Resumo
Background: Gamete cryopreservation has permitted widespread preservation of valuable genetics. However, cryopreservation of equine embryos is still not a routine procedure in the industry due to biological reasons [16] and inconsistent results in the field. Review: The basic principle of cryopreservation is to avoid ice crystal formation. Small (< 250 mm) equine embryos can be successfully frozen by slow-cooling techniques to allow slow dehydration of the embryo, thus avoiding ice crystal formation and subsequent lysis of plasma membranes, cytoskeleton disruption, and organelle dysfunction [10,14-15]. Embryos are dehydrated with glycerol, loaded in 0.25 ml straws and frozen by a programmable freezer with liquid nitrogen. Embryos are thawed in a water bath followed by serial dilutions in glycerol and sucrose [14]. Recently, a variation of the slow freezing technique [1] in field conditions produced 16 day pregnancy rates (70%), not different from controls. Vitrification is an alternative to slow-freezing and is defined as a physical state similar to glass [13]. There is no ice crystal formation or concentration of solutes. The methodology requires a high concentration of cryoprotectants, small volumes, high viscosity and rapid cooling rates. Numerous studies with different formulas and containers have resulted in successful vitrification of small equine embryos [6,8,10-11]. An economical and simple vitrification technique was developed [6] that permits in-straw dilution of embryos for direct transfer in the field. Pregnancy rates on day 16 were 65% and not different from controls. In a subsequent study [9], embryos (n=40) < 250 mm in diameter were obtained from eFSH treated mares, half cooled for 16-20 h and vitrified and the other half vitrified only. The pregnancy rate on day 16 after direct transfer under field conditions was 65 and 70% respectively. However, personal communications from practitioners around the world have found poor pregnancy rates (250 mm) have poor survival rate after cryopreservation. One explanation is that the equine embryonic capsule composed of mucin like glycoproteins [12] that replaces the zona pellucida and surrounds the embryo until embryonic attachment (16- 21 d) may impede the proper penetration of cryoprotectants, leading to intracellular ice crystal formation. Another possibility is that the small surface area to volume ratio of embryos with large amounts of fluid in the blastocoele cavity may slow addition and removal of cryoprotectants [2]. A vitrification technique was developed with a modified three-step vitrification procedure for large embryos [3]. However, the pregnancy rate on day 16 (37 %) was unacceptable for commercial use. In a recent and promising study [5] after piercing the embryonic capsule with micromanipulation tools, pregnancy rates were 70% (5/7) at day 16. In vitro produced embryos have been cryopreserved successfully. Pregnancies (~50%) after slow freezing and thawing of equine blastocysts produced after ICSI have been consistently obtained [7]. In addition, pregnancies (65%, 5/8) have been obtained after vitrification in super open pulled straws , warming and transfer of 2- to 8-cell embryos produced by ICSI [4]. However, more studies are needed in this area. Conclusions: Even though vitrification offers theoretical advantages to minimize cell damage, the learning curve and practicality of the methods need to be standardized for commercial use. Nonetheless, depending on embryological stage, both methods (ice or glass) of cryopreservation can be used successfully to produce pregnancies.
Assuntos
Animais , Criopreservação/métodos , Criopreservação/veterinária , Vitrificação , Cavalos/embriologia , Embrião de MamíferosResumo
Background: Intracytoplasmic sperm injection (ICSI) has become a useful technology to produce foals when availability of semen is limited or when in-vitro fertilization is desired, as is the need for subfertile mares. However, its application into clinical practice is challenging. The purpose of this review was to discuss some fundamental molecular aspects of oocyte maturation that should be considered when performing ICSI and to report factors of age and subfertility affecting the success of a commercial ICSI program. Review: The molecular synchrony of oocyte maturation included nuclear, epigenetic and cytoplasmic maturation. Oocyte developmental competence was found to be dependent on the ability to remain in meiotic arrest until the initiation of final maturation, requiring the adequate timing involved with follicular maturation prior to ovulation. Studies performed in cattle and humans have demonstrated that in-vivo oocyte maturation results in high pregnancy rates per oocyte fertilized. Therefore, determining precise maturation of the oocyte would be valuable for the timing of ICSI and subsequent embryo development. Reproductive aging in the mare was characterized by a decline in fertility. Using RT-PCR, quantitative and temporal differences were found in mRNA content of key regulatory maturation genes in granulosa and cumulus cells and in oocytes during in vivo maturation in young and old mares. These results suggested premature oocyte maturation in aged mares that potentially could result in subfertility. Consequently, the timing of oocyte retrival after gonadotropin administration should be carefully evaluated when performing ICSI. In a commercial program, equine patients were classified into normal mares (2.5 to 15 years), problem mares (15-23 years that had not been producing embryos or pregnancies) and old mares (>24 years). Old mares were assessed for endocrine, physical and nutritional imbalances. Follicular and oocyte maturation were induced with a dominant follicle >30 mm in diameter after a normal growth and blood flow and uterine edema with a combination of hCG and GnRH. Transvaginal oocyte retrieval was performed 20 hours after administration of gonadotropins. Oocytes were further cultured in vitro for 12 to 20 hours. Frozen semen was used for all sperm injections. Injected oocytes were further cultured in vitro for at least 24 hours. Embryos were then transferred surgically into oviducts of synchronized recipients. Oocyte recovery rate was 94% (523/557 cycles), cycles per month were 3.3, 2 and 1.3 for young, problem and old mares respectively. Cleavage rates were different (p < 0.05) between young (82 %), problem (70 %) and old (52%) mares. Pregnancy rates at day 60 were also different (p < 0.05) for young (68 %), problem (50 %) and old (23%) mares. Number of pregnancies obtained from a single straw of frozen semen ranged from 2 to 12. Reproductive senescence was observed in 10% of old mares. In addition, Cushing's disease and elevated diestrus FSH were observed in 80% of the old mares. Foaling rates were evaluated in 55 pregnancies; 5% were lost in the last trimester and the remaining foals have shown no apparent abnormalities. Conclusion: More studies are needed to further elucidate the mechanisms of oocyte maturation and activation in the horse, as well as more objective methods to determine oocyte maturity and quality. A clinical ICSI program required an understanding of gamete physiology and detailed mare reproductive management. Our clinical data demonstrated that aging affects fertility profoundly in ways that may be difficult to address with current technology; nonetheless, ICSI has provided the equine industry an alternative to produce offspring from valuable mares and stallions that are subfertile.