Introduction Reproductive science has critical roles in species conservation and management.1,2 Specifically, advances in understanding species’ reproductive biology facilitate development of repro- ductive technologies that are useful for ensuring genetic and demographic viability of ex situ wildlife populations and assist in development of strategies to control overpopulated species.1 Of the 37 Canidae family (includes domestic dog [Canis familiaris]) species, 5 are listed as ‘endangered’ or ‘critically endangered’ by the International Union of Conservation of Nature. Therefore, development of assisted reproductive technologies (ARTs) would certainly be useful for conservation and management of these threatened canids. Currently, existing knowledge on canid reproductive biology is mostly gleaned from domestic dog studies.3-7 It is well reco- gnized that reproductive biology of female canids is unique compared to other mammalian species. Specifically, female reproductive cycle is characterized by an extended proestrus followed by protracted estrus with each period lasting on average of 1 week.8 Estrous period is characterized by an estrogen peak that coincides with rising progesterone concentrations before ovulation.8 Estrus is followed by diestrus, a luteal phase avera- ging 2 months in duration irrespective of pregnancy. Diestrus is succeeded by anestrus, an extended interval of ovarian quies- cence.8 Whereas domestic dogs exhibit nonseasonal monoestrus once or twice a year,8 most wild canids breed seasonally. The onset of breeding season in wild canids varies among species Current and future perspectives of reproductive technologies in domestic and wild canids Nucharin Songsasen Center for Species Survival, Smithsonian Conservation Biology Institute National Zoological Park, Front Royal, Virginia Abstract Due to their unique reproduction, reproductive technology advances in canids have lagged behind compared to other mammalian species. Currently, semen cryopreservation and artificial insemination have been widely used in domestic dog. However, artificial insemination in wild canids is still limited due largely to poor semen quality, high susceptibility of sperm to cryopreservation procedures, and inability to noninvasively predict ovulation and timed insemination. For female canids, inability to consistently mature oocytes in vitro has limited the use of embryo technology in these species. However, birth of the first domestic dog puppies produced via in vitro fertilization has reinvigorated research interests in this area. This presentation will summarize the status of assisted reproductive technologies in domestic and wild canids and will discuss new, exciting research in fertility preservation and application of reproductive technologies in wild canid conservation. Keywords: Canids, artificial insemination, in vitro maturation and fertilization, in vitro folliculogenesis, somatic cell nuclear transfer and is dependent on environmental factors, such as latitudes and rainfalls.9,10 Furthermore, although most canids are spon- taneous ovulators, there has been evidence of induced estrus or ovulation in the Island fox (Urocyon littoralis), maned wolf (Chrysocyon brachyurus),11,12 and seasonal polyestrus in dholes (Cuon alpinus) and bush dog (Speothos venaticus), indicating diversity in reproductive mechanisms within Canidae family. Canid oocyte is also unique compared to that of other mamma- lian species. Specifically, domestic dog ovaries contain a higher proportion (7 - 11%) of polyovular follicles than those (4%) of the domestic cat.13,14 Although polyovular follicles release multiple oocytes, there is evidence that only 1 gamete is capable of undergoing maturation and fertilization.14 Another unique feature of canid oocytes is that they contain large amount of cytoplasmic lipids compared to other mammalian species, inclu- ding cat and pig.15 To date, the extent to which cytoplasmic lipids have roles in oocyte development is unknown. Nevertheless, it has been suggested that the challenges in applying conven- tional in vitro oocyte maturation systems to the dog is partly associated with the unusually large amount of cytoplasmic lipid in this species.16 Finally, the most striking feature of dog gamete biology is that the oocyte ovulates in an immature stage requiring up to 48 - 72 hours to complete nuclear maturation within the oviduct,6,15,16 and this characteristic undoubtedly contributes to challenges in developing in vitro maturation (IVM) system for dog oocytes. Clinical Theriogenology 2021; 13: 138 Due to inability to reliably mature dog oocytes in vitro, ARTs that require the use of in vitro matured gametes have lagged behind compared to other species. Nevertheless, a handful of live offspring have been produced from in vitro derived embryos produced from in vivo matured oocytes via in vitro fertilization (IVF) or somatic nuclear transfer (SCNT),17-19 and thousands of pups were born from AI with fresh and frozen-thawed sperm.20,21 This review will summarize the status of reproductive technologies in domestic and wild canids and will discuss exciting research in fertility preservation and the application of reproductive technologies in wild canid conservation. Semen collection and artificial insemination Apart from domestic dog and farmed fox species, semen collection is normally performed by electroejaculation (EEJ) in wild canids.22-27 Maned wolf,28 gray wolf (Canis lupus),29,30 and crab-eating fox (Cerdocyon thous)31 ejaculates also have been obtained using digital stimulation, although this is not a routine method. The limitation of digital stimulation technique is that it requires preconditioning of animals to physical restraint, and thereby, relies on the availability of animal trainers. However, this method does not require anesthesia and can be performed more frequently than EEJ (multiple collections per week versus once or twice during breeding season).28 Recently, urethral catherization (after medetomidine treatment) developed for felids32,33 has been successfully applied to the domestic dog34 and red wolf (Canis rufus).27 Like EEJ, this method requires anesthesia; however, it does not require specialized equipment, and therefore, can be applied to individuals living in situ or under conditions where EEJ is not feasible. Semen characteristics of domestic and wild canid ejaculates collected using various techniques are summarized (Table). A major challenge in semen collection in canids, especially via EEJ and urethral catheterization is urine contamination.24,27,34 The prevalence of urine contamination varies among species, collection method and time of the year. Specifically, this author has observed urine contamination as a common feature in the maned wolf, regardless of collection methods. For red wolf, urethral catheterization often results in urine contamination compared to EEJ.27 As a result, it is recommended that urethral catheterization semen collection method should not be used in red wolf for cryopreservation of sperm.27 Urine alters osmolarity and pH of semen samples that, in turn, increases the proportions of sperm with bent and coiled tail, decreases motility,27,35 and increases the susceptibility of sperm to osmotic stress. For the African wild dog (Lycaon pictus), urine contamination in semen samples collected via EEJ occurred more often when samples were collected from subordinate males during prebreeding season Species Volume (ml) Concentration (x 106 sperm/ml) Motility (%) Morphologically normal sperm (%) Citations Digital manipulation Domestic dog Maned wolfa Breeding season Non-breeding season Gray wolfb Blue foxb Crab-eating foxa 1-30 1.3 ± 1.2 0.4 ± 0.6 1.7 ± 0.2 0.39 ± 0.26 0.39 ±0.18 300 - 1000 73.9 ± 87.2* 6.1 ± 4.9* 290.8 ± 53.5 491.8 ± 594.4 463.7 ±84.3 70 76.1 ± 23.9 80 ±14.5 91.7 ± 1.5 N/A 86.0 ±16.9 70 36.5 ± 24.0 20.8 ±19.8 N/A 89.9 ± 4.4 2.0 ±1.0 36 28 30 37 31 Electroejaculation Domestic dog Coyoteb Red wolfa Red wolfb African wild dogb African wild dogb Breeding season Non-breeding season Maned wolfb 1.8 1.67 ± 0.4 6.15 ± 5.6 4.7 ± 0.7 0.6 ± 0.1 NA NA 2.0 ± 0.6 129.6 549.2 ± 297.7 96.7 ± 178.7 146.5 ± 25.7 212.3 ± 87.3 32.3 ± 9.2 27.4 ± 11.5 43.4 ± 18.2 30.1 90.4 ± 4.5 80.8 ±16.9 71.2 69.5 ± 3.3 47.4 ± 6.7 17.3 ± 10.2 59.8 ± 4.9 N/A 78.0 ± 13.5 46.5 ±14.1 73.6 ± 3.2 76.2 ±6.2 50.9 ± 5.2 40.6 ± 9.8 28.1 ± 4.4 38 26 27 39 25 24 22 Urethral catheterization Domestic dogb Red wolfa 0.09 ± 0.03 0.36 ± 0.08 1,186.67 ± 304.66 50.4 ± 23.5 58.3 ± 8.7 ~40% 53.2 ± 5.6 NA 34 27 Table. Seminal traits of domestic dog and wild canids *Total sperm per ejaculate aMean ± standard deviation bMean ± standard error of mean Clinical Theriogenology 2021; 13: 139 than breeding season.24 To circumvent this issue, a common practice for semen collection in wild canids is to remove urine and flush the urinary bladder with saline prior to EEJ. To date, AI with fresh, chilled and frozen-thawed sperm has been widely used in the domestic dog21 and farmed foxes.40 Offspring were produced from AI with fresh or frozen-thawed sperm in gray wolves,41,42 Mexican gray wolves,40 and red wolves.39 However, AI has not been routinely applied to genetic management of threatened and endangered canids, likely due to the lack of knowledge on species’ reproductive biology, the challenges in predicting ovulation onset and effectively manipulating female reproduction as well as poor seminal quality (Table 1).40 Therefore, future research should focus on developing a noninvasive method to predict ovulation in endangered canids. For example, the ability to assess luteinizing hormone in urine samples would be extremely useful for ovulation prediction and timed insemination when frequent blood sampling is not feasible. Sperm cryopreservation First records of live birth after AI with cryopreserved sperm were reported in 196943 for domestic dog and in 1975 for gray wolf.42 Since these first successes, canid sperm cryopreservation has been widely studied in both domestic dog21,44-46 and wild canids, including gray wolf,47,48 red wolf,27,39,49 maned wolf,22 African wild dog,25 red fox,50 and blue fox.50 Glycerol has been generally used as a cryoprotectant for canid sperm.21,25,27 However, in 1 study, dimethyl sulfoxide was superior to glycerol for cryopreservation of maned wolf sperm.22 Interestingly, dimethyl sulfoxide was toxic for dog sperm,51 indicating species differences in the response to cryoprotective additives. Like pig52,53 and horse,54 there appears to be male-to-male variations in the susceptibility of sperm to cryopreservation that is independent of the quality of fresh semen and normal fertility at natural mating.21,46,55 Whelping rate and litter size obtained from frozen-thawed dog sperm are about 23 - 30% less than fresh sample.21 Nevertheless, AI with frozen-thawed sperm has been applied in domestic dog breeding and has resulted in thousands of puppies.21 Unlike domestic dog, studies in wild canids, including African wild dog25 and red wolf have demonstrated precipitous decreases in motility and viability of frozen-thawed sperm following incubation despite acceptable viability immediately after postthaw.49 Our laboratory recently evaluated the effects of extracellular vesicles (EVs) from domestic dog oviducts on postthaw survival of red wolf sperm. EVs contain proteins, RNA, and DNA messages that deliver to neighboring cells, that in turn regulate recipient cells’ function.56 We reported that thawing red wolf sperm in medium containing dog oviductal EVs supports sperm motility and acrosomal membrane integrity after 2 hours of incubation compared to nonEVs control.57 Proteomic analysis also revealed that dog oviductal EVs contain several proteins that influence mitochondria function, plasma and acro- somal membrane integrity, and stress responses,57 indicating the useful potential of oviductal EVs in improving posthaw survival and longevity of wild canid sperm. In vitro oocyte maturation and fertilization In vitro oocyte maturation (IVM) Due to their unique reproductive and gamete biology, develop- ment of IVM systems for canids has been far from successful.16 Investigations have included impacts of stage of reproductive cycle, culture medium and protein, and hormone (gonadotropins and gonadal steroids) and growth factor supplementation (see review16). Nevertheless, on average, only 15 - 20% of cultured oocytes achieve the metaphase II (MII) stage after 48 - 72 hours of in vitro culture.16 Supplementing the culture medium with 10 mM caffeine during the first 24 of 72 hours of incubation increased maturation rates compared to unsupplemented controls (42.2 versus 25.5%) with small proportion (4%) of gametes from the former treatment developing to morula stage after IVF.58 Interestingly, incubating dog oocytes with caffeine during 24 - 48 or 72 hours of culture did not improve maturation rate. The beneficial effect of caffeine on dog IVM is likely due to activation of maturation-promoting factor and mitogen-activated protein kinase (MAPK),59 2 kinases with critical roles in chromatin reconfiguration during oocyte maturation.60 Supplementation of insulin like growth factor-1,61 growth differentiation factor-9 and bone morphogenetic protein- 1562 also enhanced nuclear maturation of dog oocytes compared to unsupplemented control. Yet, overall MII rates in those studies were still < 20%. During the past several years, interest in examining the roles of reproductive EVs in regulating gamete function has substantially increased.56 Oviductal exosomes (the smallest EVs) stimulated cumulus cell proliferation by activating epidermal growth factor receptor (EGFR)/MAPK signaling pathway.63 This indicates the potential role of oviductal EVs in regulating dog oocyte maturation, as cumulus cells are known to induce meiotic resumption and support cytoplasmic maturation in several mammalian species.64 Dog’s oviductal and cumulus cells recovered during estrus had higher levels of MAPK1 than cells recovered during anestrus and diestrus.65 Yet, coincubation of dog oocytes with oviductal cells from estrus resulted only in 10% of the cultured gametes develo- ping to MII.65 Coincubation of estrous oocytes with oviductal cells significantly improved MII rate after 72 hours IVM compared to controls (47 versus 11%).66 Furthermore, in vitro matured estrous oocytes developed to 8-cell stages (66%) following parthenoge- netic activation and in vitro culture at a rate comparable (85%) to in vivo matured gametes.66 Despite the discrepancy in the above studies, findings to date are encouraging and emphasize the need to further explore the roles of reproductive EVs (from follicle fluid and/or oviduct) in development and maturation of dog oocytes. Most IVM studies recover dog oocytes from tissues obtained during routine ovariohysterectomy. Follicle size significantly influenced developmental competence of the dog oocyte.67 Specifically, ~ 80% of oocytes from follicles > 2 mm in diameter complete nuclear maturation in vitro compared to only 16 to 38% of those from smaller (0.5 - < 2 mm) source follicles.67 Because > 2 mm diameter follicles only appear during proestrus and estrus, it is hypothesized Clinical Theriogenology 2021; 13: 140 that the overall low IVM success in canids is likely due to that the oocytes from smaller follicles have not fully acquired develop- mental competence and therefore, are not able to mature under culture conditions developed for fully grown gametes.67 Exposure of dog oocytes for 48 hours to meiotic inhibitor compounds (e.g., roscovitine and butyrolactone) inhibited meiotic resumption of dog oocytes in vitro.68 Such oocytes were able to resume meiosis at a higher rate than those not been exposed to meiotic inhibitor compounds.68 Therefore, it may be useful to explore the influence of short-term inhibition of meiotic resumption followed by coincu- bation with oviductal cells or EVs on dog oocyte development. Such studies may provide insights in mechanisms regulating gamete maturation, information that is critical for the development of an effective IVM system for this species. In vitro fertilization The initial report of IVF using in vitro matured dog oocytes was published more than 40 years69ago, although embryonic deve- lopment was not reported. Since then, several investigators have attempted to perform IVF of incubated oocytes, albeit with limited success.16,58,70-77 Thus far, there is only a single report demonstrating the production of 1 blastocyst from IVF from in vitro matured oocytes,72 and 1 non-term pregnancy after transferring in vitro derived presumptive zygotes into recipient females.73 In vitro maturation and fertilization have also been conducted in silver fox; however, embryonic development was not reported.78 To date, there have been a few studies on dog and fox IVF using in vivo matured gametes. Only 1 oocyte reached morula (144 hours after IVF) when blue fox oocytes (n = 36) were inseminated with frozen-thawed sperm.79 Approximately, 12% of oocytes collected 4 days after ovulation developed to 2-cell stage post IVF; 5 embryos developed further but arrested at the 4-cell stage. Only recently was the first litter of pups produced from cryopreserved, in vitro derived embryos.17 Two factors contributing to that success included supplementation of magnesium to the sperm capacitation medium and use of day 6 (post LH surge) oocyte for IVF.17,80 Interestingly, the presence of progesterone during IVF did not impact fertilization and embryonic development.17 Oocyte and embryo cryopreservation Large amounts of intracellular lipid within the canid oocyte presents an additional challenge in developing ARTs. To date, a handful of studies have been conducted on canid oocyte cryopreservation.81-84 Due to the lack of effective IVM systems to assess developmental competence of cryopreserved gametes, morphological assessment or vital staining have been used to determine cryopreservation success in most studies. By using the open-pull straw technique, the percentage of vitrified-warmed dog oocytes completing nuclear maturation was similar to that of fresh control, although more cryopreserved gametes were arrested at the GV stage than those of fresh counterparts.83 Approximately, 90% of blue fox oocytes vitrified using the two-step open-pulled straw method exhibited normal morphology post-warming and 11% of these gametes developed to the MII stage, comparable to the fresh control.84 Finally, 60% of dog81 and Mexican gray wolf oocytes82 maintained viability (based on vital staining) after vitrification using the cryotop technique. Successful embryo cryopreservation in canids either by vitrification85 or slow freezing method86 has been reported in the domestic dog, demonstrating a stage-dependency in the susceptibility to cryopre- servation. Specifically, blastocysts are more sensitive to vitrification than those at the earlier stages of development (1-cell to morula stages).85 Although dog blastocysts cryopreserved using the slow freezing method were able to re-expand during in vitro culture, transferring these embryos did not result in offspring production.86 To date, live births have been produced from embryos that had been frozen during cleavage stages (2 - 16 cells). For example, transfer of 77 vitrified-warmed 4 - 16-cell dog embryos resulted in the birth of 7 live offspring (9.1%).85 Furthermore, 16% birth rate were achieved after transferring 6 embryos vitrified using a closed vitrification system (Vit kit).87 This same vitrification method has been used to cryopreserve in vitro derived embryos resulting in live birth.17 In vitro folliculogenesis Each ovary contains thousands of immature follicles enclosing oocytes that are never ovulated and thus never contribute to reproduction. The ability to activate and grow immature follicles to a mature stage producing a competent oocyte would help preserve genetically valuable dog models of human diseases and endangered canids.88 During the past decade, advances have been made in development of in vitro culture system for dog ovarian tissue and isolated follicles.89-96 Compared to domestic cat, dog ovarian tissues are highly susceptible to in vitro culture, likely due to the highly rigid cortex that limits nutrient supply to the enclosed follicles.97 Studies have examined effects of growth factors, inclu- ding epidermal growth factors and vascular endothelial growth factors,92,98as well as an anti-apoptotic agent, Z-VAD-FMK99 on the activation and survival of enclosed follicles, with varying results. The current in vitro culture protocol can maintain the viability of enclosed follicles for only 7 days.92 Nevertheless, the culture system developed for dog has been applied to the maned wolf with comparable results. These findings underscore the impor- tance of the dog model for developing ARTs for threatened and endangered canids. Follicle stimulating hormone (FSH) is essential for in vitro growth of isolated dog follicles;90,93although they increase in size and produce steroids in the presence of FSH, gonadotropin supplementation does not support the growth and survival of the resident gamete.90 This was attributed to disruption of the communication between the oocyte and the surrounding granulosa cells.90 Our laboratory has demonstrated that supplementing culture medium with 100 ng/ml activin promotes dog follicle growth and antral cavity expan- sion and supports oocyte’s chromatin integrity by maintaining the transzonal projection for 12 days.100 Most recently, it was reported that supplementing culture media with 2 cAMP modulators, cilos- tamide and forskolin can sustain viability of cultured oocytes by promoting cAMP production and gap junction activity.101 Because Clinical Theriogenology 2021; 13: 141 the communication between the oocyte and surrounding granulosa cells is critical for folliculogenesis and oogenesis, future work on in vitro culture of isolated follicles should focus on developing approaches that support the maintenance of the cell-cell commu- nication during long-term incubation. Somatic cell nuclear transfer and transgenesis Since the first report on live birth in 2005, there have been several studies on SCNT in domestic dogs.19,102,103 The protocol developed for the dog has also been applied to gray wolves104,105 and coyotes,106 resulting in production of live offspring. Due to the inability to in vitro mature dog oocytes, studies to date have utilized in vivo matured gametes as the recipient cells for a variety of donor cell types, including fetal and adult fibroblasts as well as adipose derived mesenchymal stem cells.103,107 Despite the low success rate (<5% live birth from numbers of transferred embryos), cloned individuals appear to have normal health and reproductive competence.103 Specifically, health and reproductive assessment of cloned indi- viduals (n = 3 dogs) revealed that age-related hematological and serum biochemical parameters as well as circulating hormone concentrations of cloned dogs are similar to non-cloned counter- parts.103 Furthermore, cloned dog ovaries exhibit morphological changes in the same manner as non-cloned individuals. Finally, live puppies have been produced after AI of cloned females with fresh semen from a cloned male dog.108 The successful production of SCNT dog embryos has facilitated the application of transgenesis technology in this species.109-111 The first transgenic dog that expressed a red fluorescent protein gene was produced in 2009.18 Since then, there have been several reports on the production of transgenic offspring as models for studying human disorders, including type 2 diabetes112 and Alzheimer’s disease.113 Summary and future perspectives Due to their unique female reproductive characteristics, the development of ARTs in the domestic dog and their wild cousins has proven to be extremely challenging. Nevertheless, substantial progress has been made during the past decade, including the first production of IVF puppies and numerous offspring from SCNT and transgenesis. Recent studies on the influence of reproductive EVs on the function and cryosurvival of dog and wild canid gametes, and utilization of organ-on-a-chip technology in growing dog ovarian cortices and isolated follicles in vitro114 have provided encouraging results. Such studies will likely provide insights into mechanisms regulating gamete formation and function and generate information useful for development of effective tools to preserve/extend fertility of domestic and wild canids. So far, the domestic dog has served as a valuable model for establishing reproductive technologies in wild canids and dog protocols have been successfully applied to their endangered cousins. Yet, there are still needs for species-specific research due to the enormous diversity in reproductive biology within the family Canidae.10 Conflict of interest There are no conflicts of interest to declare. References 1. IUCN. The IUCN Red List of Threatened Species. Version 2020-3. https://ww.iucnredlist.org 2021.ž 2Wildt DE, Wemmer C: Sex and wildlife: the role of reproductive science in conservation. Biodiversity and Conserv 1999;8:965-976. 2. Wildt DE, Ellis S, Howard JG: Linkage of reproductive sciences: from «quick fix» to «integrated» conservation. J Reprod Fertil (Suppl 57 ) 2001;57:295-307. 3. Concannon PW, Castracane VD, Temple M, et al: Endocrine control of ovarian function in dogs and other carnivores. Anim Reprod 1999;6:172-193. 4. Concannon PW: Research challenges in endocrine aspects of canine ovarian cycles. Reprod Domest Anim 2012;6:6-12. 5. Songsasen N, Nagashima J, Thongkittidilok C: Endocrine and paracrine controls of canine follicular development and function. Reprod Domest Anim 2017;2:29-34. 6. Reynaud K, Fontbonne A, Marseloo N, et al: In vivo meiotic resump- tion, fertilization and early embryonic development in the bitch. Reproduction 2005;130:193-201. 7. Reynaud K, Fontbonne A, Saint-Dizier M, et al: Folliculogenesis, ovulation and endocrine control of oocytes and embryos in the dog. Reprod Domest Anim 2012;6,:66-69. 8. Concannon P: Endocrinolgic control of normal canine ovarian function. Reprod Domest Anim 2009;44 (Suppl 2):3-15. 9. Asa C, Valdespino C: Canid reproductive biology: an integration of proximate mechanisms and ultimate causes. Am Zool 1998;38:251-259. 10. Jewgenow K, Songsasen N: Reproduction and advances in repro- ductive studies in carnivores. Adv Exp Med Biol 2014;753:205-239. 11. Jones MK, Reiter LE, Gilmore MP, et al: Physiological impacts of housing maned wolves (Chrysocyon brachyurus) with female relatives or unrelated males. Gen Comp Endocrinol 2018;267:109-115. 12. Johnson AE, Freeman EW, Colgin M, et al: Induction of ovarian acti- vity and ovulation in an induced ovulator, the maned wolf (Chrysocyon brachyurus), using GnRH agonist and recombinant LH. Theriogenology 2014;82:71-79. 13. Telfer E, Gosden RG: A quantitative cytological study of polyovular follicles in mammalian ovaries with particular reference to the domestic bitch (Canis familiaris). J Reprod Fertil 1987;81:137-147. 14. Reynaud K, Viaris de Lesegno C, Chebrout M, et al: Follicle popu- lation, cumulus mucification, and oocyte chromatin configuration during the periovulatory period in the female dog. Theriogenology 2009;72:1120-1131. 15. Chastant-Maillard S, de Lesegno CV, Chebrout M, et al: The canine oocyte: uncommon features of in vivo and in vitro maturation. Reprod Fertil Dev 2011;23:391-402. 16. Songsasen N, Wildt DE: Oocyte biology and challenges in develo- ping in vitro maturation systems in the domestic dog. Anim Reprod Sci 2007;98:2-22. 17. Nagashima JB, Sylvester SR, Nelson JL, et al: Live births from domestic dog (Canis familiaris) embryos produced by in vitro fertilization. PLoS One 2015;10:e0143930. Clinical Theriogenology 2021; 13: 142 18. Hong SG, Kim MK, Jang G, et al: Generation of red fluorescent protein transgenic dogs. Genesis 2009;47:314-322. 19. Lee BC, Kim MK, Jang G, et al: Dogs cloned from adult somatic cells. Nature 2005;436: 641. 20. Hollinshead F, Hanlon D: Normal progesterone profiles during estrus in the bitch: A prospective analysis of 1420 estrous cycles. Theriogenology 2019;125:37-42. 21. Linde Forsberg C: Biology of reproduction of the dog and modern reproductive technology. In: Ruvinsky A, Sampson J: editors. The Genetics of the Dog. CABI ebook: 2001. p. 401-430. 22. Johnson AE, Freeman EW, Wildt DE, et al: Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensi- tivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide. Cryobiology 2014;68:361-370. 23. Asa C, Miller P, Agnew M, et al: Relationship of inbreeding with sperm quality and reproductive success in Mexican gray wolves. Anim Conserv 2007;10:326-331. 24. Van den Berghe F, Paris MCJ, Sarnyai Z, et al: Social rank does not affect sperm quality in male African wild dogs (Lycaon pictus). Reprod Fertil Dev 2019;31:875-887. 25. Johnston SD, Ward D, Lemon J, et al: Studies of male reproduc- tion in captive African wild dogs (Lycaon pictus). Anim Reprod Sci 2007;100:338-355. 26. Minter LJ, DeLiberto TJ: Seasonal variation in serum testosterone, testicular volume, and semen characteristics in the coyote (Canis latrans). Theriogenology 2008;69:946-952. 27. Franklin AD, Waddell WT, Goodrowe KL: Red wolf (Canis rufus) sperm quality and quantity is affected by semen collection method, extender components, and post-thaw holding temperature. Theriogenology 2018;116:41-48. 28. Teodoro LO, Melo-Junior AA, Spercoski KM et al: Seasonal aspects of reproductive physiology in captive male maned wolves (Chrysocyon brachyurus, Illiger 1815). Reprod Domest Anim 2012;47 (Suppl):250-255. 29. Mech LD, Christensen BW, Asa CS, et al: Production of hybrids between western gray wolves and western coyotes. PLoS One 2014;9:e88861. 30. Mitsuzuka M: Collection, evaluation and freezing of wolf semen. In: Frank H. editor. Man and Wolf: Advances, Issues, and Problems in Captive Wolf Research 1st edition. Dordrecht; Dr. W. Junk Publishers: 1987. p. 127-141. 31. Carvalho JC, Silva FED, Rizzoto G, et al: Semen collection, sperm characteristics and ultrasonographic features of reproductive tissues in crab-eating fox (Cerdocyon thous). Theriogenology 2020;155:60-69. 32. Swanson WF, Bateman HL ,Vansandt LM: Urethral catheterization and sperm vitrification for simplified semen banking in felids. Reprod Domest Anim 2017;52 (Suppl 2):255-260. 33. Araujo GR, Paula TAR, Deco-Souza T, et al: Comparison of semen samples collected from wild and captive jaguars (Panthera onca) by urethral catheterization after pharmacological induction. Anim Reprod Sci 2018;195:1-7. 34. Kuczmarski AH, Alves de Barros M, Souza de Lima LF, et al: Urethral catheterization after pharmacological induction for semen collection in dog. Theriogenology 2020;153: 34-38. 35. Platz CC, Waddell W, Smith R, et al: Semen collection in wild canidae using electroejaculation. 1st International Symposium on Assisted Reproductive Technology for Conservation and Genetic Management of Wildlife. 2001 p. 134-135. 36. Johnston SD: Performing a complete canine semen evaluation in a small animal hospital. Vet Clin North Am Small Anim Pract 1991;21:545-551. 37. Stasiak K, Janicki B, Kupcewicz B: Biological parameters of polar fox (Alopex lagopus L.) semen during the breeding season. Turk J Vet Anim Sci 2008;34:327-331. 38. Ohl DA, Denil J, Cummins C, et al: Electroejaculation does not impair sperm motility in the beagle dog: a comparative study of electroejacu- lation and collection by artificial vagina. J Urol 1994;152:1034-1037. 39. Goodrowe KL, Hay MA, Platz CC, et al: Characteristics of fresh and frozen-thawed red wolf (Canis rufus) spermatozoa. Anim Reprod Sci 1998;53:299-308. 40. Thomassen R, Farstad W: Artificial insemination in canids: a useful tool in breeding and conservation. Theriogenology 2009;71:190-199. 41. Asa CS, Bauman K, Callahan P, et al. GnRH-agonist induction of fertile estrus with either natural mating or artificial insemination, followed by birth of pups in gray wolves (Canis lupus). Theriogenology 2006;66:1778-1782. 42. Seager SWJ, Platz CC, Hodge W: Successful pregnancy using frozen semen in the wolf. Int Zoo Yearb 1975;15:140-143. 43. Seager SWJ. Successful pregnancies utilizing frozen dog semen. AI Digest 1969; 17, 6-7. 44. Farstad W: Semen cryopreservation in dogs and foxes. Anim Reprod Sci 1996;42:251-260. 45. Farstad W: Customizing semen preservation protocols for indi- vidual dogs and individual species: Sperm preservation beyond the state of the art. 7th International Symposium on Canine and Feline Reproduction 2012. 46. Farstad W: Cryopreservation of canine semen - New challenges. Reprod Domest Ani 2009;44 (Suppl 2):336-341. 47. Zindl C: Cryopreservation of Mexican Gray Wolf (Canis lupus baileyi) Semen – Evaluation of Different Times and Rates of Pre-freeze Cooling and Equex pasta® Supplementation – in Comparition with Semen of the Domestic Dog and Generic Gray Wolf (Canis lupus). Dr. med.vet. dissertation Hannover, DVG Service 2006. 194 p. 48. Zindl C, Asa CS, Gunzel-Apel A-R: Influence of cooling rates and addition of Equex pasta on cooled and frozen-thawed semen of generic gray (Canis lupus) and Mexican gray wolves (C. l. Baileyi). Theriogenology 2006;66:1797-1802. 49. Goodrowe KL, Mastromonaco GF, Walker SL, et al: In vitro maintenance, cooling and cryopreservation of red wolf (Canis rufus) spermatozoa. J Reprod Fertil (Suppl) 2001;57:387-392. 50. Farstad W. Reproduction in foxes: current research and future challenges. Ani Reprod Sci 1998;53:35-42. 51. Songsasen N, Yu I, Murton S, et al: Osmotic sensitivity of canine spermatozoa. Cryobiology 2002;44:79-90. 52. Wasilewska K, Fraser L: Boar variability in sperm cryo-tolerance after cooling of semen in different long-term extenders at various temperatures. Anim Reprod Sci 2017;185:161-173. 53. Yeste M: Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenology 2016;85:47-64. 54. Loomis PR, Graham JK: Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 2008;105:119-128. 55. England GC: Cryopreservation of dog semen: a review. J Reprod Clinical Theriogenology 2021; 13: 143 Fertil Suppl 1993;47:243-255. 56. Machtinger R, Laurent LC, Baccarelli AA: Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. Hum Reprod Update 2016;22:182-193. 57. de Almeida Monteiro Melo Ferraz M, Nagashima JB, Noonan MJ, et al: Oviductal extracellular vesicles improve post-thaw sperm function in red wolves and cheetahs. Inter J Mol Sci 2020;21:3733. 58. Fathi M, Salama A, Badr MR: Improvement of the developmental competence of canine oocyte using caffeine supplementation during IVM at different maturation time. Zygote 2018;26:162-167. 59. Lee JH, Campbell KH: Effects of enucleation and caffeine on maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities in ovine oocytes used as recipient cytoplasts for nuclear transfer. Biol Reprod 2006;74:691-698. 60. Kotani T, Yamshita M. Discrimination of the roles of MPF and MAP kinase in morphological changes that oocur during oocyte maturation. Dev Biol 2002;252:271-286. 61. Sato A, Sarentonglaga B, Ogata K, et al: Effects of insulin-like growth factor-1 on the in vitro maturation of canine oocytes. J Reprod Dev 2018;64:83-88. 62. Garcia P, Aspee K, Ramirez G, et al: Influence of growth differentiation factor 9 and bone morphogenetic protein 15 on in vitro maturation of canine oocytes. Reprod Domest Anim 2019;54:373-380. 63. Lee SH, Oh HJ, Kim MJ, et al: Exosomes derived from oviduct cells mediate the EGFR/MAPK signaling pathway in cumulus cells. J Cell Physiol 2020;235:1386-1404. 64. Tanghe S, Van Soom A, Nauwynck H, et al: Minireview: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol Reprod Dev 2002;61:414-424. 65. Lee SH, Oh HJ, Kim MJ, et al: Oocyte maturation-related gene expression in the canine oviduct, cumulus cells, and oocytes and effect of co-culture with oviduct cells on in vitro maturation of oocytes. J Assist Reprod Gen 2017;34:929-938. 66. No J, Zhao M, Lee S, et al: Enhanced in vitro maturation of canine oocytes by oviduct epithelial cell co-culture. Theriogenology 2018;105:66-74. 67. Songsasen N, Wildt DE: Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocyte. Mol Reprod Dev 2005;72:113-119. 68. Hanna C, Menges S, Kraemer D, et al: Synchronisation of canine germinal vesicle stage oocytes prior to in vitro maturation alters the kinetics of nuclear progression during subsequent resumption of meiosis. Reprod Fertil Dev 2008;20:606-614. 69. Mahi CA,Yanagimachi R: Maturation and sperm penetration of canine ovarian oocytes in vitro. J Exp Zool 1976;196:189-195. 70. Luvoni GC, Chigioni S, Allievi E, et al: Factors involved in vivo and in vitro maturation of canine oocytes. Theriogenology 2005;63:41-59. 71. Yamada S, Shimazu Y, Kawaji H, et al: Maturation, fertilization, and development of dog oocytes in vitro. Biol Reprod 1992;46:853-858. 72. Otoi T, Murakami M, Fujii M, et al: Development of canine oocytes matured and fertilised in vitro. Vet Rec 2000;146:52-53. 73. England GWW, Verstegen JP, Hewitt DA: Pregnancy following in vitro fertilisation of canine oocytes. Vet Rec 2001;148:20-22. 74. Rodrigues BA, Dos Santos LC, Rodrigues JL: Embryonic development of in vitro matured and in vitro fertilized dog oocytes. Mol Reprod Dev 2004;67:215-223. 75. Hatoya S, Sugiyama Y, Torii R, et al: Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes. Theriogenology 2006;66:1083-1090. 76. Rodrigues BA, dos Santos LC, Rodrigues JL. Effect of maturation medium on in vitro cleavage of canine oocytes fertilized with fresh and cooled homologous semen. Zygote 2007;15:43-53. 77. Songsasen N, Yu I, Leibo SP: Nuclear maturation of canine oocytes cultured in protein-free media. Mol Reprod Dev 2002;62:407-415. 78. Feng HL, Liu JM, Wen XH, et al: Follicular oocyte maturation and sperm penetration in vitro in the silver fox (Vulpes vulpes). Anim Reprod Sci 1994;36:163-170. 79. Farstad W, Hyttel P, Grondahl C, et al: Fertilization in vitro of oocytes matured in vivo in the blue fox (Alopex lagopus). J Reprod Fertil (Suppl) 1993;47:219-226. 80. Nagashima JB, Travis AJ, Songsasen N: The domestic dog embryo: in Vitro fertilization, culture, and transfer. In Herrick JR: editor. Comparative Embryo Culture: Methods and Protocols. New York, Springer; New York: 2019. p. 247-267. 81. Abe Y, Lee D-S, Kim S-K, et al: Vitrification of canine oocytes. J Mamm Ova Res 2008;28:32-36. 82. Boutelle S, Lenahan K, Krisher R, et al: Vitrification of oocytes from endangered Mexican gray wolves (Canis lupus baileyi). Theriogenology 2011;75:647-654. 83. Turatham B, Saikhun K, Sangsuwan P, et al: Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes. Reprod Biol Endocrinol 2010;8:70. 84. Zhou G-B, Ma C-B, Liu G-S, et al: Vitrification of farmed blue fox oocytes in ethylene glycol and DMSO-based solutions using open-pulled straw (OPS). CryoLetters 2009;30: 112-118. 85. Abe Y, Suwa Y, Asano, T. et al: Cryopreservation of canine embryos. Biol Reprod 2011;84:363-368. 86. Guaitolini CRF, Taffarel MO, Teixeira NS, et al: Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing. Theriogenology 2012;78:576-582. 87. Nagashima J, Kim YH, Hollinshead F, et al: IVF in the dog: recent advancements and future directions. Clin Theriogenology 2017;9:261-266. 88. Comizzoli P, Songsasen N, Wildt DE: Protecting and extending fertility options for females of wild and endangered species. Cancer Treat Res 2010;156:87-100. 89. Fujihara M, Comizzoli P, Wildt DE, et al: Cat and dog primordial follicles enclosed in ovarian cortex sustain viability after in vitro culture on agarose gel in a protein-free medium. Reprod Domest Anim 2012;47:102-108. 90. Nagashima J, Wildt DE, Travis AJ, et al: Follicular size and stage and gonadotropin concentration affect alginate-encapsulated in vitro growth and survival of pre- and early antral dog follicles. Reprod Fertil Dev 2015;29:262-273. 91. Songsasen N, Woodruff TK ,Wildt DE: In vitro growth and steroidoge- nesis of dog follicles as influenced by the physical and hormonal microenvironment. Reproduction 2011;142:113-122. 92. Thongkittidilok C, Wildt DE, Songsasen N: Responsiveness of intraovarian dog follicles in vitro to epidermal growth factor and vascular endothelial growth factor depends on ovarian donor age. Reprod Domest Anim 2017;52:114-122. 93. Serafim MK, Araujo VR, Silva GM, et al: Canine preantral follicles cultured with various concentrations of follicle-stimulating hormone Clinical Theriogenology 2021; 13: 144 (FSH). Theriogenology 2010;74:749-755. 94. Serafim MK, Duarte AB, Silva GM, et al: Impact of growth hormone (GH) and follicle stimulating hormone (FSH) on in vitro canine prean- tral follicle development and estradiol production. Growth Horm IGF Res 2015;25:85-89. 95. Serafim MK, Silva GM, Duarte AB, et al: High insulin concentrations promote the in vitro growth and viability of canine preantral follicles. Reprod Fertil Dev 2013;25:927-934. 96. Pereira LMC, Lopes MD, Songsasen N: Effect of oxygen tension on in vitro viability and development of dog follicles enclosed within the ovarian cortex. Reprod Domest Anim 2019;54:1139-1144. 97. Songsasen N, Comizzoli P, Nagashima J, et al: The domestic dog and cat as models for understanding the regulation of ovarian follicle development in vitro. Reprod Domest Anim 2012;6:13-18. 98. Abdel-Ghani MA, Shimizu T, Suzuki H: Expression pattern of vascular endothelial growth factor in canine folliculogenesis and its effect on the growth and development of follicles after ovarian organ culture. Reprod Domest Anim 2014;49:734-739. 99. Costa Pereira LM, Thongkittidilok C, Lopes MD, et al: Effect of anti-apoptotic drug Z-VAD-FMK on in vitro viability of dog follicles. Theriogenology 2018;122:124-129. 100. Nagashima JB, Wildt DE, Travis AJ, et al: Activin promotes growth and antral cavity expansion in the dog ovarian follicle. Theriogenology 2019;129:168-177. 101. Thongkittidilok C, Doriguzzi N, Nagashima J, et al: Cilostamide and forskolin maintain gap junction function of incubated dog follicles. Theriogenology 2020;142:222-228. 102. Jeong YW, Kim JJ, Hossein MS, et al: Influence of somatic cell donor breed on reproductive performance and comparison of prenatal growth in cloned canines. Theriogenology 2014;81:1207-1213. 103. Lee SH, Oh HJ, Kim MJ, et al: Dog cloning-no longer science fiction. Reprod Domest Anim 2018;53 (Suppl 3):133-138. 104. Oh HJ, Kim MK, Jang G, et al: Cloning endangered gray wolves (Canis lupus) from somatic cells collected postmortem. Theriogenology 2008;70:638-647. 105. Kim MK, Jang G, Oh HJ, et al: Endangered wolves cloned from adult somatic cells. Cloning Stem Cells 2007;9:130-137. 106. Hwang I, Jeong YW, Kim JJ, et al: Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes. Reprod Fertil Dev 2013;25:1142-1148. 107. Kim GA, Oh HJ, Park, JE et al: Species-specific challenges in dog cloning. Reprod Domest Anim 2012;47 (Suppl 6):80-83. 108. Park JE, Hong SG, Kang JT, et al: Birth of viable puppies derived from breeding cloned female dogs with a cloned male. Theriogenology 2009;72:721-730. 109. Eun K, Hong N, Jeong YW, et al: Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer. PLoS One 2020;15:e0233784. 110. Kim MJ, Oh HJ, Park JE, et al: Generation of transgenic dogs that conditionally express green fluorescent protein. Genesis 2011;49:472-478. 111. Zou Q, Wang X, Liu Y, et al: Generation of gene-target dogs using CRISPR/Cas9 system. J Mol Cell Biol 2015;7:580-583. 112. Jeong YW, Lee GC, Kim JJ, et al: Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypy- ruvate carboxykinase. Inter J Mol Med 2012;30:321-329. 113. Lee GS, Jeong YW, Kim JJ, et al: A canine model of Alzheimer’s disease generated by overexpressing a mutated human amyloid precursor protein. Inter J Mol Med 2014;33:1003-1012. 114. Nagashima J, El Assal R, Songsasen N, et al: Evaluation of an ovary- on-a-chip in large mammalian models: Species specificity and influence of follicle isolation status. J tissue Eng Regen Med 2018;12:e1926-e1935. anireprosci.2021.106698. Clinical Theriogenology 2021; 13: 145 Current and future perspectives of reproductivetechnologies in domestic and wild canids