Topic:Assisted Reproductive Techniques
Assisted Reproductive Techniques (ART) in horses encompass a range of technologies designed to aid in the breeding process. These techniques include artificial insemination, embryo transfer, intracytoplasmic sperm injection (ICSI), and oocyte transfer. ART is employed to enhance reproductive efficiency, manage genetic diversity, and support breeding programs for both commercial and conservation purposes. Artificial insemination involves the collection and deposition of semen into the mare's reproductive tract, while embryo transfer entails the collection of a fertilized embryo from a donor mare and its implantation into a recipient mare. ICSI involves the direct injection of a single sperm into an oocyte to achieve fertilization. Oocyte transfer involves the transfer of an oocyte from one mare to another for fertilization and gestation. This page compiles peer-reviewed research studies and scholarly articles that examine the methodologies, advancements, and applications of assisted reproductive techniques in equine reproduction.
Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection. Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not diff...
Equine cloning: applications and outcomes. Cloning is one of several new assisted reproductive techniques being developed for clinical use in the equine industry. Potential uses of equine cloning include: (1) the preservation of genetics from individual animals that would otherwise not be able to reproduce, such as geldings; (2) the preservation of genetic material of endangered and/or exotic species, such as the Mongolian wild horse (Przewalski's horse); and (3) because of the companion animal role that horses fill for some individuals, it is likely that some horse owners will have individual animals cloned for emotional fulfillment. ...
Effect of repeated transvaginal ultrasound-guided follicle aspiration on fertility in mares. To determine whether performance of transvaginal ultrasound-guided follicle aspiration (TVUFA) repeatedly in mares adversely affects their fertility. Methods: Historical prospective study. Methods: 23 mares that had never undergone TVUFA and 59 mares that had undergone TVUFA on 1 to 11 occasions. Methods: Mares were classified into 4 groups according to the number of TVUFA procedures previously performed on the ovary in which ovulation occurred at the time of insemination as follows: group 1, 0 TVUFAs (control group, n = 23 mares); group 2, 1 or 2 TVUFAs (40 mare-cycles); group 3, 3 or 4 TVUFA...
Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type. The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear...
Effects of recombinant human follicle stimulating hormone on follicle development and ovulation in the mare. The only gonadotrophin preparation shown to stimulate commercially useful multiple ovulation in mares is equine pituitary extract (EPE); even then, the low and inconsistent ovulatory response has been ascribed to the variable, but high, LH content. This study investigated the effects of an LH-free FSH preparation, recombinant human follicle stimulating hormone (rhFSH), on follicle development, ovulation and embryo production in mares. Five mares were treated twice-daily with 450 i.u. rhFSH starting on day 6 after ovulation, coincident with PGF(2alpha) analogue administration; five control mare...
Fertility in the mare after repeated transvaginal ultrasound-guided aspirations. Ovum pick-up (OPU) by transvaginal ultrasound guided aspiration (TUGA) is a procedure applied in equine-assisted reproduction programs such as oocyte transfer and in vitro embryo production. Despite a large number of studies reporting that it is a repeatable and safe technique, little information is available about the effect of repeated punctures on fertility of mares. Moreover, even if flushing follicles improves the oocyte recovery rate, to our knowledge the efficiency of flushing estrous and diestrous follicles has not been evaluated. The aims of the present study were (1) evaluate if repe...
Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles. This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract...
Integration of future biotechnologies into the equine industry. There has and will continue to be reproductive techniques available that have a positive impact upon the equine breeding industry. This review focuses on semen technologies that have been developed or are in the process of being developed. The use of fluorescent dyes and flow cytometry has provided the researcher and clinician with powerful tools to evaluate several sperm attributes. These procedures have been utilized to evaluate sperm viability, acrosome status, mitochondrial status, DNA integrity and stages of capacitation. Flow cytometry allows several sperm attributes to be evaluated on t...
The development and application of the modern reproductive technologies to horse breeding. Although the horse was probably the first animal to experience and benefit from artificial insemination, it trailed the field somewhat with regard to the application of embryo transfer and other oocyte and embryo-related modern breeding technologies. But with a late run it is now back in mid-field and gaining fast on the other large domestic species in the application of the many technological advances of the past 20 years to sound breeding practice. Improvements in extenders and cryoprotectants have resulted in a veritable upsurge in the transport and insemination of cooled and frozen stallio...
Update on equine ICSI and cloning. Intracytoplasmic sperm injection (ICSI) has recently become efficient enough to be considered for clinical use. With ICSI, one spermatozoa is injected into a mature oocyte. Harvesting of an oocyte ex vivo, followed by ICSI and transfer of the fertilized oocyte to the oviduct, may be applicable when semen quality is insufficient for standard insemination. Sperm injection, followed by in vitro embryo culture to the blastocyst stage, may be used in cases where multiple oocytes are to be fertilized (e.g. when oocytes are collected post-mortem). Nuclear transfer (cloning) of horses is possible but ...
Factors affecting the success of oocyte transfer in a clinical program for subfertile mares. Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2 +/- 0.4 yr of age (mean +/- S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0+/-0.1h after administration of hCG to estrous donors and cultured 16.4 +/- 0.2 h prior to transfer into recipients' oviducts. At 16 and 50 d after transfer, pregnancies were detected i...
Estrous cycle stage-independent treatment of PMSG and hCG can induce superovulation in adult Wistar-Imamichi rats. The estrous cycle influence on the number of ovulated eggs after injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was investigated in 12, 18, and 24 weeks old adult female Wistar-Imamichi (WI) rats. PMSG (150 IU/kg) was injected at metestrus, diestrus, proestrus, or estrus, followed by hCG (75 IU/kg) 55 h later. Ovulation was induced at all ages and stages of the estrous cycle. The number of ovulated eggs was not affected by stage for similarly aged rats, however, the number of ovulated eggs obtained after treatment decreased with age. These results d...
Cloned horse pregnancies produced using adult cumulus cells. The objectives of the present study were to: (1) clone horses using adult cumulus cells; and (2) determine whether the cumulus cell donor affected the outcome. In vivo-matured cumulus-oocyte complexes were obtained using transvaginal ultrasound-guided follicle aspiration; oocytes were used as cytoplasts, whereas cumulus cells (from one of three different mares) were used as donor cells. Immediately following nuclear transfer and activation procedures, cloned embryos were transferred surgically to the oviduct of recipient mares (n = 2-5 embryos per recipient) that had ovulated within 24 h prior...
Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study. The occurrence of pregnancies and births after embryo transfer (ET) of in vivo produced embryos is generally more successful compared to that of embryos produced in vitro. This difference in ET success has been observed when embryos of morphological equal (high) quality were used. The incidence of apoptosis has been suggested as an additional criterion to morphological embryo evaluation in order to assess embryo quality and effectively predict embryo viability. In this study, equine, porcine, ovine, caprine and bovine in vivo and in vitro produced morphologically selected high quality (grade-I...
Integration of sperm sexing technology into the ART toolbox. Sex-sorting of mammalian spermatozoa has applications for genetic improvement of farm animals, in humans for the control of sex-linked disease, and in wildlife as a captive management strategy and for the re-population of endangered species. Considerable research has been undertaken worldwide on the Beltsville sperm sexing technology, the only effective method for pre-selection of sex of offspring. The combination of this method with assisted reproductive technologies has resulted in the birth of offspring in a wide range of animals, including cattle, the only livestock species in which sperm ...
Low dose insemination in the mare: an update. The generally recommended minimum number of spermatozoa required for conventional artificial insemination in the mare is in excess of 200 x 10(6) progressively motile spermatozoa. Recent developments in different insemination techniques such as deep uterine, hysteroscopic and oviductal insemination, which have been designed to use significantly fewer spermatozoa, are reviewed in this paper. A number of studies have demonstrated that ultrasound guided deep uterine insemination of 5 x 10(6) fresh spermatozoa can produce satisfactory pregnancy rates. The use of hysteroscopic insemination enables ...
Intra-uterine insemination in farm animals and humans. Artificial insemination (AI) is the oldest and currently most common technique in the assisted reproduction of animals and humans. The introduction of AI in farm animals was forced by sanitary reasons and the first large-scale applications with a commercial goal were performed in cattle in the late 1930s of last century. After the Second World War, cryopreservation of semen facilitated distribution and AI was mainly performed for economic reasons, especially in dairy cattle industry. In humans however, AI was initially performed in cases of physiological and psychological sexual dysfunction, b...
Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection. This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 degrees C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61-72 vs 23-2...
Japanese Society for Animal Reproduction: award for outstanding research 2002. Cryopreservation of follicular oocytes and preimplantation embryos in cattle and horses. Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both i...
Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse. In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single mo...