HOW TO INCORPORATE ASSISTED REPRODUCTIVE TECHNOLOGIES


ultimate outcome of the IVP production of embryos. From nontoxic labware to media to temperature of the overnight shipment, all steps require vigilance to create the optimal environment. Results from experiments in the author’s labora-


tory suggest that modulation of either preIVM media or temperature, or both, to delay the sponta- neous resumption of meiosis following oocyte retrieval can improve blastocyst production. Man- agement of oocyte and cumulus cell cAMP level prior to IVM appears to be an effective means to enhance equine oocyte competence and blastocyst develop- ment. Studies in other species have demonstrated that a similar system of simulated physiological oocyte maturation, designed to reproduce the phys- iological maturation that occurs in vivo by using the cAMP modulators prior to in vitro maturation (IVM), improves oocyte developmental outcome.9 Studies to determine the optimal preIVM temper-


ature for equine oocytes suggest that the Equitainer at a temperature of 5°C is not appropriate for ship- ment of equine oocytes. In the author’s study, there was a significant drop in embryo cleavage and blastocyst development rate when compared to the storage temperature of 20°C. Earlier studies sup- port this result. Other researchers have reported that a reduced number of blastocysts formed from immature oocytes that had been held overnight at 5°C10 or 7°C11 prior to maturation; furthermore, there were no pregnancies following transfer of the few blastocysts that formed. However, the developmental competence of equine


oocytes appears to improve by cooling them over- night to temperatures below room temperature. The study from the author’s laboratory showed im- proved blastocyst development when oocytes were incubated at 16°C versus 20°C. Because there was no difference in blastocyst development rates follow- ing cleavage, these results suggest that maturation and cleavage functioned as the determining devel- opmental benchmark affected by this temperature difference. The preIVM period of immature oocytes sets the


stage for cytoplasmic and nuclear maturation, and following maturation fertilization, the development of the embryo. This paper has presented practical guidelines for the handling and shipping of equine oocytes.


Acknowledgments


Declaration of Ethics The Author has adhered to the Principles of Veter- inary Medical Ethics of the AVMA.


Conflict of Interest The Author has no conflicts of interest.


References and Footnotes


1. Foss R, Ortis H, Hinrichs K. Effect of potential oocyte trans- port protocols on blastocyst rates after intracytoplasmic sperm injection in the horse. Equine Vet J Suppl 2013;45: 39–43.


2. Hinrichs K. Assisted reproductive techniques in mares. Reprod Dom Ani 2018;53:4–13.


3. Metcalf ES, Masterson KR, Battaglia D, et al. The effect of a simulated physiological oocyte maturation system on the in vitro production of equine embryos. J Equine Vet Sci 2016; 41:65.


4. Swearman H, Koustas G, Knight E, et al. pH: the silent variable significantly impacting meiotic spindle assembly in mouse oocytes. Reprod Biomed Online 2018;37:279–290.


5. Choi YH, Heloísa S, Canesin, et al. Effects of different me- dia systems and osmolarity on equine blastocyst development after ICSI. J Equine Vet Sci 2016;41:60–61.


6. Wang WH, Keefe DL. Spindle observation in living mam- malian oocytes with the polarization microscope and its prac- tical use. Cloning Stem Cells 2002;4:269–76.


7. Martino NA, Dell’Aquila ME, Filioli Uranio M, et al. Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox poten- tial. Reprod Biol Endocrinol 2014;12:99.


8. Metcalf ES, Masterson KR Battaglia D, et al. Handling, holding and maturation of immature equine oocytes. Repro Dev Fert. In Press


9. Sugimura S, Yamanouchi T, Palmerini MG, et al. Effect of pre-in vitro maturation with cAMP modulators on the acqui- sition of oocyte developmental competence in cattle. J Re- prod Dev 2018;64:233–241.


10. Carnevale EM, Maclellan LJ, Coutinho da Silva MA. et al. Pregnancies attained after collection and transfer of oocytes of five euthanized mares. J AmVet Med Assoc 2003;222:60– 62.


11. Diaw M, Salgado RM, Canesin HS, et al. Effect of different shipping temperatures (22 °C vs. 7 °C) and holding media on blastocyst development after overnight holding of imma- ture equine cumulus-oocyte complexes. Theriogenology 2018;111:62–68.


aMila International Inc, Erlanger, KY 41042. bVigro™


48060. e


, Vetoquinol USA, Fort Worth, TX 76137. cSagent Pharmaceuticals, Schaumburg, IL 60195.


dEZ Way Filter™, Partnar Animal Health, Port Huron, MI Emcare™, ICP Bio Reproduction, Spring Valley, WI 54767.


fCooper Surgical, Trumball, CT 06611. gSigma Life Science, St Louis, MO 63118. hFisher Scientific, Waltham, MA 02451. iBemis NA, Neenah, WI 54956. jInvitrogen, Carlsbad, CA 92008.


kMicroQ Technologies, Scottsdale, AZ 85260. lHamilton BioVet, LLC, Ipswich, MA 01938.


mEquipure™, Nidacon International AB, 431 37 Mo Sweden. ¨lndal,


AAEP PROCEEDINGS  Vol. 65  2019 363


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