Resumo

Purpose: This study aimed to compare the degree of maturation and development of fetal pig segmental intestinal tissue with that of spheroids created by in-vitro reaggregation of dissociated fetal intestinal cells after transplantation into immunodeficient mice. Methods: Fetal pig small intestines were transplanted as segmental grafts into the omentum and subrenal capsules of immunodeficient mice or enzymatically treated to generate single cells. Spheroids made by in-vitro reaggregation of these cells were transplanted into the subrenal capsules of immunodeficient mice. The segmental grafts and spheroids were harvested four and eight weeks after transplantation, and the structural maturity and in-vivo development of these specimens were histologically evaluated. Results: The spheroids were engrafted and supplied blood vessels from the host mice, but an intestinal layered structure was not clearly observed, and there was almost no change in size. On the other hand, the segmental grafts formed deep crypts in the mucus membrane, the inner circular layer, and outer longitudinal muscles. The crypts of the transplanted grafts harvested at eight weeks were much deeper, and the smooth muscle layer and the enteric nervous system were more mature than those of grafts harvested at the fourth week, although the intestinal peristaltic wave was not observed. Conclusions: Spheroids created from fetal small intestinal cells could not form layered structures or mature sufficiently. Conversely, segmental tissues structurally matured and developed after in-vivo transplantation and are therefore potential grafts for transplantation.

Resumo

ABSTRACT This study evaluated the effects of long-acting injectable progesterone supplementation (P4LA) on embryonic and fetal development and birth weight in Nellore cows seven days after timed artificial insemination (TAI). One hundred and nineteen Nellore female cows became pregnant after the TAI protocol and were divided into two groups: P4LA with cows that received 150mg of P4LA, seven days after insemination, in a single dose, and the control group, with cows who did not receive any hormonal supplementation after TAI. Blood samples were collected on days 17 and 30 after TAI to determine P4 concentration. Embryonic and fetal measurements were performed at 30 and 45 days of gestation, respectively, with the aid of ultrasonography, measuring craniocaudal length and thoracic diameter. No difference was observed in P4 concentration between the groups supplemented with or without injectable P4 on days 17 (P=0.73) and 30 (P=0.62) after TAI. There was no significant difference in embryonic and fetal development or birth weight between the supplemented and non-supplemented groups (P=0.59, P=0.09, and P=0.64, respectively). Supplementation with injectable progesterone seven days after TAI did not interfere with the embryonic and fetal development of Nellore cows, nor did it affect birth weight.

RESUMO O presente estudo avaliou o efeito da suplementação de progesterona injetável de longa ação (P4LA), sete dias após a inseminação artificial em tempo fixo (IATF), em matrizes Nelores, sobre o desenvolvimento embrionário, fetal e o peso ao nascimento. Para tanto, 119 fêmeas Nelores que ficaram gestantes após protocolo de IATF foram divididas em dois tratamentos: grupo P4LA, com fêmeas que receberam 150mg de P4 injetável de longa ação, em dose única, sete dias após a IATF; grupo controle, com fêmeas que não receberam nenhuma suplementação após a IATF. Amostras de sangue foram coletadas nos dias 17 e 30 após a IATF, para determinação da concentração de P4. A mensuração embrionária e fetal foi realizada aos 30 e 45 dias de gestação, respectivamente, com o auxílio da ultrassonografia, pela mensuração do comprimento craniocaudal e o diâmetro torácico. Não foi observada diferença na dosagem de P4 entre o grupo suplementado ou não no dia 17 (P=0,73) e (P=0,62) após a IATF. Não houve diferença significativa entre o desenvolvimento embrionário e fetal dos grupos suplementados ou não (P=0,59; 0,09, respectivamente). Suplementação com progesterona injetável sete dias após a IATF não interferiu no desenvolvimento embrionário e fetal de matrizes Nelores, assim como no peso ao nascimento.

Resumo

With the development of in vitro technologies, embryos can be produced using oocytes retrieved directly from the ovaries, i.e., regardless of ovulation. This has allowed the use of different animal categories as oocyte donors, including prepubertal cattle. The advantages of using this strategy to shorten the generation interval and accelerate genetic gain over time were soon recognized, and the first offspring generated using oocytes collected from calves were born in the early 1990s. Nevertheless, embryo production from calves and prepubertal heifers remains a challenge. The oocytes collected before puberty present low in vitro developmental potential, and the subsequent blastocyst rates are consistently lower than those from pubertal females. The acquisition of developmental competence by the oocytes occurs progressively throughout the prepubertal period, which can be subdivided into an early, intermediate, and late prepubertal (or peripubertal) phases, each characterized by different physiological and endocrine features. Therefore, embryo yield increases with age but will only achieve its maximum after puberty. The most common strategy to improve oocyte developmental potential before puberty is the use of gonadotrophic stimulation prior to oocyte retrieval. The results with superstimulation, however, vary among studies, depending on the source, dose, and length of FSH treatment, as well as the age and breed of the donors. The use of calves and prepubertal heifers as oocyte donors should also consider the possible impacts of the oocyte retrieval technique (LOPU or OPU) and the use of exogenous hormones on their subsequent fertility and productive life.(AU)

Resumo

With the development of in vitro technologies, embryos can be produced using oocytes retrieved directly from the ovaries, i.e., regardless of ovulation. This has allowed the use of different animal categories as oocyte donors, including prepubertal cattle. The advantages of using this strategy to shorten the generation interval and accelerate genetic gain over time were soon recognized, and the first offspring generated using oocytes collected from calves were born in the early 1990s. Nevertheless, embryo production from calves and prepubertal heifers remains a challenge. The oocytes collected before puberty present low in vitro developmental potential, and the subsequent blastocyst rates are consistently lower than those from pubertal females. The acquisition of developmental competence by the oocytes occurs progressively throughout the prepubertal period, which can be subdivided into an early, intermediate, and late prepubertal (or peripubertal) phases, each characterized by different physiological and endocrine features. Therefore, embryo yield increases with age but will only achieve its maximum after puberty. The most common strategy to improve oocyte developmental potential before puberty is the use of gonadotrophic stimulation prior to oocyte retrieval. The results with superstimulation, however, vary among studies, depending on the source, dose, and length of FSH treatment, as well as the age and breed of the donors. The use of calves and prepubertal heifers as oocyte donors should also consider the possible impacts of the oocyte retrieval technique (LOPU or OPU) and the use of exogenous hormones on their subsequent fertility and productive life.(AU)

Resumo

The oviduct and uterus provide an optimal environment for early embryo development, where effective communication between the embryo and the maternal reproductive tract is crucial for establishing and maintaining pregnancy. Oviductal and uterine-derived EVs play pivotal roles in this maternal-embryonic communication and in facilitating early embryo development. However, despite the ability of in vitro culture methods to produce viable embryos, the lack of exchange between the embryo and the mother often results in lower-quality embryos than those derived in vivo. Therefore, there is a pressing need to increase our understanding of the physiological mechanisms underlying embryo interaction with the oviduct and endometrium through EVs and to develop models capable of mimicking the in vivo environment. This review aims to provide up-to-date insights into the communication between the mother and pre-implantation bovine embryo, exploring their applications and perspectives in the field.(AU)

Resumo

The initial stages of early embryonic development were analyzed as a function of the incubation period and age of Japanese quail breeders. A total of 203 Japanese quails housed in 29 conventional laying cages with 5 females and 2 males at 31, 39, 48, and 59 weeks of age were used, and the fertile eggs from these breeders were selected and incubated. The eggs were opened, and the embryos were isolated, fixed in a glutaraldehyde solution, analyzed and classified according to the stage of development. For after laying and the incubation periods of 24, 48, and 72 hours, the embryos presented, on average, Hamburger-Hamilton stages XI, HH 6.1, HH 12.7, and HH 18.5, respectively, with no effect of breeder age. It was also observed that, between 31 and 59 weeks of age in Japanese quail breeders, the eggs become longer and wider, with greater weight, volume, and area. Therefore, it is concluded that the age of the Japanese quail mother influences the weight, length, width, volume, and area of the eggs but does not influence the embryonic development up to 72 hours.(AU)

Resumo

During oocyte meiosis resumption, a coordinated program of transcript translation and decay machinery promotes a remodeling of mRNA stores, which determines the success of the acquisition of competence and early embryo development. We investigated levels of two genes related to mRNA translation (CPEB1 and CPEB4) and two related to mRNA degradation (CNOT7 and ZFP36L2) machinery and found ZFP36L2 downregulated in in vitro-matured bovine oocytes compared to in vivo counterparts. Thereafter, we tested the effects of a pre-IVM step with NPPC and a modified IVM with AREG on the modulation of members of mRNA translation and degradation pathways in cumulus cells and oocytes. Our data showed a massive upregulation of genes associated with translational and decay processes in cumulus cells, promoted by NPPC and AREG supplementation, up to 9h of IVM. The oocytes were less affected by NPPC and AREG, and even though ZFP36L2 transcript and protein levels were downregulated at 9 and 19h of IVM, only one (KDM4C) from the ten target genes evaluated was differently expressed in these treatments. These data suggest that cumulus cells are more prone to respond to NPPC and AREG supplementation in vitro, regarding translational and mRNA decay programs. Given the important nursing role of these cells, further studies could contribute to a better understanding of the impact of these modulators in maternal mRNA modulation and improve IVM outcomes.(AU)

Resumo

Embryo transfer in cattle is an increasingly important technique for cattle production. Full attainment of the benefits of the technology will depend on overcoming hurdles to optimal performance using embryos produced in vitro. Given its importance, embryo technology research should become a global research priority for animal reproduction science. Among the goals of that research should be developing methods to increase the proportion of oocytes becoming embryos through optimization of in vitro oocyte maturation and in vitro fertilization, producing an embryo competent to establish and maintain pregnancy after transfer, and increasing recipient fertility through selection, management and pharmacological manipulation. The embryo produced in vitro is susceptible to epigenetic reprogramming and methods should be found to minimize deleterious epigenetic change while altering the developmental program of the resultant calf to increase its health and productivity. There are widening opportunities to rethink the technological basis for much of the current practices for production and transfer of embryos because of explosive advances in fields of bioengineering such as microfluidics, three-dimensional printing of cell culture materials, organoid culture, live-cell imaging, and cryopreservation.(AU)

Resumo

Selection strategies are performed post-fertilization when the random combination of paternal and maternal genomes has already occurred. It would be greatly advantageous to eliminate meiotic uncertainty by selecting genetically superior gametes before fertilization. To achieve this goal, haploid embryonic cells and embryonic stem cell lineages could be derived, genotyped, and used to substitute gametes. On the paternal side, androgenetic development can be achieved by removing the maternal chromosomes from the oocyte before or after fertilization. We have shown that once developed into an embryo, haploid cells can be removed for genotyping and, if carrying the selected genome, be used to replace sperm at fertilization. A similar strategy can be used on the maternal side by activating the oocyte parthenogenetically and using some embryonic cells for genotyping while the remaining are used to produce diploid embryos by fertilization. Placed together, both androgenetic and parthenogenetic haploid cells that have been genotyped to identify optimal genomes can be used to produce offspring with predetermined genomes. Successes and problems in developing such a breeding platform to achieve this goal are described and discussed below.(AU)

Resumo

One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.(AU)

Resumo

This study explored the migration of follicular fluid (FF)-derived extracellular vesicles (EVs) of the uterine environment to the bloodstream and their interaction with neutrophils in vivo and in vitro. For the in vivo experiment, six Nellore heifers (Bos indicus) received an intrauterine infusion seven days after ovulation with 1X PBS only (sham group; n=1), 1X PBS stained with lipophilic dye PKH26 (control group; n=2), or FF-derived EVs stained with PKH26 (treated group; n=3). Plasma was collected at 0, 10, 30, 60-, 180-, 360-, 720-, and 1440-min post-infusion to obtained EVs for analysis by nano flow cytometry. Labeled EVs were present in the bloodstream at 30- and 60-min post-infusion in the treatment group. Additionally, plasma derived-EVs from all groups were positive for Calcein-AM, Alix, Syntenin, and Calnexin, which confirm the presence of EVs. The second experiment utilized the plasma-derived EVs from the heifers from 30 and 60 min timepoints to evaluate if neutrophils can uptake EVs in vitro. As results, it was possible to observe the presence of labeled EVs in neutrophils treated with plasma derived-EVs from the treatment group. In summary, our results suggest that labeled EVs can migrate from the uterine environment rapidly and interact with circulating immune cells in bovine.(AU)

Resumo

We evaluated the reproductive losses in three periods: period I ­ starting from the time the cows were exposed to the bulls to pregnancy diagnoses (PD, number of cows diagnosed as non-pregnant/total exposed cows × 100); period II ­ from the time of PD to calving (number of calving cows/number of cows diagnosed as pregnant × 100); and period III ­ from calving to weaning (number of weaned calves/number of calving cows × 100) in purebred Hereford (HH) and Angus (AA) cows and their crosses in beef cows under extensive production systems. Likewise, the effect of parity (nulliparous, primiparous, and multiparous) and the interaction between both factors were studied. A thirteen-year data set (2505 record) of an experimental breeding herd, maintained in an extensive production system based on natural grassland, was used. The dataset was under a complete diallel design between HH and AA breeds. Both the genetic group and parity of the cow affected the reproductive losses, but only in period I. No interaction was found. Purebred cows had higher reproductive losses than the crossbred cows, without differences between the purebred (HH and AA) or between the crossbred (AH and HA). The greatest losses were observed rather in primiparous than in nulliparous and multiparous cows without difference between the latter two. The use of crossbred cows in extensive production systems is an alternative to reduce reproductive losses and to increase calf harvest.(AU)

Resumo

For nearly 100 years the postcoital inflammatory response has been described in the female reproductive tract of rodents. Since the 1950's this observation has been made in a number of animals including humans and domestic species. Yet pregnancy can be initiated and maintained by using embryo transfer which bypasses insemination and the related postcoital inflammatory response. Thus, the role of semen exposure beyond sperm transport and subsequent postcoital inflammatory response in female reproductive tissues has yet to be given a true physiological purpose. Historically the postcoital inflammatory response of female tissues was suggested to remove spermatozoa and male derived pathogens from the female reproductive tract. More recently, semen exposure and the postcoital inflammatory response have been suggested to play a role in long-term preparation of the maternal immune system to the semi-allogeneic pregnancy, ancillary support of the preimplantation embryo, and potentially fetal programing that improves pregnancy outcomes, while the absence or inappropriate postcoital inflammation has been suggested to contribute to pregnancy complications. Although the postcoital inflammatory response has been robustly characterized, the evidence for its role in promoting positive pregnancy outcomes or reducing pregnancy complications remains tenuous. This manuscript is designed to balance the information we know regarding semen exposure and postcoital inflammation in various animal systems, with the information we perceive to be factual but perhaps not yet fully tested, along with the data we have yet to generate if we intend to postulate a physiological purpose of the postcoital inflammatory response to pregnancy outcomes.(AU)

Resumo

Over the past 40 years, assisted reproductive technologies (ARTs) have grown significantly in scale and innovation, from the bovine embryo industry's shift from in vivo derived to in vitro produced embryos and the development of somatic cell-based approaches for embryo production. Domestic animal models have been instrumental in the development of ARTs for wildlife species in support of the One Plan Approach to species conservation that integrates in situ and ex situ population management strategies. While ARTs are not the sole solution to the biodiversity crisis, they can offer opportunities to maintain, and even improve, the genetic composition of the captive and wild gene pools over time. This review focuses on the application of sperm and embryo technologies (artificial insemination and multiple ovulation/in vitro produced embryo transfer, respectively) in wildlife species, highlighting impactful cases in which significant progress or innovation has transpired. One of the key messages following decades of efforts in this field is the importance of collaboration between researchers and practitioners from zoological, academic, governmental, and private sectors.(AU)

Resumo

Embryonic stem cells (ESCs) have proven to be a great in vitro model that faithfully recapitulates the events that occur during in vivo embryogenesis, making them a unique tool to study the cellular and molecular mechanisms that define tissue specification during embryonic development. Livestock ESCs are particularly attractive and have broad prospects including drug selection and human disease modeling, improvement of reproductive biotechniques and agriculture-related applications such as production of genetically modified animals. While mice and human ESCs have been established many years ago, no significant advances were made in livestock species until recently. Nowadays, livestock ESCs are available from cattle, pigs, sheep, horses and rabbits with different states of pluripotency. In this review, we summarize the current advances on livestock ESCs establishment and maintenance along with their present and future applications.(AU)

Resumo

Assisted reproductive technologies (ART) are fundamental for cattle breeding and sustainable food production. Together with genomic selection, these technologies contribute to reducing the generation interval and accelerating genetic progress. In this paper, we discuss advancements in technologies used in the fertility evaluation of breeding animals, and the collection, processing, and preservation of the gametes. It is of utmost importance for the breeding industry to select dams and sires of the next generation as young as possible, as is the efficient and timely collection of gametes. There is a need for reliable and easily applicable methods to evaluate sexual maturity and fertility. Although gametes processing and preservation have been improved in recent decades, challenges are still encountered. The targeted use of sexed semen and beef semen has obliterated the production of surplus replacement heifers and bull calves from dairy breeds, markedly improving animal welfare and ethical considerations in production practices. Parallel with new technologies, many well-established technologies remain relevant, although with evolving applications. In vitro production (IVP) has become the predominant method of embryo production. Although fundamental improvements in IVP procedures have been established, the quality of IVP embryos remains inferior to their in vivo counterparts. Improvements to facilitate oocyte maturation and development of new culture systems, e.g. microfluidics, are presented in this paper. New non-invasive and objective tools are needed to select embryos for transfer. Cryopreservation of semen and embryos plays a pivotal role in the distribution of genetics, and we discuss the challenges and opportunities in this field. Finally, machine learning (ML) is gaining ground in agriculture and ART. This paper delves into the utilization of emerging technologies in ART, along with the current status, key challenges, and future prospects of ML in both research and practical applications within ART.

Resumo

In beef cattle operations that conduct embryo transfer, the overall success depends on the pregnancy outcome that results from every pregnancy opportunity. In this review, we dissected the main components that determine if a recipient will sustain the pregnancy after embryo transfer up to calving. Specifically, we describe the effect of the uterus on its ability to provide a receptive environment for embryo development. We then discuss the capacity of the embryo to thrive after transfer, and especially the contribution of the sire to embryo fitness. Finally, we review the interaction between the uterus and the embryo as an integrated unit that defines the pregnancy.(AU)

Resumo

Reviewing the current state of knowledge on reproductive performance and productive traits in rams has many advantages. First, the compilation of this information will serve as a literature resource for scientists conducting research around the world and will contribute to the understanding of the data collected and interpreted by researchers on the different hormonal strategies used to improve reproductive performance in rams. Second, it will allow scientists to identify current knowledge gaps and set future research priorities in ram reproduction. Rams play an important role in the global flock economy, but their reproductive analysis has been limited in the use of hormonal technologies to increase the productivity of sheep flocks. In this review, we cite the most important works on six hormones that, in one way or another, modify the hypothalamus-pituitary-gonadal axis, at different doses, in and out of the reproductive season, breeds, application methods, among other factors. The overall aim is to increase the reproductive efficiency of rams in different scenarios and, in some cases, of other species due to the lack of limited information on rams.(AU)

Resumo

In Experiment 1, PBMC were isolated from cows considered healthy or with SCE (n=6/group) on Days 0 (estrus) and 7 (diestrus) of a synchronized estrous cycle. In Experiment 2, on D21 (D0 was defined as the day of Fixed Timed Artificial Insemination (FTAI), cows were evaluated by ultrasonography to assess luteal blood perfusion and PBMC were isolated. On D32, cows were classified into: healthy pregnant (n=7), pregnant with SCE (n=4), healthy non-pregnant (n=8), and non-pregnant with SCE (n=10). Gene expression of ISGs (ISG15, OAS1, MX1 and IFI6) and proinflammatory cytokines (IL1-ß, TNF-α and IFN-γ) were determined. Expression of ISG15, MX1, IFI6, TNF-α and IFN-γ did not differ between SCE and healthy cows and between Days 0 and 7. Expression of OAS1 and IL1-ß were higher (P=0.02) on Day 7 than Day 0, regardlees of the SCE presence. In Exp.2, ISG15 abundance was 2.5-fold greater (P=0.0008), TNF-α was 2.2-fold greater (P=0.05), and IL1-ß tended (P=0.06) to be 2.4-fold higher in pregnant than non-pregnant cows. Luteal blood perfusion was greater (P=0.01) in pregnant animals. In conclusion, OAS1 and IL1-ß are transcripts upregulated in PBMC at diestrus, regardless of SCE occurrence. Proinflammatory cytokines are not affected by SCE occurrence, but IL1-ß and TNF-α are upregulated in pregnant animals on D21 of pregnancy. ISG15 abundance is a good pregnancy predictor, regardless SCE presence.(AU)

Resumo

The objective of the present study was to transpose sperm freezing methodology from domestic goat to the Tadjik markhor (Capra falconeri heptneri) and to address the feasibility to develop IVP and artificial insemination using such frozen semen. Semen of different adult markhor males were successfully recovered by electro-ejaculation and were then frozen using caprine methodology. Frozen semen showed good survival rates at thawing and good fertility rates were assessed in heterologous in vitro fertilization system with goat oocytes. LOPU/IVF was applied for Tadjik markhor females allowing the first successful blastocyst production in vitro. In an applied program, we also transposed successfully intrauterine AI method with frozen/thawed semen to the Tadjik markhor.AU)