Gene expression patterns of in vivo-derived sheep blastocysts is more affected by vitrification than slow freezing technique.

Transfer of fresh sheep embryos frequently results in higher pregnancy rate compared to cryopreserved ones, possibly due to a failure in the communication between the cryopreserved embryo and the endometrium during pre-implantation and pregnancy establishment. Thus, this study assessed the effect of sheep embryo cryopreservation (slow freezing or vitrification) on embryo survival rate and expression of genes related to trophectoderm differentiation (CDX2), pluripotency maintenance (NANOG), cell proliferation (TGFB1), mitochondrial activity (NRF1) and apoptosis (BAX and BCL2). Superovulation (n = 32 ewes) was performed and embryos were transcervically collected. One hundred good quality (Grade I and II) embryos were allocated into three groups: fresh embryos (CTL; n = 15), slow freezing (SF; n = 42) or vitrification (VT; n = 43). After thawing/warming, three pools of five blastocysts per group were used for RT-qPCR; the remaining 55 embryos were cultured in vitro in SOFaa medium at 38.5 °C and 5% CO2 (SF: n = 27 and VT: n = 28). Survival rate of SF and VT were, respectively, 29.6% (8/27) and 14.2% (4/28) at 24 h; and 48.1% (13/27) and 32.1% (9/28) at 48 h (P > 0.05). Only CDX2 was affected (up-regulated, P < 0.05) in both groups compared to CTL. The BAX transcript was upregulated in VT, compared to SF group. The VT increased (P < 0.05) the expression of all genes, except for NANOG and NRF1, when compared to the CTL. In conclusion, although in vitro survival was similar between techniques, VT led to increased changes in blastocyst gene expression compared to CTL and SF.

In Vitro Culture of Embryos from LOPU-Derived Goat Oocytes.

A high oocyte quality is the prerequisite for in vitro embryo production. Goat cumulus-oocyte complexes (COC) are mainly collected from slaughterhouse ovaries or by laparoscopic ovum pickup (LOPU) from live animals. Several features can influence the availability of good quality oocytes recovered by the LOPU technique. Interestingly, slaughterhouse and LOPU oocytes have different in vitro maturation kinetics and requirements, and thus, the IVP system must be adapted regarding the oocyte origin. Overall, the use of undefined media in the different steps makes interpretation of results more difficult, hampers their reproducibility, and introduces a sanitary risk. Thus, there is an effort worldwide to use simpler conditions for goat IVP. Although the success of IVP rates is relatively high, in vitro embryos differ from in vivo-derived ones in many aspects, resulting in lower viability. Therefore, strategies to improve in vitro embryo quality are crucial, such as the use of oviductal epithelium cells for coculture. Here we describe the main steps and culture media which can be utilized to produce embryos in vitro from LOPU or slaughterhouse oocytes in goats.

Dose and administration protocol for FSH used for ovarian stimulation affect gene expression in sheep cumulus-oocyte complexes.

The present study evaluated the effect of four ovarian stimulation protocols on the follicular population and molecular status of cumulus-oocyte complexes (COCs). Twelve Santa Inês ewes (in a cross-over design) received 80 or 120mg FSH alone in a multiple-dose (MD80 and MD120) regimen or in combination with 300IU equine chorionic gonadotrophin (eCG) in a one-shot (OS80 and OS120) protocol. The follicular population, COC recovery rate, mean COCs per ewe and the rate of brilliant Cresyl blue-positive (BCB+) COCs were similar among treatments (P>0.05). The expression of markers of oocyte competence (ZAR1, zygote arrest 1; MATER, maternal antigen that embryo requires; GDF9, growth differentiation factor 9; BMP15, bone morphogenetic protein 15; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2 associated X protein) and the steroidogenic pathway (ERα, oestrogen receptor α; LHr, LH receptor; FSHr, FSH receptor; STAR, steroidogenic acute regulatory protein) was affected by stimulation. Specifically, the expression of markers of the steroidogenic pathway was reduced with increasing FSH dose in the OS protocol. FSH at a dose of 80mg reduced the expression of FSHr and ERα in the OS versus MD protocol. Conversely, in MD protocol, only LHr was affected by increasing FSH dose. In conclusion, 80mg FSH in the MD or OS protocol was sufficient to promote the development of multiple follicles and obtain fully grown (BCB+) oocytes. The MD protocol may be more appropriate for the production of better-quality oocytes.

Dynamics of recombinant hG-CSF in transgenic goat: preliminary study in the founder during hormonally induced lactation.

This study aimed to characterize the dynamic of human granulocyte colony-stimulating factor (hG-CSF) during artificial lactation in a transgenic founder goat and to assess its potential ectopic expression and health. The female secreted 93.9 to 1,474.6 µg hG-CSF per mL of milk. Two peaks of serum hG-CSF (3,470 and 7,390 pg/mL) were detected in the first half of the lactation. Outside of the lactation, hG-CSF was absent from serum, indicating no ectopic expression. During the treatment to induce lactation, transgenic female presented increased neutrophil and lymphocyte blood counts when compared to nontransgenic female. Despite transient neutrophilia, serum biochemistry profiles indicated normal liver and renal functions. Thus, transgenic goat expressed hG-CSF in quantities sufficient for a commercial bioreactor and remained clinically healthy.