Search

In vitro, efficient communication between mammalian embryos and between embryos and 22 their environment, e.g. maternal somatic cells, implies that there is a sender, a message and a 23 receiver which is able to decode the message. Embryos are secreting a variety of autocrine and 24 paracrine factors, and among those, extracellular vesicles have recently been implicated as 25 putative messengers in embryo-embryo communication and in communication of the embryo 26 with the maternal tract. Extracellular vesicles (EVs) are membrane-bound vesicles, found in 27 biofluids and in culture media conditioned by the presence of embryos or cells, that carry and 28 transfer regulatory molecules, such as microRNAs (miRNAs), messenger RNAs (mRNA), lipids 29 and proteins. 30 Here, we conducted a systematic search of the literature to review and present the currently 31 available evidence on the possible roles of EVs in embryo communication and embryo 32 development. It is important to note that many of the biologically plausible functions of EVs in 33 embryo communication have not yet been substantiated by conclusive experimental evidence. 34 However, indirect evidence, such as the use of media conditioned by embryos or by somatic 35 cells with improved embryo development as a result, may indicate that EVs can be an 36 important asset for the development of tailor-made media allowing better embryo 37 development in vitro, even for single embryo culture.

The present study investigated the in vitro development of and cytoskeletal disruption suffered by in vivo-derived porcine blastocysts subjected to superfine open pulled straws (SOPS) vitrification. Blastocysts were either untreated prior to SOPS vitrification or were subjected to one of the following three pretreatment protocols: (1) centrifugation (12 min, 13 000g); (2) 25 min equilibration with 7.5 mu g mL(-1) cytochalasin B; or (3) equilibration with cytochalasin B followed by centrifugation. After 24 h culture, fresh (n = 32) and vitrified-warmed (n = 188) blastocysts were evaluated by stereomicroscopy, with survival and hatching rates recorded. Some blastocysts were stained with 4,6-diamidino2- phenylindole and processed for cytoskeletal evaluation. Three cytoskeletal patterns were identified: Grade I, intact cytoskeleton; Grade II, gross maintenance of integrity, but with some clumps of actin within the cytoplasm; and Grade III, a highly disrupted cytoskeleton. There were no differences in the survival, hatching and cell death rats, total cell number or cytoskeletal integrity between the different vitrification groups. Cell death was greater for vitrified blastocysts than for fresh blastocysts (3.6 +/- 0.4% v. 0.4 +/- 0.7%, respectively; P less than 0.05) and the percentage of blastocysts with a Grade I cytoskeletal pattern was lower for vitrified compared with fresh blastocysts (60.8% v. 92%, respectively; P less than 0.05). The vitrified-warmed blastocysts that hatched during culture exhibited a Grade I cytoskeletal pattern. In conclusion, successful SOPS vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation.

Di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in polyvinylchloride (PVC) products (e.g., plastic bags and medical equipment), has been reported to have toxic effects on animal reproduction and is considered an environmental hazard based, mostly, on rodent studies. However, the doses used in these studies are often considerably higher than that presumed in human exposure. In the present study we used young boars as model animals to assess the effects of pre-pubertal DEHP exposure on the ability of spermatozoa to penetrate homologous oocytes in vitro. Eight pairs of cross-bred male boar siblings were used. One brother in each pair became, at random, the test animal exposed to DEHP per os, three times a week, from 3 to 7 weeks of age while the other acted as the control, i.e., placebo-exposed. Semen was collected and frozen between 8 and 9 months of age and stored until spermatozoa were evaluated for their ability to in vitro penetrate in vitro-matured homologous oocytes post-thaw. Both the penetration rate and the number of spermatozoa per oocyte were considered within expected ranges for frozen boar semen of good quality. Penetration rate did not significantly differ (p greater than 0.05) between the groups with DEHP-exposed: 50%; control: 59%, which could be owing to a large variation between boars, and between replicates. The number of spermatozoa in the ooplasm was low and similar (p greater than 0.05) between the groups with DEHP-exposed: 1.5 and the control: 1.7. Under the conditions of the present experiment, pre-pubertal exposure to DEHP does not seem to cause a deleterious effect on the in vitro fertilizing ability of frozen spermatozoa post-puberty.

In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5943 COCs were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa froth 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2 mM), hyaluronic acid (HA; [0.5 mg/mL]) and adenosine (10 mu M), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5 mg/ml) and adenosine (0, 10, 20 and 40 mu M) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 thin or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6 h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 1215 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P less than 0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9 +/- 3.9% versus 62.7 +/- 3.9% and 1.5 +/- 3.2 versus 1.3 +/- 3.5 for 10 min or 6 h, respectively), but reduced monospermy (P les