Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification.

BACKGROUND: The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs). METHODS: Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential. RESULTS: An increase in chromosome misalignment was observed in spindles from older mares when compared to those of younger mares (P < 0.05). When MII oocytes subjected to vitrification were examined following warming, there was no difference in the percentage of oocytes displaying chromosome misalignment. Next, GV oocytes, collected from the ovaries of younger and older mares, were compared between fresh IVM and IVM following vitrification and warming. For nonvitrified samples, an age difference was again noted for spindle organization and chromosome alignment, with a higher (P < 0.05) percentage of normal bipolar meiotic spindles with aligned chromosomes observed in nonvitrified oocytes from young versus older mares. Vitrification led to a reduction of spindle length (P < 0.05) for oocytes from old mares, whether vitrified at GV or MII stages, whereas this effect was not observed in oocytes from young mares except those vitrified at GV and subjected to IVM. Oocyte developmental potential after vitrification was evaluated after ICSI of vitrified and warmed MII or GV oocytes from young mares. From 25 MII oocytes, 18 oocytes were injected with sperm, and six blastocysts were produced, which, upon transfer to mares' uteri, resulted in four pregnancies. Immature (GV) oocytes collected from live mares were also vitrified, warmed, and matured in vitro before ICSI. In this group, nonvitrified, control, and vitrified oocytes did not differ (P > 0.05) with respect to the incidence of maturation to MII, cleavage after ICSI, or blastocyst development. CONCLUSION: These findings demonstrate an effect of maternal age in an equine model at the level of meiotic spindle integrity and chromosome positioning that is influenced by both the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.

Oocyte metabolic function, lipid composition, and developmental potential are altered by diet in older mares.

Dietary supplementation is the most feasible method to improve oocyte function and developmental potential in vivo. During three experiments, oocytes were collected from maturing, dominant follicles of older mares to determine whether short-term dietary supplements can alter oocyte metabolic function, lipid composition, and developmental potential. Over approximately 8 weeks, control mares were fed hay (CON) or hay and grain products (COB). Treated mares received supplements designed for equine wellness and gastrointestinal health, flaxseed oil, and a proprietary blend of fatty acid and antioxidant support (reproductive support supplement (RSS)) intended to increase antioxidant activity and lipid oxidation. RSS was modified for individual experiments with additional antioxidants or altered concentrations of n-3 to n-6 fatty acids. Oocytes from mares supplemented with RSS when compared to COB had higher basal oxygen consumption, indicative of higher aerobic metabolism, and proportionately more aerobic to anaerobic metabolism. In the second experiment, oocytes collected from the same mares prior to (CON) and after approximately 8 weeks of RSS supplementation had significantly reduced oocyte lipid abundance. In the final experiment, COB was compared to RSS supplementation, including RSS modified to proportionately reduce n-3 fatty acids and increase n-6 fatty acids. The ability of sperm-injected oocytes to develop into blastocysts was higher for RSS, regardless of fatty acid content, than for COB. We demonstrated that short-term diet supplementation can directly affect oocyte function in older mares, resulting in oocytes with increased metabolic activity, reduced lipid content, and increased developmental potential.

Update on advanced semen-processing technologies and their application for in vitro embryo production in horses.

The increased commercialisation of intracytoplasmic sperm injection (ICSI) in horses creates more opportunities to incorporate advanced reproductive technologies, such as sex-sorted, refrozen and lyophilised spermatozoa, into a breeding program. This paper reviews the status of these semen-handling technologies in light of their use in equine ICSI programs. Pregnancies have been achieved from each of these advanced technologies when combined with ICSI in horses, but refinements in the semen-handling processes underpinning these technologies are currently being explored to produce more reliable and practical improvements in the results from equine ICSI.

In Vitro Culture of Embryos from Horses.

Establishment of optimal methods for equine embryo culture has been slow when compared to some domestic species. In part, this delay was caused by the failure of standard in vitro fertilization techniques in horses. However, the development of intracytoplasmic sperm injection (ICSI) for the assisted fertilization of equine oocytes has resulted in a renewed interest in establishing optimal methods for embryo culture. Currently, ICSI-produced equine embryos are cultured using media designed for other species or other cell cultures and, typically, with the addition of serum. Although systems specifically for horse embryo culture still have not been established, ICSI-produced embryos are developmentally competent and capable of producing live offspring.

Collection, evaluation, and use of oocytes in equine assisted reproduction.

Assisted reproductive techniques have been developed to obtain pregnancies from subfertile mares and stallions and to salvage gametes after death. In recent years, these procedures have been used for clinical cases with repeated success. Although new developments occur, the basis for the success and future development of assisted reproductive techniques is our ability to collect and handle the equine oocyte successfully. This article focuses on important clinical aspects of oocyte collection and evaluation and briefly discusses the clinical use of assisted reproductive procedures in the horse.

Use of parentage testing to determine optimum insemination time and culture media for oocyte transfer in mares.

Parentage identification was used to test the developmental competence of oocytes cultured under different conditions and fertilized in vivo after oocyte transfer. Oocytes were collected transvaginally from follicles of estrous mares approximately 22 h after administration of human chorionic gonadotropin. Oocytes were cultured for approximately 16 h in one of three media, with or without addition of hormones and growth factors. Groups of three or four oocytes, cultured in different media, were transferred into the oviduct contralateral to a recipient's own ovulation. Recipients were inseminated with semen from two different stallions at 15 h before and 2.5 h after oocyte transfer. Sixteen days after transfer, embryos were recovered from uteri and submitted for parentage testing. The percentage of oocytes resulting in embryonic vesicles was nearly identical (P >0.05) for transferred oocytes (32/44, 73%) versus ovulated oocytes of recipients (9/13, 69%). More (P <0.01) oocytes were fertilized by sperm inseminated before (35/38, 92%) versus after (3/38, 8%) oocyte transfer. Tissue culture medium (TCM)-199 was superior to equine maturation medium I (EMMI; a SOF-based medium) for culturing oocytes (P <0.05), although addition of hormones and growth factors during culture did not improve (P >0.05) development of embryos.

Pregnancies attained after collection and transfer of oocytes from ovaries of five euthanatized mares.

After euthanasia, ovaries were removed from 5 horses and shipped to a laboratory where 46 oocytes were collected. The oocytes were cultured for 24 to 30 hours, and 36 oocytes were transferred to 10 recipient mares via flank laparotomies. Recipient mares were inseminated with semen from various stallions. Sixteen days after transfer, 4 of the recipients were pregnant with at least 1 embryonic vesicle. Embryonic death occurred in 3 recipients, whereas a healthy live foal was born from 1 recipient. Ovaries from valuable mares can be a source of viable oocytes after death of the mare. For shipping to a laboratory, fluctuations in temperature should be minimized and the ovaries should not be chilled.

Differential requirement for pulsatile LH during the follicular phase and exposure to the preovulatory LH surge for oocyte fertilization and embryo development in cattle.

The requirement for pulsatile LH and the LH surge for the acquisition of oocyte fertilizing potential and embryo developmental competency was examined in Zebu heifers. Follicular growth was superstimulated using the GnRH agonist-LH protocol in which pulsatile LH and the preovulatory LH surge are blocked. In experiment 1, heifers were assigned on Day 7 of the estrous cycle to receive: group 1A (n = 5), 1.5 mg norgestomet (NOR) implant; group 1B (n = 5), GnRH agonist implant. Follicular growth was superstimulated with 2x daily injections of FSH from Day 10 (a.m.) to Day 13 (p.m.), with PGF2alpha injection on Day 12 (a.m.). Heifers were ovariectomized on Day 15 (a.m.) and oocytes were placed immediately into fertilization, without 24 h maturation. Respective cleavage and blastocyst development rates were: group 1A, 0/64 oocytes (0%) and 0/64 (0%); group 1B, 34/70 oocytes (48.6%) and 2/70 (2.9%). In experiment 2, heifers were assigned on Day 7 of the estrous cycle to receive: group 2A (n = 10), 1.5 mg NOR implant; group 2B (n = 10), GnRH agonist implant; group 2C (n = 10), GnRH agonist implant. Follicular growth was superstimulated as in experiment 1 above. Heifers in groups 2A and 2B received an injection of 25 mg LH on Day 14 (p.m.) and all heifers were ovariectomized on Day 15 (a.m.); oocytes were placed immediately into fertilization without 24 h maturation. Cleavage rates were similar for heifers in group 2A (84/175 oocytes, 48.0%), group 2B (61/112 oocytes, 54.5%) and group 2C (69/163, 42.3%). Blastocyst development rates were similar for heifers in group 2A (22/175 oocytes, 12.6%) and group 2B (25/112 oocytes, 22.3%) and lower (P < 0.05) for heifers in group 2C (9/163 oocytes, 5.5%). Oocytes obtained from heifers treated with GnRH agonist, without injection of exogenous LH, underwent cleavage indicating that neither pulsatile LH nor the preovulatory LH surge are obligatory for nuclear maturation in cattle oocytes. Exposure to a surge-like increase in plasma LH increased embryo developmental competency indicating that the preovulatory LH surge promotes cytoplasmic maturation. The findings have important implications for controlling the in vivo maturation of oocytes before in vitro procedures including nuclear transfer.