Effects of fusion-activation interval and embryo aggregation on in vitro and in vivo development of bovine cloned embryos.

Nuclear reprogramming in somatic cell cloning is one of the key factors for proper development, with variations in the protocol appearing to improve cloning efficiency. This study aimed to determine the effects of two fusion-activation intervals and the aggregation of bovine cloned embryos on subsequent in vitro and in vivo development. Zygotes produced by handmade cloning were exposed to two fusion-activation intervals (2 h or 4 h), and then cultured in microwells either individually (1 × 100%) or after aggregation of two structures (2 × 100%). Zona-intact oocytes and zona-free oocytes and hemi-oocytes were used as parthenote controls under the same fusion-activation intervals. Day-7 cloned blastocysts were transferred to synchronous recipients. Cleavage (Day 2), blastocyst (Day 7) and pregnancy (Day 30) rates were compared by the χ2 test (P < .05). Extending fusion-activation interval from 2 to 4 h reduced cleavage (91.0 vs. 74.4%) but not blastocyst (34.8 vs. 42.0%) rates. On a microwell basis, cloned embryo aggregation (2 × 100%) increased cleavage (91.5% vs. 74.4%) and blastocyst (46.0% vs. 31.3%) rates compared to controls (1 × 100%), but did not improve the overall embryo production efficiency on Day 7 (23.0% vs. 31.3%), on a per reconstructed embryo basis, respectively. Treatments had no effects on in vitro developmental kinetics, embryo quality, and in vivo development. In summary, the fusion-activation interval and/or the aggregation of cloned bovine embryos did not affect cloning efficiency based on the in vitro development to the blastocyst stage and on pregnancy outcome.

Effect of aquaporin 3 knockdown by RNA interference on antrum formation in sheep secondary follicles cultured in vitro.

SummaryThe objectives were to develop an effective protocol for transfection of ovine secondary follicles and to assess the effect of attenuating aquaporin 3 (AQP3) using a small interfering RNA (siRNA-AQP3) on antrum formation and follicular growth in vitro. Various combinations of Lipofectamine® volumes (0.5, 0.75 or 1.0 µl), fluorescent oligonucleotide (BLOCK-iT ™) concentrations (3.18, 27.12 or 36.16 nM) and exposure times (12, 14, 16, 18 or 20 h) were tested. The BLOCK-iT™ was replaced by siRNA-AQP3 in the transfection complex. Ovine secondary follicles were isolated and cultured in vitro for 6 days using standard protocols. Follicles were transfected on day 0 or 3 or on both days (0 and 3) and then cultured for an additional 3 or 6 days. As revealed by the fluorescence signal, the Lipofectamine®/BLOCK-iT™ complex (0.75 µl + 27.12 nM by 12 h of incubation) crossed the basement membrane and granulosa cell and reached the oocytes. In general, the rate of intact follicles was higher and the rate of antrum formation was lower in transfected follicles compared with control follicles. In conclusion, ovine secondary follicles can be successfully transfected during in vitro culture, and siRNA-mediated attenuation of AQP3 gene reduced antrum formation of secondary follicles.

Mid-pregnancy ewe shearing and the effects on fetus liver and muscle glycoprotein deposits.

The reason why shearing ewes in mid-pregnancy does increase the lamb birth weight is not completely clears. Therefore, we focused on the analyses of the deposition of glycogen in different fetal tissues to investigate this issue. Thirteen pregnant Australian Merino ewes, raised in native pasture, were separated in two groups. One group (n = 7) was shorn (SE) at 70 days of pregnancy, whereas another group (n = 6) remained unshorn (NSE). Cesarean section was conducted in all the ewes at near parturition, when placenta and fetuses sampling were collected. Placenta, liver and muscle samples were fixed and stained with glycoprotein-reactive acid-Schiff acid for analysis under light microscopy. The quantification of these glycoproteins was performed with the support of a program that analyzes the measurement of the intensity of staining by field. Five random fields from each sample were used, where statistical analyzes was used as normal test T. Among the analyzed regions, the deposition of glycoprotein between SE and NSE groups was statistically different in the hepatic portal vein (54,499.23 µm 2 in SE and 34,830.73 µm2 in NSE) and in the total muscle area of the sample fragment (41,128, 7 µm2 and 31,942.7 µm2 , respectively; P < 0.05). We conclude that shearing ewes at the 70th day of gestation lead to accumulation of glycoproteins in the liver and muscle of fetuses, which may be responsible for the increase in birth weights in that group.

Milk from transgenic goat expressing human lysozyme for recovery and treatment of gastrointestinal pathogens.

Lysozyme is an important non-specific immune protein in human milk, modulating the immune response against bacterial infections. The aim of this study was to characterize the milk of a transgenic goat expressing a recombinant human lysozyme (rhLZ) in the milk, also testing the in vitro antibacterial activity of the rhLZ milk against pathogens of the gastrointestinal tract. Milk samples collected from Tg and non-transgenic goats (nTg) from the 3rd to the 11th week of lactation were submitted to physicochemical analyses, rhLZ semi-quantification, and to rhLZ antimicrobial activity against Micrococcus luteus, Shiguella sonnei and Enterococcus faecalis. Viability and cell migration were studied in ileum epithelial cells (IEC-18) in absence or presence of E. faecalis, Staphylococcus aureus, Escherichia coli (EPEC) and S. sonnei. The expression of ZO-1 and IL-6 genes was evaluated in IEC-18 to evaluate the effect of rhLZ milk on intestinal barrier function and intestinal inflammation. Physicochemical parameters between goat Tg and nTg milk were similar and within normal values for human consumption, with hLZ concentrations being similar between Tg (224µg/mL) and human (226µg/mL) milk. The Tg milk had bactericidal activity against M. luteus, no bactericidal effect on S. sonnei, and relative to discrete sensitivity against E. feacalis than controls. Better migrating parameters were observed in cells in culture with nTg and Tg than controls. In the presence of pathogens, the Tg milk promoted improved migrating parameters than controls, except for S. sonnei, with lower cell numbers in the presence of nTg samples and E. faecalis and S. sonnei. No differences in ZO-1 relative expression patterns were observed in cultured cells, with increased expression in IL-6 in cells exposed to nTg milk than controls, with the Tg group being similar to all groups. In conclusion, goat milk containing rhLZ demonstrated valid evidence for its potential use as a nutraceutical for improvement of health and nutrition quality in humans.

Impact of cumulative gain in expertise on the efficiency of handmade cloning in cattle.

The aim of this study was to determine the effects of the cumulative gain in expertise in carrying out handmade cloning (HMC) procedures on embryo yield and pregnancy outcome in cattle. Results from in vitro and in vivo embryo development after HMC during three periods of 7 months, separated by 3-month intervals, were compiled and designated as P1, P2 and P3. Blastocyst yield, morphological quality and stage of development, and pregnancy per embryo transfer (ET) on Day 30 of gestation were compared. Zona-intact oocytes were activated chemically in each experiment replicate, and development of parthenogenetic blastocysts was used as a control measurement of oocyte quality and in vitro culture conditions. A total of 21,231 cumulus-oocyte complexes (COCs) were in vitro-matured, with 5,432, 10,721 and 5078 COCs used in 16, 18 and 10 replicates for P1, P2 and P3, respectively. Cloned blastocyst yields on Day 7 increased from 15.5% (124/798) in P1 to 21.6% (309/1428) and 36.6% (280/764) in P2 and P3, respectively. No differences were observed in blastocyst development of parthenogenetic embryos, which average 30.0, 37.6, and 36.4% in P1, P2, and P3, respectively. A 10-fold higher probability of obtaining cloned blastocysts at more advanced stages of development and of higher morphological grade was seen during P3 compared with P1. Pregnancy per ET on Day 30 also increased with gain in expertise, being 6.7% (2/30), 20.8% (10/48) and 40.0% (24/60) for P1, P2 and P3, respectively. The relative efficiency for the establishment of pregnancies (per total COC) increased from 0.04% (1:2716) in P1 to 0.22% (1:460) in P2, reaching 0.47% (1:212) in P3. Results demonstrated a gradual improvement in in vitro and in vivo embryo development over time after establishment of HMC procedures in the laboratory, highlighting the importance of gaining experience and technical skills on the overall cloning efficiency.

Developmental Outcome and Related Abnormalities in Goats: Comparison Between Somatic Cell Nuclear Transfer- and In Vivo-Derived Concepti During Pregnancy Through Term.

Cloning by somatic cell nuclear transfer (SCNT) is characterized by low efficiency and the occurrence of developmental abnormalities, which are rather poorly studied phenomena in goats. This study aimed at comparing overall SCNT efficiency in goats by using in vitro-matured (IVM) or in vivo-matured oocytes and fibroblast donor cells (mock transfected, transgenic, or wild type), also characterizing symptoms of the Abnormal Offspring Syndrome (AOS) in development, comparing results with pregnancies produced by artificial insemination (AI) and in vivo-derived (IVD) embryos. The SCNT group had lower pregnancy rate (18.3%, 11/60), total number of concepti (20.0%, 12/60), term births (3.3%, 2/60), and live births (1.7%, 1/60) than both the IVD (77.8%, 7/9; 155.5%, 14/9; 122.2%, 11/9; 88.8%, 8/9) and the AI (71.4%, 10/14; 121.4%, 17/14; 100%, 14/14; 78.5%, 11/14) groups, respectively (p < 0.05). No SCNT pregnancies reached term using IVM oocytes, but in vivo-matured oocytes resulted in two term transgenic cloned kids. The proportion fetal membrane (FM) weight/birth weight reflected an increase in FM size and cotyledonary enlargement in clones, for disproportionally bigger newborns in relation to cotyledonary numbers. Overall, goat cloning showed losses and abnormality patterns similar to the AOS in cloned cattle and sheep, which have not been previously well recognized in goats.

Transient Expression of Functional Glucocerebrosidase for Treatment of Gaucher's Disease in the Goat Mammary Gland.

Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.

Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes.

Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.