A diet enriched in linoleic acid compromises the cryotolerance of embryos from superovulated beef heifers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

Assessment of the reproductive parameters, laparoscopic oocyte recovery and the first embryos produced in vitro from endangered Caninde goats (Capra hircus)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro Maturation of Oocytes from Santa Ines Ewes Subjected to Consecutive Sessions of Follicular Aspiration by Laparoscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efficiency of OPU-IVEP-ET of Fresh and Vitrified Embryos in Buffaloes

The present study aims to report ovum pickup (OPU), in vitro embryo production (IVEP) and embryo transfer (ET) outcomes of fresh and vitrified buffalo embryos. For this purpose, 36 buffalo donors were submitted to 11 OPU sessions (n = 201). A total of 998 oocytes (5.0 +/- 0.5/donor/session) and 584 viable oocytes (2.9 +/- 0.3/donor/session) were recovered. Viable oocytes (grades 1, 2 and 3) were subjected to IVM, IVF (D0) and IVC. On D2, 54.5% of cleavage rate was obtained. Embryo yield on D7 was 44.9% (grade 1: 229 embryos, grade 2: 5 embryos and grade 3: 28 embryos). From this total, 115 fresh (grades 1 to 3) and 70 vitrified embryos (only grade 1) were transferred into recipients previously synchronized with fixed time embryo transfer (FTET) protocol. Vitrification was performed using the cryotop method. Pregnancy diagnosis in fresh and in vitrified groups were, respectively: 43.5% (50/115) and 37.1% (26/70) on 30 days after embryo transfer, and 41.7% (48/115) and 31.4% (22/70) on 60 days after embryo transfer. In conclusion, our results demonstrate the possibilities for commercial use of the techniques of OPU, IVEP and ET of fresh and vitrified embryos in buffaloes.

The role of chromosome variation in the speciation of the red brocket deer complex: the study of reproductive isolation in females

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito de dose reduzida de somatotropina bovina recombinante associada a protocolos de superovulação na fertilidade de embriões transferidos para receptoras em lactação

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Control of oocyte maturation

Oocyte maturation is a complex process involving nuclear and cytoplasmic maturation. The nuclear maturation is a chromosomal segregation and the cytoplasmic maturation involves the reorganization of the cytoplasmic organelles, mRNA transcription and storage of proteins to be used during fertilization and early embryo development. The mechanism of oocyte maturation in vivo and in vitro still are not totally understood. However it is generally accepted that the second messenger cyclic adenosine monophosphate (cAMP) plays a critical role in the maintenance of meiotic blockage of mammalian oocytes. A relative increase in the level of cAMP within the oocyte is essential for maintaining meiosis block, while a decrease in cAMP oocyte concentration allows the resumption of meiosis. The oocyte cAMP concentration is regulated by a balance of two types of enzymes: adenylate cyclase (AC) and phosphodiesterases (PDEs), which are responsible for the synthesis and degradation of cAMP, respectively. After being synthesized by AC in cumulus cells, cAMP are transferred to the oocyte through gap junctions. Thus, specific subtypes PDEs are able to inhibit or attenuate the spontaneous meiotic maturation of oocytes with PDE4 primarily involved in the metabolism of cAMP in granulosa cells and PDE3 in the oocyte. Although the immature oocytes can resume meiosis in vitro, after being removed from antral follicles, cytoplasmic maturation seems to occur asynchronously with nuclear maturation. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of in vitro embryo production and to develop treatments for various forms of infertility. This review will present current knowledge about the maintenance of the oocyte in prophase arrest, and the resumption of meiosis during oocyte maturation, focusing mainly on the changes that take place in the oocyte.

Preservation of wild feline semen by freeze-drying: experimental model

According to the Convention on International Trade in Endangered Species, 36 wild feline species are threatened by extinction or severely endangered, and to save them is the target of several conservation programs. This study aimed to assess the viability of the freeze-drying technique for domestic cat sperm cells, with the ultimate goal of transferring this technology to the wild feline species. The domestic cat is an excellent experimental model for wild felids. It is in this scenario that the freeze-drying process (low-temperature vacuum dehydration) of sperm cells shows its value in preserving male cats' germplasm. Results from membrane and DNA integrity analysis are promising and validates the use of frozen-dried sperm samples in intracytoplasmic sperm injections (ICSIs). Further studies are still necessary to evaluate the ICSI embryo production using domestic cat frozen-dried sperm and the possibility of using such technology with wild felines.

Oocyte maturation in bitches

The canine species has been used as an experimental model for preservation of endangered species. Biotechnologies of reproduction, such as in vitro maturation (IVM), have been used to meet this objective. Several protocols for in vitro embryo production (IVEP) in swine and bovine species have been adapted for canids. However, the highest rate reported for in vitro maturation in canids is only 39%, which is still lower than those in other species. Therefore, current research on assisted reproduction in canids have focused on several IVM protocols, including the addition of proteins, hormones, meiosis inhibitors, growth factors and antioxidants to the maturation media and the determination of suitable timing for culture, so that variables involved in the process can be fine-tuned. This review has the main objective of describing major developments and limitations in the process of oocyte maturation in bitches.

Biotecnologias aplicadas à reprodução de éguas velhas

The acceptance of biotechnology for the most equine breeders association had a significant effect in the horse industry, gaining popularity around the world, because the increasing on the genetic gain, allowing the use of sub fertile mares and stallions with high genetics value on reproduction. The embryos in vitro production of human and cattle has been used with success, however in vitro embryo production is not efficient in the horse, as oocyte transfer (OT) and intracytoplasmatic sperm injection (ICSI). The oocyte transfer has been used especially in subfertile old mares presenting reproductive pathologies as: endometrite, cervical and uterine adhesions, blocked oviduct, perineal laceration and ovulation failures. During oocyte recovery process, the oocytes must be collected from immature follicles that need be matured in vitro or in vivo matured oocytes from pre-ovulatory follicles through the transvaginal aspiration guided by ultrasound. The recovered oocyte is transferred to a previously inseminated recipient mare, through the flank laparotomy. The intracytoplasmatic sperm injection (ICSI) is a procedure of in vitro fertilization that needs only one sperm that is aspirated and injected inside the oocyte. The oocytes used, can be from mature and immature follicles. Fresh, cooled and frozen semen can be used, because the procedure not requires a functional sperm. The use of Piezo drill resulted in a breakthrough the pellucid zone, allowing the vibration per minute provided in the sperm injection pipette, a major result of cleaved oocytes, due to a better sperm injection in the oocyte. The embryo transfer can be straight inside the oviduct, as also transcervical transferred after embryo culture produced in vitro. In conclusion both procedures (OT and ICSI) are effective to be used on equine assisted reproduction, getting results even lower than expected, but satisfactory from animal genetically superior

Dinâmica folicular ovariana e bases para a aplicação das biotécnicas da reprodução em bovinos

In the last years Brazil has achieved a leading position in worldwide cattle breeding. This country is consolidating the practice of an intensive and technical level cattle breeding, due to greater emphasis on reproductive efficiency and genetic improvement. The attention given to reproductive efficiency became to enhance the genetic improvement process, by exploiting the full reproductive potential of the animals. The biotechniques applied to reproduction arised to expand the reproductive potential for greater economic return. Are the most important reproductive biotechniques the FTAI and in vivo and in vitro embryo production for embryo transfer. Different hormonal treatments are consolidating to interfere with normal physiology in order to achieve the desired goals. The FTAI works to increase the reproductive rates of cattle through pre-booked artificial insemination in all the females in reproduction, without the need for estrous detection. The in vivo and in vitro embryo production aims to distribute the genetics of males and females genetically improved by transferring their embryos to genetically inferior females. Superovulation treatments designed to prevent the mechanism of dominance, which results in many follicles ovulating simultaneously for in vivo embryo production. To the in vitro embryo production, the technique of ovarian puncture guided by transvaginal ultrasound allows to aspirate a number of quality oocytes. The application of biotech is based on knowledge of the female and ovarian reproductive physiology and knowledge of their acting in different breeds and environments.

Morfometria e compactação da cromatina espermática de touros Nelore de acordo com a idade e sua influência na produção de embriões in vitro

Pós-graduação em Medicina Veterinária - FCAV

Controle e estimulação de crescimento folicular em doadoras da raça holandesa para a produção in vitro de embriões

Pós-graduação em Medicina Veterinária - FCAV