Assisted reproductive technologies used in small animal veterinary practice - possibilities, obstacles and future

Assisted Reproductive Technologies (ART) are currently used in animals usually in three main situations: 1. As a form of treatment of subfertility and infertility in females and/or males, 2. As a method to obtain genetically valuable progeny in relatively short time in healthy fertile animals, 3. As a modern smart tool to obtain progeny in endangered animal species in programs of rescue of wild animals threatened extinction. Generally the efficiency of reproductive biotechniques in dogs and cats is lower in comparison to obtained in farm animals and in human. Independently of the aim of their use, there are some techniques, which are better developed in dogs and some others seems to be better developed in cats. It may be assumed that simple, clinical techniques are well elaborated and more frequently used in dogs while more advanced techniques are better developed in cats. The level of effectiveness of ART is conditioned by anatomical and physiological factors specific for species, general demands for their use in veterinary practice and research, and general interest of breeders and scientific community in such activity.(AU)

Importance of the study of reproduction in small animals and preservation of species / Importancia del estudio de la reproducción en animales pequeños y la preservación de especies / Importância do estudo da reprodução em pequenos animais e preservação das espécies

Although reproduction in small animals is relatively current, its objectives are very varied. Initially they were used as a study model in physiology and various pathologies. In the same way, many small animals are produced as food and finally, in the last 20 years, the importance of pets and the intensification in the selection of canine and feline breeds, has caused an important impulse in the development of new scientific information. Although currently technical resources have advanced substantially, there are still many basic and important issues that have not yet been elucidated by formal science. Global threats facing wild species include the consequences of climate change, population growth, urbanization, air and water pollution, and the release of chemicals into the environment, causing, on average, 25% of animals are in danger of extinction. Wild animal populations are small and dispersed in their habitat with little or no opportunity for genetic exchange, which has generated in recent years many programs to preserve wild species and design appropriate strategies that lead to sustainable populations. Fortunately, recent and inspiring advances in the science of wildlife reproduction have been reported that will set directions for future research and will surely lead to further successes in conservation biology.(AU)

A interface entre a saúde humana e a reprodução assistida em primatas não humanos / The interface between human health and assisted reproductive technologies in non-human primates

Os primatas não-humanos (PNHs) são tidos como importantes modelos para estudos biomédicos devido à sua grande similaridade biológica com os seres humanos. As técnicas de reprodução assistida (TRAs) constituem uma importante ferramenta para realização de estudos que envolvam infertilidade, desenvolvimento de contraceptivos, gestação e desenvolvimento fetal, preservação da fertilidade em pacientes com câncer, geração de modelos experimentais por meio de técnicas de edição gênica, entre outros. O objetivo dessa revisão é discutir as principais TRAs utilizadas em PNHs e suas aplicações. Dentre as técnicas mais utilizadas em PNHs, podem-se citar colheita e criopreservação de sêmen, monitoramento do ciclo ovariano, estimulação ovariana controlada e aspiração de folículos ovarianos, fecundação in vitro, injeção intracitoplasmática de espermatozoides, inseminação artificial, microinjeção para injeção gênica, biopsia embrionária, criopreservação de embriões, transferência de embriões, diagnóstico de gestação, enxerto de gônadas e clonagem. Os estudos apresentados nesta revisão mostram a evolução das TRAs e suas diferentes aplicações. Particularmente, os estudos de edição gênica e clonagem representam um grande avanço na utilização combinada de diversas TRAs para geração de modelos biomédicos para doenças humanas, demostrando o papel dessas técnicas para avanços científicos no que diz respeito à saúde humana.

Nonhuman Primates (NHPs) are considered important models for biomedical studies due to their high biological proximity with humans. The Assisted Reproductive Technologies (ARTs) represent an important tool to perform studies related to infertility, development of contraceptives, gestation and fetal development, fertility preservation in cancer patients, generation of experimental models through gene editing techniques, among others. The objective of this review is to discuss the main ARTs used in NHPs and their application. Among the most used techniques in NHPs, we can include collection and cryopreservation of semen, ovarian cycle monitoring, controlled ovarian stimulation and follicle aspiration, in vitro fertilization, intracytoplasmic sperm injection, artificial insemination, microinjections for gene editing, embryo biopsy, embryo cryopreservation, embryo transfer, pregnancy diagnosis, grafting of gonadal tissue, and cloning. The studies cited in this review illustrate the evolution of the ARTs and their applications. The gene editing and cloning studies, in particular, represent the great advancements on the combined use of several different ARTs for the generation of biomedical models for human diseases, demonstrating the role of these techniques in the scientific advancement with regards to human health.

Defining the male contribution to embryo quality and offspring health in assisted reproduction in farm animals

Assisted reproductive technologies such as artificial insemination have delivered significant benefits for farm animal reproduction. However, as with humans, assisted reproduction in livestock requires the manipulation of the gametes and preimplantation embryo. The significance of this ‘periconception’ period is that it represents the transition from parental genome regulation to that of the newly formed embryo. Environmental perturbations during these early developmental stages can result in persistent changes in embryonic gene expression, fetal organ development and ultimately the long-term health of the offspring. While associations between maternal health and offspring wellbeing are well-defined, the significance of paternal health for the quality of his semen and the post-conception development of his offspring have largely been overlooked. Human and animal model studies have identified sperm epigenetic status (DNA methylation levels, histone modifications and RNA profiles) and seminal plasma-mediated maternal uterine immunological, inflammatory and vascular responses as the two central mechanisms capable of linking paternal health and post-fertilisation development. However, there is a significant knowledge gap about the father’s contribution to the long-term health of his offspring, especially with regard to farm animals. Such insights are essential to ensure the safety of widely used assisted reproductive practices and to gain better understanding of the role of paternal health for the well-being of his offspring. In this article, we will outline the impact of male health on semen quality (both sperm and seminal plasma), reproductive fitness and post-fertilisation offspring development and explore the mechanisms underlying the paternal programming of offspring health in farm animals.

Defining the male contribution to embryo quality and offspring health in assisted reproduction in farm animals

Assisted reproductive technologies such as artificial insemination have delivered significant benefits for farm animal reproduction. However, as with humans, assisted reproduction in livestock requires the manipulation of the gametes and preimplantation embryo. The significance of this ‘periconception period is that it represents the transition from parental genome regulation to that of the newly formed embryo. Environmental perturbations during these early developmental stages can result in persistent changes in embryonic gene expression, fetal organ development and ultimately the long-term health of the offspring. While associations between maternal health and offspring wellbeing are well-defined, the significance of paternal health for the quality of his semen and the post-conception development of his offspring have largely been overlooked. Human and animal model studies have identified sperm epigenetic status (DNA methylation levels, histone modifications and RNA profiles) and seminal plasma-mediated maternal uterine immunological, inflammatory and vascular responses as the two central mechanisms capable of linking paternal health and post-fertilisation development. However, there is a significant knowledge gap about the fathers contribution to the long-term health of his offspring, especially with regard to farm animals. Such insights are essential to ensure the safety of widely used assisted reproductive practices and to gain better understanding of the role of paternal health for the well-being of his offspring. In this article, we will outline the impact of male health on semen quality (both sperm and seminal plasma), reproductive fitness and post-fertilisation offspring development and explore the mechanisms underlying the paternal programming of offspring health in farm animals.(AU)

Low invasive estrous synchronization protocol for wild animals: an example with melengestrol acetate in brown brocket deer (Mazama gouazoubira)

Deer are sensitive to stressful stimuli by handling and their reproductive physiology could be altered by these procedures, making it necessary to develop less invasive protocols for ART. Melengestrol acetate (MGA), a synthetic progestin administered orally, appears as an alternative for estrous synchronization protocols (ESP), such as reported in cattle. Firstly, we compared two MGA doses (0.5 and 1.0 mg/day/animal), which would have suppression effect in estrous behavior (EB). Eight females were randomly and equally distributed in Group 1 (G1) and Group 2 (G2), which received 0.5 and 1.0 mg/day/animal respectively for 15 days (D1 to D15). Two cloprostenol (CP) applications were performed on D0 and D11. Estrus detection (ED) was performed every day. All females from G1 displayed estrus during treatment period, whereas all females from G2 displayed estrus after treatment, suggesting a suppressive effect of 1.0 mg in the EB. Once the suppressive MGA dose (1.0 mg) was defined, we used this dose for assessing ESP. The same eight females received 1.0 mg/animal for eight days (D-8 to D-1), followed by 0.25 mg of estradiol benzoate on D-8 and 265 g of CP on D0. Feces for fecal progesterone metabolites (FPM) measurement were collected from D0 until seven days after the last day of estrus. Seven females displayed estrus between 12 and 72 h after CP application, which was followed by a significant increase in FPM levels (except female MG6), suggesting the formation of corpus luteum. After ED, females were placed with a fertile male to assess the fertility of the protocol. Pregnancy was confirmed by ultrasound 30 days after mating in 3/6 individuals. Although the low effectiveness of MGA protocol, it should be considered as a promising alternative in deer ESP since this protocol has less stressful effect on the animal during reproductive management when compared to other ESP.

Low invasive estrous synchronization protocol for wild animals: an example with melengestrol acetate in brown brocket deer (Mazama gouazoubira)

Deer are sensitive to stressful stimuli by handling and their reproductive physiology could be altered by these procedures, making it necessary to develop less invasive protocols for ART. Melengestrol acetate (MGA), a synthetic progestin administered orally, appears as an alternative for estrous synchronization protocols (ESP), such as reported in cattle. Firstly, we compared two MGA doses (0.5 and 1.0 mg/day/animal), which would have suppression effect in estrous behavior (EB). Eight females were randomly and equally distributed in Group 1 (G1) and Group 2 (G2), which received 0.5 and 1.0 mg/day/animal respectively for 15 days (D1 to D15). Two cloprostenol (CP) applications were performed on D0 and D11. Estrus detection (ED) was performed every day. All females from G1 displayed estrus during treatment period, whereas all females from G2 displayed estrus after treatment, suggesting a suppressive effect of 1.0 mg in the EB. Once the suppressive MGA dose (1.0 mg) was defined, we used this dose for assessing ESP. The same eight females received 1.0 mg/animal for eight days (D-8 to D-1), followed by 0.25 mg of estradiol benzoate on D-8 and 265 g of CP on D0. Feces for fecal progesterone metabolites (FPM) measurement were collected from D0 until seven days after the last day of estrus. Seven females displayed estrus between 12 and 72 h after CP application, which was followed by a significant increase in FPM levels (except female MG6), suggesting the formation of corpus luteum. After ED, females were placed with a fertile male to assess the fertility of the protocol. Pregnancy was confirmed by ultrasound 30 days after mating in 3/6 individuals. Although the low effectiveness of MGA protocol, it should be considered as a promising alternative in deer ESP since this protocol has less stressful effect on the animal during reproductive management when compared to other ESP.(AU)

State of the art of assisted human reproduction

Background: Infertility is a disease observed in approximately 10% of the reproductive age population (20-44 years old), and is defined as the failure to conceive after twelve months of regular sexual intercourse, without contraception; in women older than 35 years old, this period is reduced to 6 months. The main causes of infertility are tubal, ovarian and uterine and sperm abnormalities, endometriosis, and those with undetermined causes. Over the past 30 years, several techniques were developed to overcome these factors including gamete cryopreservation, controlled ovarian stimulation, intra-uterine insemination, in vitro fertilization, intracytoplasmatic sperm injection). Review: Despite advances in assisted reproductive technologies (ART), treatment success is still strongly dependent on oocyte and sperm quality, and resulting embryo viability. The most promising advance on oocyte quality assessment is the evaluation of the ovarian reserve by the quantification of the anti-müllerian hormone (AMH). Since ovarian reserve is closely related to oocyte quality, AMH levels could be an indicator of both oocyte production capacity and the potential of these oocytes to generate a viable embryo. On the other hand, despite the development of techniques to overcome male factor infertility, attention has been paid on the semen evaluation, since routine sperm evaluation techniques are known to be ineffective, especially in those cases of unexplained infertility. Therefore, techniques were developed to assess acrosome and membrane integrity, mitochondrial potential, DNA integrity, and fertilizing capacity of sperm. However, further studies are necessary to evaluate sperm DNA integrity without damaging the cell, allowing the injection of a spermatozoon with an intact DNA when using ICSI. Regarding embryo quality, even with a good quality oocyte (as assessed by the current techniques) and an apparently normal sperm, there are still chances of generating an embryo with genetic abnormalities. In such cases, and in cases of recurrent failures, women over 35 years of age, and couples with a pre-existing genetic risk, the preimplantation genetic diagnosis (PGD) appears to be an important tool to improve the odds of pregnancy and avoid abortions or the conception of fetuses with genetic abnormalities. The technique of PGD, usually performed with PCR or FISH, has gained a powerful tool with the development of the Comparative Genomic Hybridization (CGH). However, recent studies aiming to identify markers of oocyte and sperm quality and embryo viability are in course using mass spectroscopy. With this sensitive technique applied to body fluids (i.e., blood, follicular fluid, seminal plasma), granulosa cells, sperm, and culture media, researches are being conducted to non-invasively identify biomarkers that will help understand reproductive mechanisms and to efficiently predict the outcome of ARTs. Conclusion: Significant advances in ART have been observed in the last few years, yet, failures still occur with high frequency. This review will focus on techniques to assess oocyte quality, sperm function and embryo viability, aiming to provide tools for a precise prognosis when treating infertile couples.

Is ART changing the epigenetic landscape of imprinting?

Imprinted genes, which are epigenetically modified such that only a single parental allele is expressed, are often regulated by imprinting control regions (ICRs). ICRs typically are DNA methylated in the male or female germline and this DNA methylation is subsequently maintained, even when the genome is reprogrammed after fertilization. Many of the manipulations associated with Assisted Reproductive Technologies (ART) occur during periods of epigenetic reprogramming and it should, therefore, not be surprising that animal data show that loss of imprinting and loss of DNA methylation of ICRs is associated with procedures such as superovulation, in vitro fertilization and embryos culture and transfer. Less clear is whether there is an increase in loss of imprinting disorders associated with ART. Here we review the human and animal literature and discuss what it is necessary to address current controversies.

Is ART changing the epigenetic landscape of imprinting?

Imprinted genes, which are epigenetically modified such that only a single parental allele is expressed, are often regulated by imprinting control regions (ICRs). ICRs typically are DNA methylated in the male or female germline and this DNA methylation is subsequently maintained, even when the genome is reprogrammed after fertilization. Many of the manipulations associated with Assisted Reproductive Technologies (ART) occur during periods of epigenetic reprogramming and it should, therefore, not be surprising that animal data show that loss of imprinting and loss of DNA methylation of ICRs is associated with procedures such as superovulation, in vitro fertilization and embryos culture and transfer. Less clear is whether there is an increase in loss of imprinting disorders associated with ART. Here we review the human and animal literature and discuss what it is necessary to address current controversies.(AU)