APERFEIÇOAMENTO EM MÉTODO PARA CULTIVO IN VITRO DE EMBRIÕES BOVINOS

Description

1 / 7 IMPROVEMENT IN METHOD FOR IN VITRO CULTURE OF EMBRYOS CATTLE Field of invention

[0001] The present invention relates to the field of biotechnology applied to animal reproduction, introducing improvements in the technique of in vitro production of bovine embryos. The invention may also be useful to the agricultural sciences sector, to the genetic improvement of species of economic interest and to veterinary medicine, with direct application in animal genetic improvement and reproduction programs. Fundamentals of the invention

[0002] In vitro production of bovine embryos (IVP) is a biotechnology of great importance to livestock farming, allowing animals with superior genetics to produce numerous offspring, and facilitating the transfer of this genetics to other herds. To produce embryos in vitro, it is necessary to cultivate them in an incubator, in plates containing media produced in the laboratory. The media and supplements aim to offer the embryos ideal cultivation conditions so that they reach the blastocyst stage and can be transferred to recipient females ("surrogate mothers").

[0003] The known media used have a common base formula, such as SOF (Synthetic Oviduct Fluid), whose basic composition is in the public domain (Duszewska et al., 2012). However, supplements can be added to this base medium to improve embryo quality and, therefore, increase the efficiency of IVF. Several supplements have been described and tested for bovine embryo culture, such as supplements associated with increased... Petition 870250006309, dated 01 / 27 / 2025, page 10 / 18 2 / 7 of metabolism (De La Torre-Sanchez, et al., 2006; BarcelóFimbres & Seidel, 2007; Dias et al., 2017) and lipid reduction (De La Torre-Sanchez et al., 2006; Leão et al., 2017).

[0004] To reach the blastocyst stage and be transferred to recipients, correct initial embryonic development must occur involving successive cleavages of blastomeres (embryonic cells). Up to the 8-cell stage, embryos use the maternal stock (derived from the egg) of messenger RNA for protein synthesis, essential for the cellular machinery to function properly (Khan et al., 2012). However, in bovine species, between the 8 and 16-cell stages, blastomeres must activate their own genome to produce messenger RNAs, a process called embryonic genome activation (Badr et al., 2007). This process requires changes in chromatin architecture, mediated by covalent modifications of histones, which are necessary for genome activation and subsequent embryonic development (Oliveira et al., 2012).Many bovine embryos fail to make this zygotic transition (in the 8-to-16-cell cycle), and their development is blocked at 8 cells (Meirelles et al., 2004). This is a significant problem in the in vitro production of bovine embryos, contributing to the estimated low production efficiency: only about 30% of embryos reach the desired stage, the blastocyst.

[0005] Covalent modifications of histones are crucial for chromatin condensation or relaxation, and are mediated by enzymes. In the case of histone acetylation, a modification that relaxes chromatin and favors gene transcription, the Petition 870250006309, dated 01 / 27 / 2025, page 11 / 18 3 / 7 acetyl groups are inserted by the enzyme histone acetyltransferase (HAT), and removed by the enzyme histone deacetylase (HDAC). In bovine embryos, enzymes such as HDACs 1, 2, 3, 7 and HAT1 have been identified and show interdependent activity (McGraw et al., 2003).

[0006] In bovine embryos, after fertilization, the embryonic genome is condensed, with very low levels of gene expression (Dean et al., 2001). At the time of embryonic genome activation, histone acetylation reaches a peak, consistent with global elevations in gene expression levels, while in 16-cell embryos and morulae, acetylation levels are reduced (Maalouf et al., 2008).

[0007] Studies indicate that histone modifications are influenced by embryo manipulation and in vitro culture (Santos et al., 2003). Histone acetylation undergoes significant variations during the embryonic development of embryos cultured in the laboratory (Kang et al., 2002). Experiments conducted by the inventor found two distinct populations of in vitro produced embryos at the 8-16 cell stage: one showing similar profiles between blastomeres and the other showing notable alterations between blastomeres.

[0008] Trichostatin A (TSA) is a molecule that reversibly inhibits HDAC, increasing histone acetylation levels (Zupkovitz et al., 2006). Studies by the inventor in in vitro produced bovine embryos (IVP) have shown that TSA does not affect blastocyst rates and promotes increased histone acetylation levels when used at the indicated concentrations and on the indicated days. Furthermore, studies Petition 870250006309, dated 01 / 27 / 2025, p. 12 / 18 Studies using nuclear transfer techniques (NDT) have shown that NDT increases the efficiency of epigenetic reprogramming in somatic cells (Ding et al., 2008; Enright et al., 2003; Wee et al., 2007) and in embryos (Ding et al., 2008; Iager et al., 2008), indicating the possibility of increasing the percentage of competent embryos that activate their genomes.

[0009] To increase histone acetylation levels, an event with the potential to increase blastomere genome activation, supplements are not available in the prior art, which has been provided by the present invention.

[00010] Preliminary results from research conducted by the inventor showed that trichostatin A can be used in the culture of bovine embryos during the 8-16 cell stage without affecting development rates. Furthermore, a histone hyperacetylation effect is observed at the appropriate dosage. In low-quality embryos with fewer cells, the addition of trichostatin A reduces programmed cell death rates, demonstrating a beneficial effect for this population. Description of the invention

[00011] The present invention developed a method for producing in vitro embryos cultured in a medium supplemented with trichostatin A (TSA), which promotes histone hyperacetylation. The method, detailed below, used bovine ovaries collected from a slaughterhouse and processed within 2 hours of slaughter. The reagents used were acquired from Sigma, except where otherwise specified. The ovaries were washed in saline solution (37°C) and follicles 3 to 8 mm in diameter were aspirated with an 18G needle attached to a 20 mL syringe. The follicular fluid was deposited in a 50 mL conical tube. Petition 870250006309, dated 01 / 27 / 2025, page 13 / 18 5 / 7 mL were used for sedimentation for 20 min at 37°C, and then 10 mL of the sediment were collected and transferred to 100 mm diameter polystyrene plates. Cumulus-oocyte complexes (COCs) exhibiting at least three layers of cumulus cells and homogeneous cytoplasm were selected under a stereomicroscope. The COCs were washed in HEPES-buffered TCM-199 (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Cripion Biotecnologia, Andradina, SP, Brazil), 16 μg / mL sodium pyruvate, and 83.4 mg / mL amikacin (Instituto Biochimico, Rio de Janeiro, RJ, Brazil).

[00012] Groups of 15 to 20 CCOs were transferred to 100 μL drops of TCM-199 supplemented with 10% FBS, 1.0 μg / mL of FSH (Folltropin™, Bioniche Animal Health, Belleville, ON, Canada), 50 μg / mL hCG (Profasi™, Serono, São Paulo, SP, Brazil), 1.0 mg / mL estradiol, 16 μg / mL sodium pyruvate, and 83.4 mg / mL amikacin, covered with sterile mineral oil (Dow Corning Co., Midland, MI, USA), and incubated for 24 ha at 38.5 °C in a 5% CO2 atmosphere in saturated humidity air for in vitro maturation (IVM).

[00013] After IVM, groups of 15 to 20 oocytes were washed twice and transferred to 80 μL drops of TALP-IVF medium supplemented with 0.6% BSA, 10 μg / mL heparin, 18 mM penicillamine, 10 μM hypotaurine, and 1.8 μM epinephrine, and covered with sterile mineral oil. Semen straws (CRV, Lagoa, Sertãozinho, SP) were thawed and used for in vitro fertilization (IVF). After thawing the straw at 35.5 °C, the contents were placed in a discontinuous 45 / 90 Percoll gradient for centrifugation for 7 min at 3600 x g. The sediment was collected (100 μL) and resuspended in 700 μL of Petition 870250006309, dated 01 / 27 / 2025, p. 14 / 18 6 / 7 TALP-FIV medium, and centrifuged again for 5 min at 520 x g. After centrifugation, 30 μL of the medium containing the sediment was collected from the bottom of the tube and homogenized in a conical tube. The suspension was adjusted to a final concentration of approximately 10,000 motile spermatozoa per oocyte. The plates were incubated at 38.5 °C for 20 h in a 5% CO2 atmosphere in saturated humidity air.

[00014] After in vitro fertilization, the prospective zygotes were denuded from the cumulus cells by vigorous pipetting. The embryos were washed three times and transferred in groups of 15 to 20 for culture in 100 μL drops of SOF medium supplemented with 5 mg / mL BSA and 2.5% FBS. The plates were then incubated in a 5% CO2 atmosphere in atmospheric air under saturated humidity at 38°C.

[00015] For preparation of the TSA supplement, the lyophilized product, with a molecular weight of 303.37, was diluted in 100% ethanol in a stock solution at the ratio: 0.151685 mg in 10 mL of ethanol. The stock solution was stored in a freezer (-20°C). At the time of use, an aliquot of stock solution was prepared, containing 1 mL of SOF + 10 μL of the TSA stock solution. After homogenization, the stock solution was added to the final culture medium at the ratio 10 μL: 1 mL (5 nM concentration) and 30 μL: 1 mL (15 nM concentration).

[00016] Embryos 70 h post-IVF were washed and transferred to 100 μL drops of SOF medium supplemented with trichostatin A (TSA) at concentrations of 5 nM and 15 nM for 12, 24, and 48 h. After the determined periods, the embryos were washed and transferred to 100 μL drops of TSA-free SOF medium. Half of the medium (50 μL) from each drop was replaced every 48-72 h. Petition 870250006309, dated 01 / 27 / 2025, page 15 / 18 7 / 7 Examples of embodiments of the invention

[00017] The invention method can be used to increase histone acetylation levels in bovine embryos, showing superior results to the traditional method, which does not use TSA supplementation, called the Control group. Thus, use starting 70 h after in vitro fertilization ensures that embryos between 8-16 cells are reached to assist in chromatin remodeling, loosening the interaction of histones with DNA and enabling gene transcription from blastomere DNA. Preferably, a concentration of 5 nM should be used, since assays performed by the inventor demonstrated significant increases (2.35 times) in histone acetylation levels at this concentration. For more intense stimuli, a concentration of 15 nM can be applied. The application time can be 12, 24 or h, depending on the selected objective.If the goal is to separate delayed embryos and apply more intense stimulation, a longer treatment period may be appropriate. For lower stimulation rates in the embryo group as a whole, shorter periods may be preferable. The use of 15 nM for 48 h does not affect embryo production rates, but significantly increases programmed cell death rates (Control = 3.19%, 5 nM TSA = 3.40%, 15 nM TSA = 7.56%*). (p<0.05). Petition 870250006309, dated 01 / 27 / 2025, page 16 / 18

Claims

1 / 1 - CLAIMS - 1. IMPROVEMENT IN A METHOD FOR IN VITRO CULTURE OF BOVINE EMBRYOS, the method consisting of culturing probable zygotes between 15 and 18 hours old obtained through in vitro maturation of oocytes followed by in vitro fertilization of oocytes, the probable zygotes being stripped of the cells surrounding them by vigorous pipetting, followed by washing and transfer to microdrops of 50 to 100 microliters of SOF medium, prepared in polystyrene plates with a diameter between 35 and 60 mm and covered with mineral oil and kept in an incubator at 38.5 °C in an atmosphere of 20% oxygen in atmospheric air for between 147 and 162 hours, the improvement characterized by the addition of histone deacetylase enzyme inhibitors between 52 and 80 hours after the The start of cultivation, equivalent to 70 to 98 hours after the start of in vitro fertilization.

2. IMPROVEMENT IN METHOD FOR IN VITRO CULTURE OF BOVINE EMBRYOS, according to claim 1, characterized in that the inhibiting agent is trichostatin A.

3. IMPROVEMENT IN METHOD FOR IN VITRO CULTURE OF BOVINE EMBRYOS, according to claim 2, characterized in that trichostatin A is present in the embryonic culture medium at a concentration ranging from 5 nM to 15 nM.

4. IMPROVEMENT IN METHOD FOR IN VITRO CULTURE OF BOVINE EMBRYOS, according to claim 1, characterized by also containing the ingredients 5 mg / mL of bovine serum albumin and 2.5% fetal bovine serum in SOF base medium. Petition 870250006309, dated 01 / 27 / 2025, pp. 17 / 18