Why choose the rabbit to work in reproductive technology?

Rabbits have played a significant role in both livestock production and the advancement of reproductive scientific research. Their unique biological traits, including induced ovulation and a reproductive process that closely mirrors that of humans, have been pivotal in their use as a model. Moreover, their body size is perfectly aligned with the 3Rs principles: Replacement, Reduction, and Refinement. Consequently, techniques for gamete collection and embryo recovery, followed by their use in artificial insemination or embryo transfer, are characterized by being minimally invasive. However, refining in vitro fertilization and embryo culture techniques continues to present challenges. The incorporation of cutting-edge genomic editing tools, such as CRISPR/Cas9, has reestablished rabbits as essential models in genetic and biomedical research, driving scientific progress. This review aims to describe the most effective reproductive biotechnologies for both male and female rabbits and how these methodologies are in line with the 3Rs principles-Replacement, Reduction, and Refinement-highlighting their significance in conducting ethical research.

Advanced assisted reproduction technologies in endangered mammalian species.

A new synergistic approach of classical conservation strategies combined with advanced assisted reproduction technologies (aART) allows for protection and rescue of endangered keystone species at the brink of extinction, which can help to safeguard complex ecosystems. Reproduction biology and management in mammal species is not only challenging in regards to their diverging sizes, anatomy, and often unknown physiology; it also requires customized training or chemical restraint protocols for safe handling. Besides these general challenges, there are several new assisted reproduction techniques (ART) specifically tailored to critically endangered mammals. The current portfolio of ART in these mammalian taxa is ranging from sexual cycle characterization and manipulation, semen collection and cryopreservation, artificial insemination, biobanking of living cells, oocyte collection, in vitro fertilization (IVF), and embryo production, embryo transfer as well as stem cell-derived in vitro gametogenesis for generating gametes in culture. The article covers advanced assisted reproduction technologies (aART), success and challenges, as well as ethical implications.

Equine ART and antral follicle count: Can we deepen our understanding to improve outcomes?

Assisted reproductive technologies (ARTs) are performed worldwide in the equine industry to produce genetically valuable foals. Among them, ovum pick up (OPU) combined with intra-cytoplasmic sperm injection (ICSI) can now be more efficient than embryo transfer (ET) under optimal conditions. However, OPU is not a benign procedure for the mare and the process is costly. Improved efficiency is therefore in the interest of everyone, maximizing mare welfare and optimizing economics for the client. One of the key factors of success is the antral follicle count (AFC) at the time of OPU and subsequently the number of oocytes obtained. Variations in AFC are reported between individuals and between geographical areas. This leads to a significant increase in numbers of embryos produced per session in some countries compared to others, independent of the laboratory efficiency. This article revisits the basics of folliculogenesis involved in establishment of the antral follicle population and explores work in other species given the paucity of equine research in this area. The aim of the review is to elucidate interesting areas of further research that could generate essential information for clinicians and clients about the management and selection of the donor mare for OPU and potentially identify pharmacological targets for manipulation.

PERSPECTIVE: Semen additives for improving frozen-thawed buffalo and cattle semen - a review.

This comprehensive review delves into the evolving landscape of assisted reproductive technologies (ARTs) in bovine species, particularly focusing on the pivotal roles of semen additives in the cryopreservation of buffalo and cattle semen. In developing nations, where ARTs are still emerging, these techniques significantly influence bovine reproductive strategies. In contrast, developed regions have embraced them as primary approaches for dairy buffalo and cattle breeding. Semen cryopreservation, while offering advantages like extended storage and genetic propagation, also presents challenges. These include diminished sperm quality due to reactive oxygen species (ROS) production, alterations in sperm structure, and temperature fluctuations. Further, the effect of cryopreservation differs between cattle and buffaloes, with the latter exhibiting poorer semen viability and fertility due to inherent lipid composition susceptibilities. The generation and implications of ROS, especially hydrogen peroxide, contribute significantly to sperm DNA damage and functional impairments. To counteract these challenges, research has intensified on semen additives, aiming to bolster semen quality and protect against oxidative stress-induced damage. As the field advances, the review emphasizes the need for optimized cryopreservation techniques and tailored antioxidant strategies to harness the full potential of ARTs in bovine breeding programs. Doi.org/10.54680/fr24410110112.

Economic viability of using OvSynch and fixed timed artificial insemination protocol in breeding improvement of pastoral herds in the rangelands.

Though using Assisted Reproductive Technologies (ARTs) can improve oestrus detection, conception and pregnancy success, thus benefit breeding program implementation, empirical evidence of their economic viability is lacking to inform investment decisions in pastoral herds. This study assessed economic viability of using OvSynch and fixed Timed Artificial Insemination (TAI) protocol in Sahiwal upgrading breeding program under two hypothetical cases of best and worst in activity-based money allocations when pastoral herds deploy either optimal or low input husbandry practices. From herd owners' assessment of milk production, best-case scenarios attain on average 10 L/cow/day with optimal husbandry and 5 L/cow/day with low input husbandry. The worst-case scenarios attain 5 L/cow/day with optimal husbandry and 1 L/cow/day with low input husbandry. Benefit- Cost Analysis (BCA) estimated Net Present Value (NPV), Benefit-Cost ratio (BCR) and Internal Rate of Return (IRR) to establish economic viability of using OvSynch and TAI Protocol in pastoral breeding programs. Both best-case scenarios retuned positive NPVs (82,028 and 6,912), BCR values (1.68 and 1.08) and IRR (27.46% and 8.08%) while worst-case scenarios returned negative NPVs (-135,855 and -141,025), BCR values of below 1 (0.87 and 0.66) and IRR values below the minimum rate of returns. These economic parameters were sensitive to price changes in inputs and outputs, under both optimal and low input husbandry practices. Results indicate that using OvSynch and TAI Protocol is a profitable and economically viable investment under optimal husbandry practices but not under low input husbandry practices. By implications, use of OvSynch and TAI Protocol in Sahiwal upgrading breeding programs need be accompanied with improved husbandry practices and de-risking pastoral herd owners from price changes in input and output markets.

The oocyte: the key player in the success of assisted reproduction technologies.

The ovulation of a mature oocyte at metaphase II of meiosis, with optimal potential to undergo fertilisation by a sperm cell, complete meiosis and sustain the switch to mitotic division, and support early embryo development, involves a protracted and disrupted/delayed series of processes. Many of these are targeted for exploitation in vivo , or recapitulation in vitro , by the livestock industry. Reproductive technologies, including AI, multiple ovulation embryo transfer, ovum pick-up, in vitro embryo production, and oestrus and ovulation synchronisation, offer practitioners and producers the opportunity to produce offspring from genetically valuable dams in much greater numbers than they would normally have in their lifetime, while in vitro oocyte and follicle culture are important platforms for researchers to interrogate the physiological mechanisms driving fertility. The majority of these technologies target the ovarian follicle and the oocyte within; thus, the quality and capability of the recovered oocyte determine the success of the reproductive intervention. Molecular and microscopical technologies have grown exponentially, providing powerful platforms to interrogate the molecular mechanisms which are integral to or affected by ART. The development of the bovine oocyte from its differentiation in the ovary to ovulation is described in the light of its relevance to key aspects of individual interventions, while highlighting the historical timeline.

Assisted reproductive technologies (ARTs) in Mithun (Bos frontalis): What progress has been made so far? An overview.

Mithun, a unique bovine species, endemic to parts of North East India and plays an important role in the socioeconomic, cultural and religious fabrics of the local tribal population. To date, Mithuns are reared in a traditional free-range system by communities and increased deforestation, agricultural commercialization, disease outbreaks and indiscriminate slaughtering of elite Mithun for table purposes have significantly decreased its habitat and the elite Mithun population. Greater genetic gain is achieved with the implementation and effective use of assisted reproductive technologies (ARTs); however, presently it is limited to organized Mithun farms. At a slow pace, Mithun farmers are adopting semi-intensive rearing systems and interest in the use of ARTs is gradually escalating in Mithun husbandry. This article reviews the current status of ARTs such as semen collection and cryopreservation, estrus synchronization and timed artificial insemination (TAI), multiple ovulation and embryo transfer and in vitro embryo production and future perspectives in Mithun. Mithun semen collection and cryopreservation have been standardized, and estrus synchronization and TAI are suitable technologies that can be easily implemented under field conditions in near future. The establishment of an open nucleus-breeding system under community participatory mode along with the introduction of the ARTs is an alternative to the traditional breeding system for rapid genetic improvement of Mithun. Finally, the review considers the potential benefits of ARTs in Mithun and future research should include the use of these ARTs which will provide additional opportunities for improved breeding regimens in Mithun.

Common goals, different stages: the state of the ARTs for reptile and amphibian conservation.

Amphibians and reptiles are highly threatened vertebrate taxa with large numbers of species threatened with extinction. With so many species at risk, conservation requires the efficient and cost-effective application of all the tools available so that as many species as possible are assisted. Biobanking of genetic material in genetic resource banks (GRBs) in combination with assisted reproductive technologies (ARTs) to retrieve live animals from stored materials are two powerful, complementary tools in the conservation toolbox for arresting and reversing biodiversity decline for both amphibians and reptiles. However, the degree of development of the ARTs and cryopreservation technologies differ markedly between these two groups. These differences are explained in part by different perceptions of the taxa, but also to differing reproductive anatomy and biology between the amphibians and reptiles. Artificial fertilisation with cryopreserved sperm is becoming a more widely developed and utilised technology for amphibians. However, in contrast, artificial insemination with production of live progeny has been reported in few reptiles, and while sperm have been successfully cryopreserved, there are still no reports of the production of live offspring generated from cryopreserved sperm. In both amphibians and reptiles, a focus on sperm cryopreservation and artificial fertilisation or artificial insemination has been at the expense of the development and application of more advanced technologies such as cryopreservation of the female germline and embryonic genome, or the use of sophisticated stem cell/primordial germ cell cryopreservation and transplantation approaches. This review accompanies the publication of ten papers on amphibians and twelve papers on reptiles reporting advances in ARTs and biobanking for the herpetological taxa.

Reptile assisted reproductive technologies: can ART help conserve 300 million years of evolution by preserving extant reptile biodiversity?

Biodiversity loss is the greatest environmental problem threatening ecosystem, animal, and human health. Anthropogenic induced changes to climate, habitat, disease, species distributions, poaching, and unsustainable trade have accelerated extinction rates in all vertebrates, including reptiles. Preventing reptile extinctions will require humans to acknowledge these losses and develop ex situ and in situ plans to preserve them. Assisted reproductive technologies (ART) are management tools used to protect numerous vertebrate taxa; however, progress in developing ART for reptiles has lagged. Creating functional and sustainable reptile ART will strengthen our conservation capacity by capturing genetic material from select individuals to overcome natural or manmade boundaries. Utilising short-term gamete storage and genome resource banking, in conjunction with timed artificial insemination (AI) or ex ovo incubation, could lead to profound advances in reptile conservation, mitigating the loss of reptile biodiversity. In this article, we review ART reptile research completed since the 1970s. Topics include AI, hormonal control of reproduction, gamete collection, gamete storage, and genome resource banking. Additionally, we review the potential application of advanced reproductive methodologies, including in vitro /ex ovo fertilisation, intracytoplasmic sperm injection, cloning (somatic cell nuclear transfer), and genetic editing.

Development of assisted reproductive technologies for Mus spretus†.

The genus Mus consists of many species with high genetic diversity. However, only one species, Mus musculus (the laboratory mouse), is common in biomedical research. The unavailability of assisted reproductive technologies (ARTs) for other Mus species might be a major reason for their limited use in laboratories. Here, we devised ARTs for Mus spretus (the Algerian mouse), a commonly used wild-derived Mus species. We found that in vitro production of M. spretus embryos was difficult because of low efficacies of superovulation with equine chorionic gonadotropin or anti-inhibin serum (AIS) (5-8 oocytes per female) and a low fertilization rate following in vitro fertilization (IVF; 15.2%). The primary cause of this was the hardening of the zona pellucida but not the sperm's fertilizing ability, as revealed by reciprocal IVF with laboratory mice. The largest number of embryos (16 per female) were obtained when females were injected with AIS followed by human chorionic gonadotropin and estradiol injections 24 h later, and then by natural mating. These in vivo-derived 2-cell embryos could be vitrified/warmed with a high survival rate (94%) using an ethylene glycol-based solution. Importantly, more than 60% of such embryos developed into healthy offspring following interspecific embryo transfer into (C57BL/6 × C3H) F1 female mice. Thus, we have devised practical ARTs for Mus spretus mice, enabling efficient production of embryos and animals, with safe laboratory preservation of their strains. In addition, we have demonstrated that interspecific embryo transfer is possible in murine rodents.

Invited review: Use of assisted reproduction techniques to accelerate genetic gain and increase value of beef production in dairy herds.

The contribution of the calf enterprise to the profit of the dairy farm is generally considered small, with beef bull selection on dairy farms often not considered a high priority. However, this is likely to change in the future as the rapid rate of expansion of the dairy herd in some countries is set to plateau and improvements in dairy herd fertility combine to reduce the proportion of dairy breed calves required on dairy farms. This presents the opportunity to increase the proportion of beef breed calves born, increasing both the value of calf sales and the marketability of the calves. Beef embryos could become a new breeding tool for dairies as producers need to reassess their breeding policy as a consequence of welfare concerns and poor calf prices. Assisted reproductive technologies can contribute to accelerated genetic gain by allowing an increased number of offspring to be produced from genetically elite dams. There are the following 3 general classes of donor females of interest to an integrated dairy-beef system: (1) elite dairy dams, from which oocytes are recovered from live females using ovum pick-up and fertilized in vitro with semen from elite dairy bulls; (2) elite beef dams, where the oocytes are recovered from live females using ovum pick-up and fertilized with semen from elite beef bulls; and (3) commercial beef dams (≥50% beef genetics), where ovaries are collected from the abattoir postslaughter, and oocytes are fertilized with semen from elite beef bulls that are suitable for use on dairy cows (resulting embryo with ≥75% beef genetics). The expected benefits of these collective developments include accelerated genetic gain for milk and beef production in addition to transformation of the dairy herd calf crop to a combination of good genetic merit dairy female calves and premium-quality beef calves. The aim of this review is to describe how these technologies can be harnessed to intensively select for genetic improvement in both dairy breed and beef breed bulls suitable for use in the dairy herd.

Peripheral action of kisspeptin at reproductive tissues-role in ovarian function and embryo implantation and relevance to assisted reproductive technology in livestock: a review.

Kisspeptin (KISS1) is encoded by the KISS1 gene and was initially found to be a repressor of metastasis. Natural mutations in the KISS1 receptor gene (KISS1R) were subsequently shown to be associated with idiopathic hypothalamic hypogonadism and impaired puberty. This led to interest in the role of KISS1 in reproduction. It was established that KISS1 had a fundamental role in the control of gonadotropin releasing hormone (GnRH) secretion. KISS1 neurons have receptors for leptin and estrogen receptor α (ERα), which places KISS1 at the gateway of metabolic (leptin) and gonadal (ERα) regulation of GnRH secretion. More recently, KISS1 has been shown to act at peripheral reproductive tissues. KISS1 and KISS1R genes are expressed in follicles (granulosa, theca, oocyte), trophoblast, and uterus. KISS1 and KISS1R proteins are found in the same tissues. KISS1 appears to have autocrine and paracrine actions in follicle and oocyte maturation, trophoblast development, and implantation and placentation. In some studies, KISS1 was beneficial to in vitro oocyte maturation and blastocyst development. The next phase of KISS1 research will explore potential benefits on embryo survival and pregnancy. This will likely involve longer-term KISS1 treatments during proestrus, early embryo development, trophoblast attachment, and implantation and pregnancy. A deeper understanding of the direct action of KISS1 at reproductive tissues could help to achieve the next step change in embryo survival and improvement in the efficiency of assisted reproductive technology.

New directions in assisted breeding techniques for fish conservation.

Fish populations continue to decline globally, signalling the need for new initiatives to conserve endangered species. Over the past two decades, with advances in our understanding of fish germ line biology, new exsitu management strategies for fish genetics and reproduction have focused on the use of germ line cells. The development of germ cell transplantation techniques for the purposes of propagating fish species, most commonly farmed species such as salmonids, has been gaining interest among conservation scientists as a means of regenerating endangered species. Previously, exsitu conservation methods in fish have been restricted to the cryopreservation of gametes or maintaining captive breeding colonies, both of which face significant challenges that have restricted their widespread implementation. However, advances in germ cell transplantation techniques have made its application in endangered species tangible. Using this approach, it is possible to preserve the genetics of fish species at any stage in their reproductive cycle regardless of sexual maturity or the limitations of brief annual spawning periods. Combining cryopreservation and germ cell transplantation will greatly expand our ability to preserve functional genetic samples from threatened species, to secure fish biodiversity and to produce new individuals to enhance or restore native populations.

Maternal periconceptional and first trimester protein restriction in beef heifers: effects on maternal performance and early fetal growth.

This study evaluated the effect of protein restriction during the periconception (PERI) and first trimester (POST) periods on maternal performance, physiology and early fetal growth. Yearling nulliparous heifers (n=360) were individually fed a diet high or low in protein (HPeri and LPeri respectively) beginning 60 days before conception. From 24 to 98 days post-conception (dpc), half of each treatment group changed to the alternative post-conception high- or low-protein diet (HPost and LPost respectively), yielding four groups in a 2×2 factorial design with a common diet until parturition. Protein restriction was associated with lower bodyweight subsequent to reduced (but positive) average daily weight gain (ADG) during the PERI and POST periods. During the POST period, ADG was greater in LPeri than HPeri heifers and tended to be greater in LPost than HPost heifers during the second and third trimester. Bodyweight was similar at term. The pregnancy rate did not differ, but embryo loss between 23 and 36 dpc tended to be greater in LPeri than HPeri heifers. Overall, a greater proportion of male fetuses was detected (at 60 dpc 63.3% male vs 36.7% female). Protein restriction altered maternal plasma urea, non-esterified fatty acids, progesterone, leptin and insulin-like growth factor 1 at critical stages of fetal development. However, profiles varied depending on the sex of the conceptus.

Transforming growth factor-ß superfamily and interferon-τ in ovarian function and embryo development in female cattle: review of biology and application.

Survival of the embryo and establishment of a pregnancy is a critical period in the reproductive function of female cattle. This review examines how the transforming growth factor-ß (TGFB) superfamily (i.e. bone morphogenetic protein (BMP) 15, growth differentiation factor (GDF) 9, anti-Müllerian hormone (AMH)) and interferon-τ (IFNT) affect ovarian function and embryo development. The oocyte in a primary follicle secretes BMP15 and GDF9, which, together, organise the surrounding granulosa and theca cells into the oocyte-cumulus-follicle complex. At the same time, the granulosa secretes AMH, which affects the oocyte. This autocrine-paracrine dialogue between the oocyte and somatic cells continues throughout follicle development and is fundamental in establishing the fertilisation potential and embryo developmental competency of oocytes. The early bovine embryo secretes IFNT, which acts at the uterine endometrium, corpus luteum and blood leucocytes. IFNT is involved in the maternal recognition of pregnancy and immunomodulation to prevent rejection of the embryo, and supports progesterone secretion. Manipulation of BMP15, GDF9, AMH and IFNT in both invivo and invitro studies has confirmed their importance in reproductive function in female cattle. This review makes the case that a deeper understanding of the biology of BMP15, GDF9, AMH and IFNT will lead to new strategies to increase embryo survival and improve fertility in cattle. The enhancement of oocyte quality, early embryo development and implantation is considered necessary for the next step change in the efficiency of natural and assisted reproduction in cattle.

Development of assisted reproductive technologies in small animal species for their efficient preservation and production.

Assisted reproductive technologies (ARTs) are widely used in the animal industry, human clinics, and for basic research. In small laboratory animal species such as mice, ARTs are essential for the production of animals for experiments, the preservation of genetic resources, and for the generation of new strains of genetically modified animals. The RIKEN BioResource Research Center (BRC) is one of the largest repositories of such animal bioresources, and maintains approximately 9,500 strains of mice with a variety of genetic backgrounds. We have sought to devise ARTs specific to the reproductive and physiological characteristics of each strain. Such ARTs include superovulation, in vitro fertilization (IVF), the cryopreservation of embryos and spermatozoa, transportation of cryopreserved materials and embryo transfer (ET). Of these, superovulation likely has the most influence on animal production because it determines the quantity of starting material for other ARTs. Superovulation using anti-inhibin serum combined with estrous synchronization has resulted in approximately a three-fold increase in production efficiency with IVF-ET in the C57BL/6J strain. Wild-derived strains are important as genetically diverse resources for murine rodents (Genus Mus), and many are unique to the BRC. We have also successfully developed ARTs for more than 50 wild-derived strains, which have been cryopreserved for future use. Our work to improve and develop ARTs for mice and other small laboratory species will contribute to the cost-effectiveness of routine operations at repository centers, and to the provision of high quality animals for research use.

Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification.

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7 µM ionomycin in TCM 199 medium (5 min) followed by 2 mM 6-DMAP in TCM 199 supplemented with 10% FBS (3 hr), then cultured and evaluated every 24 hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.

Extracellular Vesicles and the Oviduct Function.

In mammals, the oviduct (or the Fallopian tube in humans) can be divided into the infundibulum (responsible for oocyte pick-up), ampulla (site of fertilization), isthmus (where preimplantation embryos develop), and uterotubal junction (where embryos transit to the uterus). The oviductal fluid, as well as extracellular vesicles produced from the oviduct epithelial cells, referred to as oEVs, have been shown to improve the fertilization process, prevent polyspermy, and aid in embryo development. oEVs contain molecular cargos (such as miRNAs, mRNAs, proteins, and lipids) that can be delivered and fuse to recipient cells. oEVs produced from the ampulla appear to be functionally distinct from those produced from the isthmus. In multiple species including mice, cats, dogs, pigs, and cows, oEVs can be incorporated into the oocytes, sperm, and embryos. In this review, we show the positive impact of oEVs on gamete function as well as blastocyst development and how they may improve embryo quality in in vitro conditions in an assisted reproductive technology setting for rodents, domestic animals, farm animals, and humans.

Assisted reproductive technologies for endangered species conservation: developing sophisticated protocols with limited access to animals with unique reproductive mechanisms.

Assisted reproductive technologies (ARTs) have been proposed as a means of overcoming the significant challenges of managing small, isolated populations of endangered species in zoos. However, efficient protocols for ARTs do not exist for most endangered species. This review will focus on research efforts to characterize unique reproductive mechanisms and develop species-specific ARTs. Central to these studies are assays to measure steroid metabolites in urine or feces and/or training programs to allow unrestrained blood collections and ultrasound evaluations. The resulting information about estrous cycle dynamics, combined with studies of semen collection and processing, provides the foundation for the development of artificial insemination (AI). In vitro fertilization and embryo transfer are also discussed in relation to the advantages these techniques could provide relative to AI, as well as the significant challenges involved with technologies that require oocytes and embryos. Finally, an argument is made for additional research of nontraditional model species (e.g., domestic cats and dogs) and the development of novel models representing unique taxa. Whether these species are studied by zoo-based researchers with the expressed intent of developing ARTs for conservation or academic scientists interested in basic biology, the resulting information will provide a unique, evolutionary perspective on reproduction that could have wide-reaching benefits. The more information we have available, the better our chances will be of developing effective ARTs and making a difference in conservation efforts for endangered species.

Perspectives on the development and incorporation of assisted reproduction in the equine industry.

Marked changes in equine breeding technologies have occurred over the past 25 years. Although there have been numerous reviews on assisted reproduction techniques for horses, few publications include the acceptance and impact of these techniques on the horse industry. In this review, several techniques are discussed, with an emphasis on how they developed in the horse industry and altered equine reproductive medicine. Embryo transfer has become a widely used technology, allowing multiple foals to be produced per year. Embryos can be collected, cooled or frozen, and shipped to a distant facility for transfer into recipient mares. Failure to obtain embryos from some mares stimulated the development of oocyte collection and transfer. Oocyte technologies became more practical when intracytoplasmic sperm injection was developed in the early 2000s. There are now facilities across the world that routinely produce embryos invitro. Cryopreservation of oocytes has lagged because of limited success, but embryo cryopreservation is commonplace. Techniques such as sex-sorted semen, superovulation and genetic diagnosis of embryos are not widely used, and they will require more development before they are established in the horse industry in a cost-efficient manner.