Studies on Gene Expression and Developmental Competence of Bovine Embryos Produced Under Different Conditions of Heat Stress

Gene expression is required in all steps of embryonic development and therefore heat stress is known to reduce developmental competence after direct exposure of oocytes and embryos to different conditions of heat shock, by decreasing protein synthesis. Moreover, as in somatic cells, the heat stress befuddles the integration of RNA and posttranscriptional modification of RNA, the assumption was that during meiotic maturation heat shock may mutate RNA within oocytes, with the possibility of altering the surrounding cumulus cells, causing, thus, reductions in development. Heat shock proteins (HSP) are among the first proteins produced during embryonic development and are crucial to cell function. The HSP70 (HSPA14 gene) is an important part of the cell’s machinery for folding, unfolding, transport, localization of proteins and differentiation, regulation of the embryonic cell cycle and helping to protect cells from stress. Therefore, HSPA14 is an apoptotic gene induced by heat shock is associated with embryonic loss, playing an important role of control mechanism of processes involved in growth, cellular differentiation, and embryonic development. In addition the connexin proteins (e.g. Cx43), related to gap junctions, are expressed in numerous tissues including gonads, act as a mediator of heat stress effect on cells. In the present review, the effect of heat stress on bovine embryonic development in a physiologic and genetic point of view is fully discussed

Crossbreeding effect of double-muscled cattle on in vitro embryo development and quality

Nowadays, several developing countries have started to breed double-muscled cattle to their autochthonous cattle to improve meat production. However, the developmental competence of the resultant crossbreeding embryos is unknown. The objective of this study was to evaluate the effect of crossbreeding double-muscled (Belgian Blue; BB) semen with beef (Limousin; LIM) and dairy (Holstein-Friesian; HF) derived oocytes on embryo development and quality, using purebred BB as a control (BB oocytes fertilized by BB sperm). A single ejaculate of a BB bull was evaluated by Computer Assisted Sperm Analysis before using for in vitro fertilization. Ovaries from each breed were collected at the local slaughterhouse (n = 1,720 oocytes). All statistical analyses were performed using R-core (P < 0.05). Embryo quality was evaluated via differential-apoptotic staining of day 8 blastocysts. Cleavage (48 h post insemination) and day 8 blastocyst rates were greater (P < 0.05) for LIM (82.9 ± 6 and 27 ± 4.3%, respectively) than for BB (69.8 ± 8.5 and 19.6 ± 3.1%, respectively) and HF (45.1 ± 10 and 12.3 ± 2.2%, respectively). Holstein-Friesian presented lower cleavage and day 8 blastocyst rates than BB (P < 0.05). Limousin blastocysts presented a higher number (P < 0.05) of inner cell mass cells (ICM; 68 ± 7.8) than HF (40.4 ± 8.2). In conclusion, crossbreeding double-muscled cattle by in vitro fertilization with LIM oocytes yielded better embryo compared with the purebred combination, while the combination with HF oocytes produced the lowest rate of blastocysts

Emerging role of extracellular vesicles in communication of preimplantation embryos <em>in vitro</em>

International audienceIn vitro, efficient communication between mammalian embryos in groups or between embryos and cocultured somatic cells implies that there is a sender, a message and a receiver that is able to decode the message. Embryos secrete a variety of autocrine and paracrine factors and, of these, extracellular vesicles have recently been implicated as putative messengers in embryo–embryo communication, as well as in communication of the embryo with the maternal tract. Extracellular vesicles (EVs) are membrane-bound vesicles that are found in biofluids and in culture media conditioned by the presence of embryos or cells. EVs carry and transfer regulatory molecules, such as microRNAs, mRNAs, lipids and proteins. We conducted a systematic search of the literature to review and present the currently available evidence regarding the possible roles of EVs in in vitro embryo communication and embryo development. It is important to note that there is limited information available on the molecular mechanisms and many of the biologically plausible functions of EVs in embryo communication have not yet been substantiated by conclusive experimental evidence. However, indirect evidence, such as the use of media conditioned by embryos or by somatic cells with improved embryo development as a result, may indicate that EVs can be an important asset for the development of tailor-made media, allowing better embryo development in vitro, even for single embryo culture

Short communication: Morphometric characterization of Lidia cow (Bos taurus) reproductive apparatus

To study Lidia cow reproductive apparatus traits, a total of 90 organs were collected after slaughtering the cows from different Bos taurus breeds: (i) Lidia cattle breed - Brava dos Açores population (n=10) and Domecq lineage (n=11); (ii) Holstein Friesian females – 10-14-month-old heifers (n=15); 15-20-month-old heifers (n=10), 21-19-month-old heifers (n=18), and (iii) cows ≥ 30 months (n=26). The length and width were measured for five portions of the female reproductive apparatus (vulva and vagina, cervix, uterine body, uterine horns and ovaries). One-way ANOVA was performed with Tukey test. The level recognized to assume differences was p<0.05 to less. Differences were not shown between Lidia groups. In general, the Lidia cow reproductive apparatus was small in size that that of the matured cows in terms of all traits, with the exception of cervix rings (5.10 ± 0.17 rings) with p≤0.01 for all the groups (averages ranged from 3.33 ± 0.11 rings to 3.50 ± 0.15 rings). The vulva and vagina (L= 27.31 ± 0.53 cm; W=2.07 ± 0.14 cm), the uterine body width (3.01 ± 0.18 cm) and the uterine horns (L= 12.24 ± 0.32; W= 1.13 ± 0.10) showed were smaller in size than those of the evaluated heifers from HF breed that ranged in age from 10 to 14 months (p≤0.01). This study was the first to perform a morphometric characterization on the Lidia cow reproductive apparatus, and the results provide useful information for understanding reproductive approaches to be used with this breed

Short communication. Morphometric characterization of Lidia cow (Bos taurus) reproductive apparatus

To study Lidia cow reproductive apparatus traits, a total of 90 organs were collected after slaughtering the cows from different Bos taurus breeds: (i) Lidia cattle breed - Brava dos Açores population (n=10) and Domecq lineage (n=11); (ii) Holstein Friesian females – 10-14-month-old heifers (n=15); 15-20-month-old heifers (n=10), 21-19-month-old heifers (n=18), and (iii) cows ≥ 30 months (n=26). The length and width were measured for five portions of the female reproductive apparatus (vulva and vagina, cervix, uterine body, uterine horns and ovaries). One-way ANOVA was performed with Tukey test. The level recognized to assume differences was p<0.05 to less. Differences were not shown between Lidia groups. In general, the Lidia cow reproductive apparatus was small in size that that of the matured cows in terms of all traits, with the exception of cervix rings (5.10 ± 0.17 rings) with p≤0.01 for all the groups (averages ranged from 3.33 ± 0.11 rings to 3.50 ± 0.15 rings). The vulva and vagina (L= 27.31 ± 0.53 cm; W=2.07 ± 0.14 cm), the uterine body width (3.01 ± 0.18 cm) and the uterine horns (L= 12.24 ± 0.32; W= 1.13 ± 0.10) showed were smaller in size than those of the evaluated heifers from HF breed that ranged in age from 10 to 14 months (p≤0.01). This study was the first to perform a morphometric characterization on the Lidia cow reproductive apparatus, and the results provide useful information for understanding reproductive approaches to be used with this breed

Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts

Extracellular vesicles (EVs) and their cargo microRNAs (miRNAs) are important regulators of embryo development to the blastocyst stage and beyond. Before implantation can take place, hatching of blastocysts from their zona pellucida is required. However, underlying mechanisms by which blastocyst formation and hatching are initiated remain largely unknown. Here, we provide evidence that embryonic EVs containing bta-miR-378a-3p play a crucial role in blastocyst hatching, using a bovine model. A customized procedure was used to isolate EV-miRNAs from culture droplets conditioned by individual bovine embryos that either developed to the blastocyst stage or did not (nonblastocyst). RNA sequencing identified 69 differentially expressed miRNAs between EVs derived from blastocyst conditioned medium (CM) and nonblastocyst CM. Among the miRNAs up-regulated in blastocyst CM, we selected bta-miR-378a-3p for further validation by functionality testing on developing in vitro embryos by means of mimics and inhibitors. Supplementing the embryo culture medium with miR-378a-3p mimic significantly improved blastocyst quality, with higher cell numbers and reduced apoptosis, and improved hatching, while the opposite was found after supplementation with miR-378a-3p inhibitor (P < 0.01). Transcriptomic analysis of embryos treated with miR-378 mimic/inhibitor showed differential expression (P < 0.01) of genes associated with embryo development and implantation, including RAP1GAP, ARFGEF2, SLC7A6, CENPA, SP1, LDLR, PYCR1, MYD88, TPP1, and NCOA3. In conclusion, miR-378a-3p is up-regulated in EVs secreted by embryos that develop to the blastocyst stage, and this EV-derived miR-378a-3p increases blastocyst quality and regulates embryo hatching, which is essential for embryo implantation