Assisted Reproduction Technologies: which ones can be proposed to pet owners?

Assisted reproduction technologies (ARTs) are auseful mean to improve reproductive efficiency, in both humans and animals. They can be applied to tackle infertility issues or to increase the reproductive potential of animals. In any case, the final aim of the application of ARTs is to obtain healthy progeny. Questions to ask are: which ART can be proposed to pet owners with a reasonable rate of success, and which is still experimental and not yet applicable in clinical practice? How to select the most suitable and applicable technique? A wide array of techniques is currently available, but different procedures imply different levels of complexity, invasiveness, and chances of success. Mostly driven by the progresses made in human ARTs, veterinary ARTs have also evolved. While some are routinely applied in farm animals, where the economic interests are stronger, some are also commonly employed for small animals. However, whereas a few, traditional procedures, such artificial insemination are now routinary techniques, others are still experimental. To guide clinicians and safeguard the patients, in human assisted procreation ARTs are often classified in three levels, based on the etiology and severity of the infertility problem, as well as on the invasiveness of the procedure, with the first level being the least invasive and simplest techniques and the third level being the most advanced, complex and impactful. When possible, the lowest level ARTs should always be chosen first. On the other hand, the cause of the infertility problem might also be used to choose the best ART to be applied in every case. Causes can be due to the male animal (i.e., male factor), to the female (i.e., female factor), to both of them (i.e., male + female factor), or sometimes cannot be distinguished or identified (i.e., idiopathic infertility). The infertility might be caused by impotentia coeundi or generandi due to problems of different origin - anatomical, hormonal, functional, behavioral etc. A gynecological examination of the bitch and estrous monitoring by means of vaginal cytology and blood progesterone assays should be routinely carried out. Firstly, it should be excluded that the infertility is only “apparent” and due to an incorrect breeding management. Instead, in the case of irregular estrous cycles, anestrus or ovulation failure or when anatomical anomalies of the vaginal tract or behavioral attitudes prevent mating, first level ARTs as medical treatments for estrous or ovulation induction or artificial insemination can be taken into account. For the male, as well as andrological examination, semen analysis is currently performed in many clinics. A base spermiogram, which should include, at least, the evaluation of motility, concentration and morphology, could show that there is a sufficient number of motile, good quality spermatozoa, and first level ARTs can be applied, such as artificial insemination. Intravaginal or intrauterine, or less commonly surgical, insemination can be used, depending on semen quality and conservation status (fresh or chilled or frozen) (Hollinshead & Hanlon, 2017; Linde-Forsberg & Forsberg, 1989; D. Zambelli, Bini, & Cunto, 2015; Daniele Zambelli & Cunto, 2005). Other evaluations of the semen, besides a base spermiogram, can also be done. For instance, the hypo-osmotic swelling test (England & Plummer, 1993), that is feasible in every environment equipped with amicroscope allows the evaluation of spermatozoa membrane integrity and viability. With a more sophisticated microscope, provided with fluorescence lamp and filters, acrosome integrity and ability to undergo acrosome reaction could be evaluated with the use of specific dyes such as the peanut agglutinin (PNA) conjugated with fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining (Cheng et al., 1996). asperm function test could also be performed, such as the zona binding assay (Ström Holst, Larsson, Linde-Forsberg, & Rodriguez-Martinez, 2000), and could suggest the ability of the spermatozoa to bind to the oocytes, giving aclue for the choice of the best fertilization method. With decreased semen quality, second level ARTs could be applied, such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). However, these techniques require a specialized, equipped lab, and the application in small animal clinics is still limited. Oocytes can be collected by laparotomy or laparoscopy and then co-incubated in a dish with motile spermatozoa for IVF, or they can be injected by means of a micromanipulator with a single, selected spermatozoon in ICSI. Therefore, ICSI is the technique of choice for severe male factors, since ejaculated, epididymal and even testicular sperm can be used, even if immotile. However, while IVF and ICSI are successful in the cat (Pope, Johnson, McRae, Keller, & Dresser, 1998), they are still tentative in the dog, where few successes have been achieved (Nagashima et al., 2015). In any case, both of these techniques can work only joint to another ART, which is embryo transfer (ET). Indeed, after IVF or ICSI and the beginning of embryo development in vitro, embryos need to be transferred to recipient animals to establish pregnancy and hopefully to give birth to progeny. Unfortunately, in cats and dogs the uterine lumen is not as easily accessible from the outside as in large animals, therefore embryo transfer must be surgical rather than transcervical. In case of early-stage embryos intratubal surgical transfer must be performed. When the quality of the ejaculate is severely compromised, in case of azoospermia, or when an ejaculate cannot be obtained, third level ARTs could be employed. After causes for obstructive azoospermia have been ascertained and, in case, surgically removed, the retrieval of epididymal or testicular spermatozoa can be attempted. Techniques derived from the human medicine can be employed. For example, microsurgical epididymal sperm aspiration (MESA) or percutaneous epididymal sperm aspiration (PESA) can be applied (Varesi, Vernocchi, Faustini, & Luvoni, 2013), as well as collection from the testis by testicular fine needle aspiration (TEFNA), testicular extraction (TESE) or microsurgical testicular sperm extraction (MicroTESE). However, these techniques, that need trained operators, might cause side effect as focal lesions and they cannot be performed for repeated semen collections. It is worth to mention that spermatozoa can also be retrieved only once from isolated gonads after castration or death, by mincing the tissues in a dish or by squeezing the content of epididymis and vas deferens in amedium drop. In conclusion, proposing an infertility treatment to pet owners is still challenging, especially in small clinics which are not equipped with specialized lab and trained personnel, and the application of advanced second or third level ARTs remains almost exclusively prerogative of those environments that can count on both clinical and research staff and facilities. While estrous monitoring, as well as ovulation control or induction, and artificial insemination can be safely performed with good expectations, thanks to high success rates currently achieved worldwide, other ARTs remain more complex to employ. In cats, IVF and ICSI could be an option to think about in specific cases, whereas in dogs this kind of effort would be useless. Third level ARTs remain experimental, although they deserve further investigations, and they are still not ready to be proposed in the clinical practice. The economic cost of ARTs for pet owners has not been discussed in this abstract, but it could be often alimiting factor for the application of advanced ARTs in dogs and cats. References: Cheng, F., Fazeli, A., Voorhout, W., Marks, A., Bevers, M., & Colenbrander, B. (1996). Use of peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome reaction in stallion spermatozoa. Journal of Andrology, 17(6), 674–682. England, G., & Plummer, J. (1993). Hypo-osmotic swelling of dog spermatozoa. Journal of Reproduction and Fertility - Supplement, 47, 261–270. Hollinshead, F. K. F., & Hanlon, D. W. D. (2017). Factors affecting the reproductive performance of bitches: aprospective cohort study involving 1203 inseminations with fresh and frozen semen. Theriogenology, 101, 62–72. https://doi.org/10.1016/j.theriogenology.2017.06.021 Linde-Forsberg, C., & Forsberg, M. (1989). Fertility in dogs in relation to semen quality and the time and site of insemination with fresh and frozen semen. Journal of Reproduction and Fertility Supplement, 39, 299–310. Nagashima, J. B., Sylvester, S. R., Nelson, J. L., Cheong, S. H., Mukai, C., Lambo, C., … Travis, A. J. (2015). Live births from domestic dog (Canis familiaris) embryos produced by in vitro fertilization. PLoS ONE, 10(12), e0143930. https://doi.org/10.1371/journal.pone.0143930 Pope, C. E., Johnson, C. A., McRae, M. A., Keller, G. L., & Dresser, B. L. (1998). Development of embryos produced by intracytoplasmic sperm injection of cat oocytes. Animal Reproduction Science, 53(1–4), 221–236. https://doi.org/10.1016/S0378-4320(98)00115-8 Ström Holst, B., Larsson, B., Linde-Forsberg, C., & Rodriguez-Martinez, H. (2000). Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes. Journal of Reproduction and Fertility, 119, 77–83. https://doi.org/10.1530/jrf.0.1190077 Varesi, S., Vernocchi, V., Faustini, M., & Luvoni, G. C. (2013). Quality of canine spermatozoa retrieved by percutaneous epididymal sperm aspiration. The Journal of Small Animal Practice, 54(2), 87–91. https://doi.org/10.1111/JSAP.12020 Zambelli, D., Bini, C., & Cunto, M. (2015). Endoscopic transcervical catheterization in the domestic cat. Reproduction in Domestic Animals, 50(1), 13–16. https://doi.org/10.1111/rda.12442 Zambelli, Daniele, & Cunto, M. (2005). Transcervical artificial insemination in the cat. 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