions and maintained at 4 or 20 degrees C for 6, 12, or 18 h. Preantral follicles were evaluated by histology and transmission electron microscopy. Storage of ovarian pieces at 20 degrees C for 12 or 18 h significantly reduced the percentage of morphologically normal follicles compared to controls. In contrast, conservation at 4 degrees C for up to 18 h and at 20 degrees C for up to 6 h kept the percentage of normal follicles similar to controls. However, the type of solution that the ovaries were immersed in had little effect on the results. Decreased cellular metabolism probably accounted for better preservation of preantral follicles at 4 degrees C. In conclusion, zebu cow ovaries were successfully stored at 4 degrees C for up to 18 h with no morphological damage to preantral follicles. However, at 20 degrees C, ovaries could only be stored for 6 h.

Influence of stage of maturation of bovine oocytes at time of vitrification on the incidence of diploid metaphase II at completion of maturation

The present study was to verify the incidence of bovine diploid oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughterhouse and then divided into five groups: control group (unvitrified oocytes), 0 h group (composed of oocytes vitrified before the onset of maturation) and 8, 12, and 22 h groups (vitrified respectively at 8, 12 and 22 h after the onset of maturation). The oocytes remained vitrified for 24 h, and then were thawed. In all groups, the oocytes completed 24 h of maturation. Subsequently, the cumulus cells were removed, and the denuded oocytes fixed on slides and stained with aceto-orcein. No differences (P>0.05) in the incidence of diploid metaphase II oocytes were observed between the control, non-vitrified group (2.4%, 1/41) and oocytes vitrified at 12 h (6.9%, 3/43) or 22 h (2%, 1/50). However, significantly (P<0.05) more diploid oocytes were detected after vitrification at 0 h (28.5%, 10/35) or 8 h (35.4%, 11/31) of maturation. These results suggest that the nuclear stage at which bovine oocytes are vitrified may affect the incidence of diploid oocytes, especially in oocytes vitrified before maturation or 8 h after the onset of maturation.

Effect of follicular wave synchronization on in vitro embryo production in heifers.

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150microg d-cloprostenol. On Day 7, all follicles >3mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14d. Each experiment had three treatment groups. In Experiment 1 (n=12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28d. Heifers from Group 1X-EB also received 2mg EB i.m. immediately after each OPU session. In Experiment 2 (n=11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.

Post-hatching development of bovine embryos in vitro: the effects of tunnel preparation and gender

The objective of this study was to compare morphological characteristics, kinetics of development, and gene expression of male and female IVP embryos that were cultured until day (D)15 (fertilization = D0), using either phosphate-buffered saline (PBS) or Milli-Q water (MQW) to dilute the agarose gel used for tunnel construction. On D11, embryos (n = 286) were placed in agarose gel tunnels diluted in PBS and MQW. Embryos were evaluated for morphology, and embryo size was recorded on D11, D12.5, D14 and D15. Then, embryos were sexed and used for gene expression analyses (G6PD, GLUT1, GLUT3, PGK1, PLAC8, KRT8, HSF1 and IFNT). The percentage of elongated embryos at D15 was higher (p < 0.05) in the PBS (54%) than in the MQW (42%) gel. However, embryos produced in MQW were bigger (p < 0.05) and had a lower expression of GLUT1 (p = 0.08) than those cultured in PBS. There was a higher proportion of male than female embryos at D15 in both treatments, MQW (65% vs. 35%; p < 0.05) and PBS (67% vs. 33%; p < 0.05); however, embryo size was not significantly different between genders. Moreover, D15 female embryos had greater expression of G6PD (p = 0.05) and KRT8 (p = 0.03) than male embryos. In conclusion, the diluent used for tunnel construction affected embryo development in the post-hatching development (PHD) system, and the use of MQW was the most indicative measure for the evaluation of embryo quality. Male and female embryos cultured from D11 to D15, either in an MQW or PBS agarose gel, demonstrated similar development but different gene expression.

Morphology, sex ratio and gene expression of Day 14 in vivo and in vitro bovine embryos

The present study was designed to compare Day 14 bovine embryos that were produced entirely in vitro using the post-hatching development (PHD) system with in vivo-derived embryos without or with transient PHD culture from Day 7 to Day 14. Embryos on Day 14 were used for sex determination and gene expression analysis of PLAC8, KRT8, CD9, SLC2A1, SLC2A3, PGK1, HSF1, MNSOD, HSP70 and IFNT using real-time quantitative (q) polymerase chain reaction (PCR). First, Day 7 in vivo- and in vitro-produced embryos were subjected to the PHD system. A higher rate of survival was observed for in vitro embryos on Day 14. Comparing Day 14 embryos produced completely in vivo or completely in vitro revealed that the mean size of the former group was greater than that of the latter (10.29±1.83 vs 2.68±0.33mm, respectively). Expression of the HSP70 and SLC2A1 genes was down- and upregulated, respectively, in the in vitro embryos. The present study shows that in vitro embryos cultured in the PHD system are smaller than in vivo embryos and that of the 10 genes analysed, only two were differentially expressed between the two groups. These findings indicate that, owing to the poor survival rate, the PHD system is not reliable for evaluation of in vitro embryo quality.

Avanços metodológicos na produção in vitro de embriões

O desenvolvimento da metodologia de producao in vitro de embrioes (PIV) para maximizar o ganho genetico e a producao animal a um custo baixo e um desafio de pesquisa cientifica ha varios anos. A possibilidade de produzir, em escala comercial, embrioes a partir de oocitos puncionados dos foliculos ovarianos pode aumentar os beneficios do uso da inseminacao artificial devido ao seu decisivo papel no aumento do progresso genetico entre geracoes de acordo com as necessidades de cada programa de melhoramento genetico ou producao animal. Diferentes rotas tecnologicas foram desenvolvidas a partir da PIV em especies de interesse zootecnico ou especies ameacadas de extincao.