Effect of shading on the physiological and reproductive parameters in Nelore cows.

This study aimed to evaluate the physiological and reproductive parameters of Nelore cows under various microclimatic conditions. The experiment was performed in Central Brazil, which has a tropical climate with dry winters and rainy summers. The experimental groups were divided into an area with shading (5.8 trees/ha; n = 42) and an area with little shading (0.4 trees/ha; n = 42). The physiological (heart rate, respiratory rate, rectal temperature, surface body temperature, and vaginal temperature) and reproductive (estrus detection, conception rate, and rate of pregnancy loss) parameters were then assessed. The rectal temperature (p = 0.002); surface body temperature on the flanks, forehead, and shoulder (p < 0.001); and vaginal temperature (p < 0.001) of cows in the group with shading were significantly lower than those of cows in the group without shading. Estrus was higher (p = 0.08) in the shaded group; however, no effect was observed on conception and pregnancy loss rates. In conclusion, shading (5.8 trees/ha) provided better thermal comfort for Nelore animals in the tropical climatic region. However, this did not affect their performance after fixed-time artificial insemination.

Lipid Metabolism in Bovine Oocytes and Early Embryos under In Vivo, In Vitro, and Stress Conditions.

Lipids are a potential reservoir of energy for initial embryonic development before activation of the embryonic genome and are involved in plasma membrane biosynthesis. Excessive lipid droplet formation is detrimental to cryotolerance and is related to alterations in mitochondrial function, which likely affects lipid metabolism. Increased lipid accumulation in in vitro produced embryos is a consequence of the stress during in vitro embryonic development process. There are several open questions concerning embryo lipid metabolism and developmental potential. Oocyte maturation and embryo development in vivo and in vitro may vary if the donors are subjected to any type of stress before follicle puncture because crucial changes in oocyte/embryonic metabolism occur in response to stress. However, little is known about lipid metabolism under additional stress (such as heat stress). Therefore, in this review, we aimed to update the information regarding the energy metabolism of oocytes and early bovine embryos exhibiting developmental competence, focusing on lipid metabolic pathways observed under in vivo, in vitro, and stress conditions.

Shading effect on physiological parameters and in vitro embryo production of tropical adapted Nellore heifers in integrated crop-livestock-forest systems.

The aim of this study was to evaluate the impact of increased shadow supply in integrated crop-livestock-forest systems on in vitro embryonic development and physiological parameters related to stress response in Nellore heifers (Bos indicus). For the study, animals (n = 16) were randomly divided into two groups and kept in areas with different afforestation systems, the integrated crop-livestock-forest (ICLF) and the integrated crop-livestock (ICL) system. The microclimate of the ICLF system provided better comfort conditions than ICL. No differences of respiratory rate, rectal temperature, cortisol, T3, T4, oocyte quality, and cleavage rate between the systems were verified. A higher blastocyst rate was observed in the ICLF (p < 0.05). The results demonstrate that Nellore heifers managed in ICLF during summer in Midwest of Brazil showed higher production of in vitro embryos, without typical changes in its physiological parameters. The results observed in the present study indicate that zebu females are able to respond satisfactorily to the intense heat conditions; however, we believe that the long period to which these animals are exposed to these conditions interferes in the oocyte competence and embryo development.

Trimethylation of histone 3 at lysine 4 in cryopreserved bovine embryos produced in vivo with sexed semen.

The production rates of viable embryos using sexed semen through the conventional methodologies of multiple ovulation and embryo transfer are generally not satisfactory. However, the cryopreservation of these embryos is considered efficient. Knowledge of epigenetics can provide new tools or allow for adapting new protocols that could enhance the efficiency of reproductive biotechnologies. The aim of this study was to characterize the pattern of trimethylation of histone 3 at lysine 4 (H3K4me3) in bovine embryos produced in vivo with sexed semen that were submitted to cryopreservation. Bos taurus × Bos indicus cows (n = 5) were superovulated and inseminated with sexed (two sessions) or conventional (two sessions) semen. A portion of the embryos collected on Day 7 was immediately stored in paraformaldehyde (3%) and another portion was stored in paraformaldehyde after cryopreservation/thawing. All embryos from the four groups (fresh, conventional semen; fresh, sexed semen; cryopreserved, conventional semen; and cryopreserved, sexed semen; 15 embryos per group) were evaluated by immunofluorescence under confocal microscopy to identify and quantify the H3K4me3 status. In total, 190 embryos were recovered, 100 of which were produced with conventional semen and 90 with sexed semen. The use of conventional semen after superovulation yielded 72% (72 of 100) viable embryos, which were mostly (81%; 59 of 72) in advanced stages of development (blastocysts and expanded blastocysts). Embryos produced with sexed semen had a lower viability rate (36.7%; 33 of 90), and most of them were collected at earlier stages of development (morulae and early blastocysts; P < 0.05). The H3K4me3 signal was similar among groups; however, there was a difference between morulae and blastocysts. A high intensity of H3K4me3 was observed in bovine embryos produced in vivo, and this pattern did not vary using sexed semen and the slow cryopreservation process. The lower viability of bovine embryos produced with sexed semen could be not explained by differences in H3K4me. Cryopreservation did not alter the pattern of H3K4me3; in this sense, we suggest that it is a process that exerts minimal damage to the embryos.

Donor cell lines considerably affect the outcome of somatic nuclear transfer in the case of bovines.

Since the first successful nuclear transfer (NT) experiments were carried out, various somatic cell types have been used as donor cells for production of cloned animals. In most experiments, fibroblasts are used since they only need to be isolated and cultivated. Recently, some researchers have shown that different cell cultures from different sources possess different capacities to support preimplantation development of NT embryos. The blastocyst rates obtained in our previous studies varied and were as high as 45% in relation to the number of reconstructed embryos. This led us to question whether the origin and culture conditions of the defined male and female fibroblast lines could be responsible for the differences in developmental potency. Taking all our results into consideration, we conclude that different fibroblast lines recovered from the same tissue and cultivated under equal culture conditions could produce dramatically different blastocyst rates. The influence of cell line itself is higher than the influence of passage number. The observed effects of cell cycle stage, chromosomal aberrations, and diminished vitality are important but not sufficient to discriminate well-qualified nuclear donor cells. We speculate that some epigenetically regulated deviations in the gene expression program are responsible for these phenomena. Explanation of the underlying mechanisms should contribute to better understanding of epigenetic reprogramming and may ultimately assist reprogramming in the laboratory.

Developmental competence of HMC(TM) derived bovine cloned embryos obtained from somatic cell nuclear transfer of adult fibroblasts and granulosa cells.

To enable us to handle a large number of oocytes at a given time and to have an increased throughput of cloned embryos, we attempted the Handmade cloning (HMC) technique, a zona-free method of bovine somatic cell nuclear transfer. Our objective was to study the developmental competence of the HMC derived embryos obtained using different types of somatic cells. A total of 6,874 cumulus-oocyte-complexes were used with either 7th or 11th passage fibroblasts (1st and 2nd groups, respectively), which were prepared from male animals, or granulosa cells (3rd group) as nuclei donors. The average cleavage rate was 65%, accompanied by a blastocyst rate of just 2% for the cleaved products and 5% for the >8-cell embryos, and there was no significant difference between the three groups. Out of 27 blastocysts recovered, 22 blastocysts were transferred to 22 recipients, resulting in two pregnancies. One pregnancy was lost after the fourth week while the other progressed to full term with the birth of a male calf. This first successful cloning of a male calf with the HMC technique in Europe indicates the successful adoption and establishment of this technique in our laboratory, and that this technique can be successful in producing viable embryos.