Risk of Chlamydia abortus transmission via embryo transfer using in vitro produced early bovine embryos.

The objectives of this study were to determine (i) whether Chlamydia (C.) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. The experimentation was made twice. For each replicate 100 (8-16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 × 107Chlamydia/ml of AB7 strain. After incubation for 18 h at 37 °C in an atmosphere of 5% CO2, the embryos were washed in accordance with the IETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13,000×g. Each batch of washed embryos and each wash pellets were tested using PCR. C. abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia-DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia-DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 ±â€¯9 × 103 bacteria/mL) than for batches of ZP-intact embryos (0.47 ±â€¯0.19 × 103 bacteria/mL). These results demonstrate that C. abortus adheres to the ZP as well as the early embryonic cells of in vitro produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS fails to remove it.

Docosahexaenoic acid mechanisms of action on the bovine oocyte-cumulus complex.

BACKGROUND: Supplementation of bovine oocyte-cumulus complexes during in vitro maturation (IVM) with 1 µM of docosahexaenoic acid (DHA), C22:6 n-3 polyunsaturated fatty acid, was reported to improve in vitro embryo development. The objective of this paper was to decipher the mechanisms of DHA action. RESULTS: Transcriptomic analysis of 1 µM DHA-treated and control cumulus cells after 4 h IVM showed no significant difference in gene expression. MALDI-TOF mass spectrometry analysis of lipid profiles in DHA-treated and control oocytes and cumulus cells after IVM showed variations of only 3 out of 700 molecular species in oocytes and 7 out of 698 species in cumulus cells (p < 0.01). We showed expression of free fatty acid receptor FFAR4 in both oocytes and cumulus cells, this receptor is known to be activated by binding to DHA. FFAR4 protein was localized close to the cellular membrane by immunofluorescence. Functional studies demonstrated that supplementation with FFAR4 agonist TUG-891 (1 µM or 5 µM) during IVM led to an increased blastocyst rate (39.5% ± 4.1%, 41.3% ± 4.1%), similar to DHA 1 µM treatment (39.2% ± 4.1%) as compared to control (25.2% ± 3.6%). FFAR4 activation via TUG-891 led to beneficial effect on oocyte developmental competence and might explain in part similar effects of DHA. CONCLUSIONS: In conclusion, we suggested that low dose of DHA (1 µM) during IVM might activate regulatory mechanisms without evident effect on gene expression and lipid content in oocyte-cumulus complexes, likely through signaling pathways which need to be elucidated in further studies.

N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle.

The positive effect of n-3 polyunsaturated fatty acids (FAs) on fertility in ruminants seems to be partly mediated through direct effects on the oocyte developmental potential. We aimed to investigate whether supplementation with physiological levels of docosahexaenoic acid (DHA, C22:6 n-3 polyunsaturated fatty acids) during IVM has an effect on oocyte maturation and in vitro embryo development in cattle. We reported that DHA (0, 1, 10, or 100 µM) had no effect on oocyte viability or maturation rate after 22-hour IVM. Incubation of oocyte-cumulus complexes with 1-µM DHA during IVM significantly increased (P < 0.05) oocyte cleavage rate as compared with control (86.1% vs. 78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (39.1% vs. 29.7%, respectively). Supplementation with 1 µM DHA during IVM also induced a significant increase in the blastocyst rate at Day 7 after IVF as compared with control (30.6% vs. 17.6%, respectively) and tended to increase the number of cells in the blastocysts (97.1 ± 4.9 vs. 81.2 ± 5.3, respectively; P = 0.08). On the contrary, 10-µM DHA had no effects, whereas 100-µM DHA significantly decreased the cleavage rate compared with control (69.5% vs.78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (19.5% vs. 29.7%). As was shown by real-time polymerase chain reaction, negative effects of 100-µM DHA were associated with significant increase of progesterone synthesis by oocyte-cumulus complexes, a three-fold increase in expression level of FA transporter CD36 and a two-fold decrease of FA synthase FASN genes in cumulus cells (CCs) of corresponding oocytes. Docosahexaenoic acid at 1 and 10 µM had no effect on expression of those and other key lipid metabolism-related genes in CC. In conclusion, administration of a low physiological dose of DHA (1 µM) during IVM may have beneficial effects on oocyte developmental competence in vitro without affecting lipid metabolism gene expression in surrounding CCs, contrarily to 100 µM DHA which diminished oocyte quality associated with perturbation of lipid and steroid metabolism in CC.