Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries.

BACKGROUND AND AIMS: Ovum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions. METHODS: MSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 106 AD-MSCs (G2), MSC-EVs produced by 2.5 × 106 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 106 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4). RESULTS: Characterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions. CONCLUSIONS: MSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo.

A longer period of epididymal sperm interaction with extender components during cryopreservation improves sperm quality, decreases the size of sperm distal cytoplasmic droplets, and changes the number of nanoparticles in the extender.

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.

Outstanding Gir oocyte donors: How does individual factor affect in vitro embryo production efficiency?

The oocyte donor plays a pivotal role in bovine in vitro embryo production (IVP) success. The individual factor affects blastocyst/oocyte ratio and determine the existence of outstanding performing animals. The aim of this study was to assess the extent of individual factor effect to IVP efficiency, in a population of Gir oocyte donors. Extreme (high or low IVP efficiency based on blastocyst/oocyte ratio) animals were selected out of a population of 250 oocyte donors (1,734 observations) to form high (>0.48, n = 40), average (0.17-0.48, n = 168), and low (<0.17, n = 42) efficiency donor groups. Cumulus-oocyte complex indicators (total number, IVF-grade number, and IVF-grade/total ratio) were lower (p < 0.05) in high efficiency donors. The number of blastocysts per OPU was analyzed for highest performing bull, and an increase (p < 0.05) in high efficiency donors and a decrease (p < 0.05) in low efficiency donors were noticed, compared to average efficiency donors. The number of pregnancies obtained per OPU was affected (p = 0.017) by donor's efficiency (low: 0.60 ± 0.09 $$ 0.60\pm 0.09 $$ , average: 1.17 ± 0.07 $$ 1.17\pm 0.07 $$ , high: 2.57 ± 0.26 $$ 2.57\pm 0.26 $$ ), being 4.3-fold higher in high than in low efficiency donors. We conclude that producing embryos from high efficiency blastocyst/oocyte ratio donors increases blastocyst and pregnancy numbers by OPU, being an important indicator for donor selection in IVP programs.