HOW TO INCORPORATE ASSISTED REPRODUCTIVE TECHNOLOGIES


tion microscope at 10–20 magnification. If the oocyte is found, it can be washed and placed in holding media immediately, and if not it is recom- mended the person scraping continues to attempt collection on the same follicle. Once all palpable follicles are explored, the process of “bread-loafing” the ovary begins. The author typically begins by systematically making a sagittal incision along the long axis of the ovarian tissue, pausing, and scrap- ing any identifiable follicles as described above. Upon fileting open the two halves, transverse inci- sions are made along both sides at 5 mm-intervals until all identifiable antral follicles have been ex- plored. Once complete, the ovary is aggressively “power-washed” with flush media in a syringe to encourage any last-minute oocytes to be collected in the large Petri dish utilized for holding. It is rec- ommended to also maintain one or a portion of one ovary frozen for potential identification require- ments at a later date. Handling these oocytes is similar to the process described above for immature oocytes. We utilize either a sterile 0.25-mL strawo with adapterp (if large expanded cumulus cells are evident) or a pi- pettor with appropriate tip size (if compact cumulus cells are evident). In addition to searching individ- ual small Petri dishes or the entire conical tube volume, always search the large Petri dish utilized for holding and processing the ovary, as often an oocyte or several are found in this loca- tion. Oocytes are placed immediately into a media for in vitro maturation or held overnight. If being shipped to a facility offering ICSI services, oocytes are maintained during transport within either a commercial or lab prepared embryo-holding media as described previously for rinsing purposes13 or premade lab EM medium (40% M199 with Hanks salts and HEPES or 40% M199 with Earles salts and 20% fetal bovine serum with 25 g–1 gentamycin). Many labs utilize a 1-mL borosilicate glass vials secured with Parafilmt filled with either medium to either hold or transport the collection of oocytes. Another option is for private practitioners to simply ship the entire conical tube of collected scrapings at room temperature if no microscope or trained per- sonnel are available to locate individual oocytes.5 Additional methods for harvesting oocytes from


equine ovaries have been described, especially when larger volumes of ovaries need to be processed. For example, when research on slaughterhouse specimens was available. Direct follicular aspira- tion with a syringe and needle or a needle attached to a pump increases efficiency and represents amore “closed” system when contamination is a concern. However, reports have indicated this method leads to stripping of the majority of cumulus cells which can lead to difficulty in oocyte identification.28 The scraping method has been directly compared to aspirating follicles with an 18-gauge needle and sy- ringe, showing no difference in maturation or fertil- ization rates.29,30 However, a needle and syringe is


358 2019  Vol. 65  AAEP PROCEEDINGS


not recommended for aspiration as the oocytes are often completely denuded of cumulus cells with this technique.29,30 The utilization of ultrasound to identify and aspirate follicles (using the in vivo col- lection setup) prior to dissection and subsequently separating the two populations throughout the col- lection process, may represent an alternative method, increase efficiency and maintain a stricter level of hygiene (Metcalf L, Sherwood, OR, personal communication, 2018).


4. Discussion


As more researchers and clinicians become aware of and are interested in ART procedures in the horse, more questions will undoubtedly surface. The equine industry worldwide is participating in these procedures and striving to find answers to these questions and creative ways of handling pitfalls. Ultimately, dedicated clinicians and researchers de- pend upon the foundational knowledge amplified within the past two decades to build upon and im- prove the safety, efficacy, and efficiency of these procedures for greater application.


Acknowledgments


Declaration of Ethics The Author has adhered to the Principles of Veteri- nary Medical Ethics of the AVMA.


Conflict of Interest The Author has no conflicts of interest.


References and Footnotes


1. Morris LHA. The development of in vitro embryo production in the horse. Equine Vet J 2018;50:712–720.


2. Galli C, Duchi R, Colleoni S, et al. Ovum pick up, intracy- toplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: From the research laboratory to clinical practice. Theriogenology 2014;81:138–151.


3. Blue BJ, McKinnon AO, Squires EL, et al. Capacitation of stallion spermatozoa and fertilisation of equine oocytes in vitro. Equine Vet J 1989;21:111–116.


4. Palmer E, Be ´zard J, Magistrini M, et al. In vitro fertiliza-


tion in the horse. A retrospective study. J Reprod Fertil Suppl 1991;44:375–384.


5. Hinrichs K. Application of assisted reproductive technolo- gies (ART) to clinical practice, in Proceedings. Am Assoc Equine Pract 2010;56:195–206.


6. Transvaginal ultrasonically guided follicular aspiration of equine oocytes: Preliminary results. J Equine Vet Sci 1992; 12:204–207.


7. Meintjes M, Bellow MS, Paul JB, et al. Transvaginal ultra- sound-guided oocyte retrieval from cyclic and pregnant horse and pony mares for in vitro fertilization. Biol Reprod 1995; 52:281–292. ´lski A, Be


8. Oko


´zard J, Duchamp G, et al. Successive puncture


of the dominant follicle followed by ovulation and fertiliza- tion: A new experimental model for the study of follicular maturation in the mare. Biol Reprod 1995;52:385–392.


9. Carnevale EM, Maclellan LJ. Collection, evaluation, and use of oocytes in equine assisted reproduction. Vet Clin North Am Equine Pract 2006;22:843–856.


10. Duffy DM, VandeVoort CA. Maturation and fertilization of nonhuman primate oocytes are compromised by oral admin- istration of a cyclooxygenase-2 inhibitor. Fertil Steril 2011; 95:1256–1260.


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