Three experiments were designed to evaluate methods of bisecting equine embryos. Viability of demi-embryos was compared to whole embryos in vitro and in vivo. In Experiment 1, Day 6 embryos were bisected either using a standard micromanipulation technique or a manual split of embryos that were adhered to a petri dish. Viability was assessed by culture in vitro. Bisected embryos in both groups increased in size during culture and developed normally. In general, manual bisection was accomplished with less difficulty and time than the micromanipulation method. Eleven additional embryos were bisected manually and both halves transferred surgically into the same recipient (Experiment 2). More pregnancies were obtained from transfer of twin whole embryos (16/22) compared to bisected, demi-embryos (5/22). In Experiment 3, 30 embryos were bisected manually; 15 were encapsulated in agar chip. Both halves were transferred non-surgically to each recipient. Fifteen whole embryos were transferred non-surgically to recipients as controls. The number of vesicles per original embryo was greater for whole embryos than for demi-embryos in agar chips.
"A unique and vital feature of equine embryos is the formation of an acellular capsule at days 6-7 after ovulation ; the capsule is necessary for embryonic survival . However, equine blastocysts do not form a capsule in vitro  . The embryos fail to assemble capsular glycoproteins into a normal and complete capsule, indicating that some aspect of the uterine environment is necessary, the microenvironment is unsuitable, cell numbers are insufficient to adequately produce the glycoproteins, or the glycoproteins disperse into the microenvironment through the hole in the zona pellucida from the ICSI procedure . "
[Show abstract][Hide abstract] ABSTRACT: The development of methods to produce embryos in vitro in the horse has been delayed compared with other domestic species. Oocytes can be collected from excised ovaries or from the small or preovulatory follicles of live mares. Intracytoplasmic sperm injection is the only reliable method to fertilize equine oocytes in vitro. Intracytoplasmic sperm injection-produced embryos can be transferred into the oviducts of recipient mares or cultured to the morula or blastocyst stage of development for nonsurgical embryo transfers into recipients' uteri. Embryos cultured in vitro have some morphological differences compared with embryos collected from the mares' uteri. Most notably, the embryonic capsule does not form in culture, and the zona pellucida fails to expand completely. However, embryo produced in vitro can result in viable pregnancies and healthy offspring.
"The experimental production of monozygotic horse twins by splitting the embryo has also proven technically difficult compared to other species (Allen and Pashen, 1984), due to the combination of the long oviducal transport time of the equine embryo (Battut et al., 1997), the small size of the inner cell mass of the equine blastocyst (Skidmore et al., 1989), and the toughness and elasticity of the glycoprotein capsule that surrounds the equine conceptus between days 6.5 and 23 after ovulation (Betteridge, 1989). Consequently very few pairs of identical twin horses have been generated (Allen and Pashen, 1984; Skidmore et al., 1989; McKinnon et al., 1989). Nevertheless , by using such identical twin mares for reference family production, it is possible to double the number of full-sibling offspring produced in a given time, since the offspring from each twin can be regarded genetically as full-siblings. "
[Show abstract][Hide abstract] ABSTRACT: Two 3-generation full-sibling reference families have been produced and form a unique resource for genetic linkage mapping studies in the horse. The F(2) generations, now comprising 61 individuals, consist of 28- to 32-day-old embryos removed nonsurgically from two pairs of identical twin mares. The same stallion sired all F(2)s such that the two full-sibling families are half-sibling with respect to each other. The families are crossbred to maximize levels of heterozygosity and include Arabian, Thoroughbred, Welsh Cob, and Icelandic Horse breeds. Milligram quantities of DNA have been isolated from each embryo and from blood samples of the parents and grandparents. The families have been genotyped with 353 equine microsatellites and 6 biallelic markers, and 42 linkage groups were formed. In addition, the physical location of 85 of the markers is known, and this has allowed 37 linkage groups to be anchored to the physical map. The inclusion of dams in the genotyping analysis has allowed the generation of a genetic map of the X chromosome. Markers have been assigned to all 31 autosomes and the X chromosome. The average interval between markers on the map is 10.5 cM, and the linkage groups collectively span 1780 cM. The results demonstrate the benefits for horse linkage mapping studies of genotyping on these unique full-sibling families, which comprise relatively few individuals, by the generation of a comprehensive low-density map of the horse genome.
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