V. J. F. Freitas

Production of transgenic goat (Capra hircus) with human Granulocyte Colony Stimulating Factor (hG-CSF) gene in Brazil

In order to produce transgenic goats with hG-CSF, a total of 24 adult Saanen and 48 adult undefined breed goats were used as donors and recipients, respectively. Donors were estrus-synchronized with vaginal sponges and superovulated by a treatment with 200 mg FSH given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced by injecting 100 microg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors were mated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine alphas1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.

In vitro production of small ruminant embryos: Late improvements and further research

Beyond the potential use of in vitro production of embryos (IVP) in breeding schemes, embryos are also required for the establishment of new biotechnologies such as cloning and transgenesis. Additionally, the knowledge of oocyte and embryo physiology acquired through IVP techniques may stimulate the further development of other techniques such as marker assisted and genomic selection of preimplantation embryos, and also benefit assisted procreation in human beings. Efficient in vitro embryo production is currently a major objective for livestock industries, including small ruminants. The heterogeneity of oocytes collected from growing follicles by laparoscopic ovum pick up or in ovaries of slaughtered females, remains an enormous challenge for IVM success, and still limits the rate of embryo development. In addition, the lower quality of the IVP embryos, compared with their in vivo-derived counterparts, translates into poor cryosurvival, which restricts the wider use of this promising technology. Therefore, many studies have been reported in an attempt to determine the most suitable conditions for IVM, IVF, and in vitro development to maximize embryo production rate and quality. This review aims to present the current panorama of IVP production in small ruminants, describing important steps for its success, reporting the recent advances and also the main obstacles identified for its improvement and dissemination.

Embryo recovery rate in Santa Inês ewes subjected to successive superovulatory treatments with pFSH

The effect of two successive superovulatory treatments was assessed in 15 Santa Inewes synchronized with 60 mg medroxyprogesterone acetate sponges for 14 days and superovulated with 200 IU of porcine FSH (pFSH) in decreasing doses at 12 h intervals during days 12-14 of progestagen treatment. Estrus was observed from 12 h after sponge removal and the ewes in estrus were hand mated with Santa Inrams of proven fertility. All embryos were recovered by laparotomy 6-7 days after estrus. Treated ewes showed estrus from 24 to 72 h after sponge removal. The time of onset of estrus after sponge removal and the length of estrus were not affected ( P> 0.05) by the repetition of treatment. Ewes that failed to superovulate in the first and the second treatment ( P> 0.05) were 27 and 40%, respectively. The mean ovulation rate for those ewes responding to treatment was similar ( P> 0.05) between the first (9 .9 ± 1.1) and the second (11.3 ± 1.9) treatment. There was no significant difference in fertility between the first (53%) and second (40%) treatment. Among those fertilized, viable embryos were detected for 78% in the first treatment and 64% in the second treatment ( P> 0.05). It was concluded that Santa Inewes respond to superovulatory regimes with pFSH and the treatment may be repeated for at least two times.

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up–derived oocytes have different kinetics and requirements regarding maturation media

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.

Assessment of the reproductive parameters, laparoscopic oocyte recovery and the first embryos produced in vitro from endangered Canindé goats (Capra hircus)

The Canindé breed of goats (Capra hircus) is currently endangered. The aims of this study were to characterize the estrus behavior, ovulatory responses and progesterone profiles, and to evaluate the in vitro embryo production (IVP) in this breed. In Experiment 1, ten nulliparous and seven pluriparous females received medroxyprogesterone acetate (MAP)-containing sponges (60mg) plus 75μg d-cloprostenol for estrus synchronization and their reproductive parameters were evaluated. In Experiment 2, oocytes obtained by laparascopy from hormonally stimulated females (n=15) were used for IVP. There was no difference (p>0.05) between nulliparous and pluriparous goats in terms of estrus response (40.0% vs. 85.7%), time from progestagen sponge removal to the onset of estrus (62.0±15.5 vs. 50.7±19.2h; mean±SEM), duration of estrus (25.0±16.1 vs. 30.0±15.1h), percentage of ovulating animals (60.0% vs. 85.7%), number of ovulations (1.2±0.4 vs. 1.3±0.8), and diameter of the preovulatory follicle (5.8±0.5 vs. 6.1±0.3mm). Progesterone concentrations were also similar (p>0.05) in both groups. During laparoscopic recovery, there were average 12.2 aspirated follicles and 9.1 oocytes per goat, resulting in a high recovery rate (74.3%, 182/245). A total of 78 embryos were produced (51.0%). The mean number of cells in the blastocysts at day 7 of in vitro culture was 170.3±12.5. In conclusion, nulliparous and pluriparous Canindé goats exhibited similar reproductive profiles. It was possible to produce embryos in vitro, allowing the instigation of an embryo bank for preservation of this breed.

Influence of heparin or the presence of cumulus cells during fertilization on the in vitro production of goat embryos

Considerable research has been focused on in vitro production (IVP) of goat embryos to improve its efficiency. In Experiment 1, the effect of the cumulus cells by comparing slaughterhouse-oocytes denuded on purpose (DOP) prior to IVF to intact COC, and the effect of heparin during IVF were assessed. In Experiment 2, oocytes that were already denuded at collection (DOC), DOP and intact COC were studied. Three treatments used oocytes denuded at collection: DOC oocytes were cultured alone for both IVM and IVF; DOC and COC were cultured together for both IVM and IVF or DOC were IVM alone and then mixed with COC for IVF. In other treatments, COC were allocated to four IVF treatments: Intact COC; COC were denuded prior to IVF; COC were denuded and IVF with added cumulus cells; COC were denuded and IVF mixed with intact COC giving two sub-treatments: Denuded oocytes that were IVF with COC; and COC that were IVF with denuded oocytes. After fertilization, all presumptive zygotes were cultured for 8 days. In Experiment 1, the yield of blastocysts as a proportion of total oocytes was greater (P<0.05) for COC that were IVF in the presence of heparin (54%) than without heparin (42%) or oocytes already denuded at collection that were IVF with or without heparin (41%; 38%; respectively). In Experiment 2, the developmental potential of oocytes denuded at collection was reduced (cleavage and blastocyst rates calculated from total oocytes: 34%; 11%, respectively) as compared to COC (77%; 59%, P<0.05). However, when equal numbers of both were mixed at the start of IVM, the rates were not significantly different to COC alone (68%; 45%), but when both were mixed equally only for IVF, the rates were reduced (57%; 40%, P<0.05). Denuded oocytes co-cultured with cumulus cells were not significantly different to intact COC (76%; 55%). The effect of adding COC during IVF to oocytes denuded after IVM was similar to adding cumulus cells to the same type of oocytes. In conclusion, both the use of heparin and the association of oocytes with cumulus cells, either detached or in intimate contact, during IVM and/or IVF significantly improve IVP of goat embryos. Furthermore, some oocytes that are already denuded at collection will develop satisfactorily to blastocysts when matured and fertilized with intact COC.

Production of recombinant proteins in milk of transgenic and non-transgenic goats

Among all the transgenic mammalians produced so far, goats have represented an excellent model of transgenesis when considering the factors such as the market demand for protein, volume of milk produced per lactation and reproductive rate. Various recombinant proteins have been obtained from the transgenic and non-transgenic goats, and among these, human antithrombin, produced by the transgenic goats, was the first recombinant protein of animal origin to be released as a drug for the clinical use in humans. This review reports the aspects inherent to the production of recombinant proteins in the goats, from the production of the animal bioreactors up to the expression of these proteins in their milk.

In vitro embryo production in small ruminants

This paper reviews the technical bases of in vitro embryo production in small ruminants with special attention to the results obtained by our group in Northeastern Brazil. The laparoscopic oocyte recovery in hormonally treated live animals indicates a promising future for the application of this technique to genetic improvement program. New molecular biology tools should provide information to improve the efficiency of in vitro maturation. Furthers efforts have to be made to improve the oocyte maturation and to standardize the semen-capacitating process.

Embryo production in superovulated goats treated with insulin before or after mating or by continuous propylene glycol supplementation.

Seventeen adult and cyclic Moxoto goats were synchronized using 60 mg MPA vaginal sponge for 11 days and 50 mug cloprostenol, 48 h before sponge removal, and superovulated with 120 mg pFSH i.m. in decreasing doses at 12 h intervals for three consecutive days. In seven goats, 0.2 IU/kg BW/day of long acting insulin was subcutaneously injected at same time as pFSH, and in the other five goats, the same dose of insulin was injected for three consecutive days starting 24 h after mating. Finally, five goats were supplemented with an oral dose of 80 ml/goat/day of propylene glycol continuously during the experiment. The animals were flushed at 7 days after mating and the embryos were classified based on International Embryo Transfer Society criteria. Blood samples were collected every 3 days for insulin assay. Administration of insulin raised the insulin levels of the goats (p < 0.05), whereas in the group treated with propylene glycol, insulin rate was different only between FSH treatment and after mating (p < 0.05). Similar rates of recovery for total (80.05 +/- 9.78%) or transferable structures (61.03 +/- 15.13%) were obtained. Treatment was not influenced (p > 0.05) by responsiveness to superovulation, which averaged 64%. By contrast, insulin treatments were shown to increase the number of embryos considered excellent with respect to goats supplemented with propylene glycol (p < 0.05). When insulin was given before mating, a strong relationship (r = 0. 90) (p < 0.05) between number of transferable embryo and ovulations was observed in the animals. In conclusion, superovulated goats treated with low doses of exogenous insulin resulted in an enhancement in embryo quality, which was related to changes in circulating insulin concentrations.

Cell-penetrating peptides (CPPs): From delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis

Cell-penetrating peptides (CPPs) have been studied for their capacity to translocate across the lipid membrane of several cell types. In membrane translocation, these peptides can remarkably transport biologically active hydrophilic molecules, such as pharmaceuticals, nucleic acids (DNA and RNA) and even high-molecular-weight proteins, Fig. 3 into the cell cytoplasm and organelles. The development of CPPs as transduction agents includes the modification of gene and protein expression, the reprogramming and differentiation of induced pluripotent stem cells and the preparation of cellular vaccines. A relatively recent field of CPP application is the transduction of plasmid DNA vectors and CPP-fusion proteins to modify genomes and introduce new traits in cells and organisms. CPP-mediated transduction of components for genome editing is an advantageous alternative to viral DNA vectors. Engineered site-specific nucleases, such as Cre recombinase, ZFN, TALENs and CRISPR associated protein (Cas), have been coupled to CPPs, and the fused proteins have been used to permeate targeted cells and tissues. The functionally active fusion CPP-nucleases subsequently home to the nucleus, incise genomic DNA at specific sites and induce repair and recombination. This review has the objective of discussing CPPs and elucidating the prospective use of CPP-mediated transduction technology, particularly in genome modification and transgenesis.