Reproductive development of dairy heifers in an integrated livestock-forest system during the summer.

This study aimed to assess the cortisol, body and reproductive development of prepubertal Holstein and Holstein-Gir ¾ heifers at 27 months of age maintained in an integrated livestock-forest (ILF) system for 60 summer days compared to the monoculture system in full sun (FS). The ILF system promoted changes (P=0.02) in the cortisol levels of Holstein-Gir ¾ heifers and did not affect weight gain in any of the breed groups studied. Animals in ILF system presented a lower (P=0.006) vulvar development for the rima height parameter and similar for the vulva width parameter. The ovarian follicular population of Holstein-Gir ¾ heifers in the ILF system was lower (P=0.004); however, for the Holstein heifers, no statistical difference was found, and numbers were higher (P=0.08) in the ILF system. None of the other ovarian parameters studied had any changes, and we also found important racial differences. Weight gain (P=0.003), vulvar development (P<0.001), and mean follicular size (P=0.008) were higher in the Holstein-Gir ¾ animals. Based on such results, the effect of the ILF system at 27 months of age on stress and reproductive parameters in the Holstein breed is considered positive, although negative effects have been detected on reproductive parameters in the Holstein-Gir ¾ breed.

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.

Imputation accuracy for genomic selection using embryo biopsy samples in Gir.

The aim of this study was to establish a platform for genomic selection of in vitro-fertilized (IVF) Gir embryos. Multiple displacement amplification (MDA)-based embryo biopsy samples were genotyped, and genomic estimated breeding values (GEBV) for milk yield (305MY) were calculated. The concordance of GEBV and accuracy between embryo biopsies and the respective liveborn were assessed. Imputation was performed using two panels (Z-Chip and Bovine HD, Illumina) based on a database of 73,110 lactating cow's database and pedigree files from 147,131 animals. Biopsied embryos had similar pregnancy rates (39% vs 40%), pregnancy loss rates (18% vs 20%), and pregnancy length compared to Control embryos. After genotyping, low call rate means were detected for biopsy samples compared to the respective calf samples (0.80 vs 0.98). Imputation presented 0.83 (Z-Chip) and 0.96 (HD) accuracy (CORRanim). Embryo GEBV accuracy levels were higher in BovineHD imputation (0.82) than Z-Chip imputation (0.55) or no imputation (0.62), and the correlation between embryo/calf pairs' accuracy was 0.85 for BovineHD imputation, 0.11 for Z-Chip imputation, and 0.02 for no imputation. GEVB estimates correlation between embryo/calf pairs was 0.87 for BovineHD imputation, 0.80 for Z-Chip imputation, and 0.41 before imputation. The call rate of embryo samples did not affect the correlation between embryo/calf pairs for accuracy and GEBV before and after BovineHD imputation. Embryos obtained on the same farm presented GEBV 305MY differences of up to 800 kg, emphasizing the expected impact of embryo genomic selection for the Gir breed.

Challenges in the use of nanostructures as carriers of nucleic acids in clinical practice.

The delivery of nucleic acids to cells is considered a crucial step for the success of genetic modifications aimed at therapeutic purposes or production of genetically modified animals. In this context, nanotechnology is one of the most promising fields of science, with the potential to solve several existing problems. Nanostructures have desirable characteristics to be used as carriers, such as nanometric size, large surface area, cell internalization capacity, prolonged and controlled release, among others. Genetically modified animals can contribute to the production of biopharmaceuticals, through the expression of high-associated-value molecules. The production of these animals, also known as biofactories, further enhances Brazilian agribusiness, since it allows adding value to the final product, and favors the integration between the agricultural market and the pharmaceutical sector. However, there is a growing concern about the safety and possible harmful effects of nanostructures, since data on the safe use of these materials are still insufficient. The objective of this review was to address aspects of the use of nanostructures, mainly carbon nanotubes as nucleic acid carriers, aiming at the production of genetically modified animals, with the certainty that progress in this field of knowledge depends on more information on the mechanisms of interaction between nanostructures, cells and embryos, as well as on its toxicity.

Challenges in the use of nanostructures as carriers of nucleic acids in clinical practice

ABSTRACT The delivery of nucleic acids to cells is considered a crucial step for the success of genetic modifications aimed at therapeutic purposes or production of genetically modified animals. In this context, nanotechnology is one of the most promising fields of science, with the potential to solve several existing problems. Nanostructures have desirable characteristics to be used as carriers, such as nanometric size, large surface area, cell internalization capacity, prolonged and controlled release, among others. Genetically modified animals can contribute to the production of biopharmaceuticals, through the expression of high-associated-value molecules. The production of these animals, also known as biofactories, further enhances Brazilian agribusiness, since it allows adding value to the final product, and favors the integration between the agricultural market and the pharmaceutical sector. However, there is a growing concern about the safety and possible harmful effects of nanostructures, since data on the safe use of these materials are still insufficient. The objective of this review was to address aspects of the use of nanostructures, mainly carbon nanotubes as nucleic acid carriers, aiming at the production of genetically modified animals, with the certainty that progress in this field of knowledge depends on more information on the mechanisms of interaction between nanostructures, cells and embryos, as well as on its toxicity.

Daily vaginal temperature in Girolando cows from three different genetic composition under natural heat stress.

The present trial evaluated the effect of crossbred composition and Temperature and Humidity Index (THI) on vaginal temperature (VT) of Girolando dairy cows maintained under tropical pasture during warm seasons. The VT was monitored along 41 to 96 h in 615 Girolando cows with different Holstein (H) × Gir genetic composition (1/2 H = 284, 3/4 H = 248, and 7/8 H = 83) from six Brazilian farms in the summer of 2016 and 2017. VT of each cow at each hour of the day and the respective THI were averaged per hour across all monitoring days to generate an averaged value for VT and THI during 24 h. A linear mixed model with repeated measures using restricted maximum likelihood (REML) method for (co)variance components estimation procedure was employed. The final model adjusted the VT for the effects of cow, time, THI, farm, year, pregnancy status, body condition score (BCS), milk yield, genetic composition, and genetic composition*time interaction. Fixed effects were evaluated by ANOVA and tested with Tukey test in R software version 3.6.1 (R Core Team, 2019). Overall mean of VT, air temperature (AT), and THI were 39.06 ± 0.52 °C, 25.63 ± 0.40 °C, and 75.06 ± 3.96, respectively. VT had moderate positive correlation with THI (r² = 0.45, P < 0.001) and AT (r² = 0.46, P < 0.001). The VT had estimated linear increase of 0.05 °C for each THI unit increase (P < 0.001). Least square mean of VT varied among the farms (P < 0.001), pregnancy status (P < 0.001), and BCS (P < 0.05) but not for Milk yield (P > 0.05). The daily average VT was affected by genetic composition (P < 0.001) with highest temperature for 3/4 H (39.08 ± 0.06 °C a) and 7/8 H (39.09 ± 0.06 °C a) and lowest temperature for 1/2 H (38.95 ± 0.06 °C b). The difference of VT among the three crossbred groups varied in function of the time of the day, from 12:00 to 20:00 h (P < 0.001), with 3/4 Holstein and 7/8 Holstein cows reaching similar VT, above to the upper limit 39.1 °C and higher than 1/2 Holstein cows during all this period. In conclusion, Girolando cows are sensitive to heat stress in tropical condition during warm seasons. Moreover, Girolando cows with genetic composition higher than 3/4 Holstein display reduced thermoregulatory efficiency. Therefore, Girolando cows in tropical dairy farms require strategies to mitigate heat stress according to their genetic composition.

Heat shock during in vitro maturation of bovine oocytes disturbs bta-miR-19b and DROSHA transcripts abundance after in vitro fertilization.

While microRNAs (miRNAs) are a class of non-coding RNAs important for embryo development, the relationship between them and heat stress during oocyte maturation has not yet been established. This study investigated the effect of heat shock during in vitro maturation (IVM) on the abundance of bta-miR-20a, -27b, -103, -21-5p, -19b, -1246 miRNAs and DROSHA and DICER1 mRNAs, previously reported for being involved in oocyte maturation, response to heat stress and miRNA biogenesis. Oocytes were exposed for 12h to heat shock during IVM, fertilized in vitro and the presumptive zygotes cultured for eight days. The relative quantification of miRNAs and mRNAs was performed by real-time PCR in vitro-matured oocytes and 8-cell stage embryos. Progression of meiosis, embryonic development and apoptotic indices was also evaluated. Heat shock compromised (p < .05) oocyte nuclear maturation, cleavage and embryo development, with a higher (p < .05) embryonic apoptotic index than the control group. The abundance of bta-miR-19b increased (p < .05) whereas the abundance of DROSHA transcripts decreased (p < .05) in embryos derived from heat-shocked oocytes. In conclusion, heat shock during IVM influences the abundance of bta-miR-19b and DROSHA in pre-implantation embryos, indicating a persistent effect of heat shock that can be associated with impaired embryo development.

Chromium supplementation improves glucose metabolism and vaginal temperature regulation in Girolando cows under heat stress conditions in a climatic chamber.

This study aimed to evaluate chromium supplementation on productive, reproductive, and metabolic parameters at lactating Girolando cows subjected to heat stress conditions in a climatic chamber. Thirty-six lactating Girolando cows were subjected to two sequential trials. In trial 1 (thermoneutral environment), the effect of chromium supplementation was evaluated (0 vs. 0.50 mg/kg of dry matter). In trial 2, the cows were fed the same diets, but they were divided into three environmental conditions: heat stress conditions in climatic chamber, fed ad libitum (HS); thermoneutral environment, fed ad libitum (TN); and thermoneutral environment, pair-fed (PF). In thermoneutral conditions, chromium supplementation did not affect productive or metabolic parameters, although supplemented cows had lower viability of oocytes (65.11 ± 0.08% vs. 76.86 ± 0.08%). During heat stress, chromium supplementation lowered plasma glucose levels (61.17 ± 1.90 vs. 67.11 ± 1.90 mg/dL), and increased the insulin:glucose ratio (0.39 ± 0.04 vs. 0.27 ± 0.04). Cows fed the control diet in the HS group had higher vaginal temperature values (39.40 ± 0.10 °C) than the cows in the TN group and PF group (38.89 ± 0.10 °C and 38.85 ± 0.11 °C, respectively). However, supplemented cows heat-stressed maintained the same vaginal temperature as cows in thermoneutral conditions. In conclusion, chromium supplementation improved glucose metabolism and prevented body temperature increases under heat stress conditions.

Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone.

The aim of this study was to evaluate the dose-response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus-oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus-oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.

Post-biopsy bovine embryo viability and whole genome amplification in preimplantation genetic diagnosis.

OBJECTIVE: To evaluate the effect of the biopsy of 8-cell to 16-cell bovine embryos on their subsequent development and the effect of whole genome amplification (WGA) on removed blastomeres. DESIGN: Randomized study. SETTING: Molecular genetics and animal reproduction laboratories. PATIENT(S): Cow ovaries obtained from slaughterhouses. INTERVENTION(S): The ovaries were punctured, and the oocytes were matured and fertilized in vitro. On the fourth day after fertilization, 8-cell to 16-cell bovine embryos were biopsied, one quarter of each embryo being removed. The blastomeres were submitted to WGA followed by polymerase chain reaction (PCR). The embryos were returned to culture for evaluation of their development. MAIN OUTCOME MEASURE(S): Subsequent rate of blastocyst development, embryo cell number, WGA efficiency, and sex determination. RESULT(S): A total of 92 embryos were submitted to biopsy. The blastocyst production was 53.3%, with 44.9% of hatching rate. These results were similar to those of the control group (66.0% and 42.6%) of 103 embryos. Overall, no impact was detected on embryo quality in blastocyst cell number between the two groups. Removed blastomeres were submitted to WGA, resulting in 98.2% of efficiency. However, only 59% of the samples were sexed by PCR. CONCLUSION(S): Biopsy of 8-cell to 16-cell bovine embryos did not affect their subsequent development. WGA was successful in removed blastomeres.

Avanços tecnológicos da criopreservação de ovócitos / Technological advances in oocytes criopreservation

Apresenta-se uma revisão atualizada sobre a criopreservação de ovócito e sua importância em reprodução assistida. O presente trabalho tem como ênfase vantagens e desvantagens dos métodos utilizados para congelamento de gametas, efeito dos crioprotetores intracelulares e extracelulares, além da aplicabilidade da técnica na fertilidade feminina e na manutenção da biodiversidade animal.

It presents an updated review of oocyte cryopreservation and its importance in assisted reproduction. Th is work has an emphasis on advantages and disadvantages of the methods used for freezing gametes, the eff ects of intracellular and extracellular cryoprotectants, and the applicability of the technique in female fertility and in maintaining animal biodiversity