Comparative genomics uncovers the evolutionary history, demography, and molecular adaptations of South American canids.

The remarkable radiation of South American (SA) canids produced 10 extant species distributed across diverse habitats, including disparate forms such as the short-legged, hypercarnivorous bush dog and the long-legged, largely frugivorous maned wolf. Despite considerable research spanning nearly two centuries, many aspects of their evolutionary history remain unknown. Here, we analyzed 31 whole genomes encompassing all extant SA canid species to assess phylogenetic relationships, interspecific hybridization, historical demography, current genetic diversity, and the molecular bases of adaptations in the bush dog and maned wolf. We found that SA canids originated from a single ancestor that colonized South America 3.9 to 3.5 Mya, followed by diversification east of the Andes and then a single colonization event and radiation of Lycalopex species west of the Andes. We detected extensive historical gene flow between recently diverged lineages and observed distinct patterns of genomic diversity and demographic history in SA canids, likely induced by past climatic cycles compounded by human-induced population declines. Genome-wide scans of selection showed that disparate limb proportions in the bush dog and maned wolf may derive from mutations in genes regulating chondrocyte proliferation and enlargement. Further, frugivory in the maned wolf may have been enabled by variants in genes associated with energy intake from short-chain fatty acids. In contrast, unique genetic variants detected in the bush dog may underlie interdigital webbing and dental adaptations for hypercarnivory. Our analyses shed light on the evolution of a unique carnivoran radiation and how it was shaped by South American topography and climate change.

New Insights in Canine Reproduction.

Reproduction in canids has been characterized by having some peculiar aspects, such as the extended reproductive cycle and ovulation of immature oocytes [...].

First assessment of hoary fox (Lycalopex vetulus) seasonal ovarian cyclicity by non-invasive hormonal monitoring technique.

Reproduction is key to species survival, and reproductive physiology represents a high priority investigative area for conservation biology, as it provides a basic understanding of critical life-history traits, information that is helpful for the establishment of management strategies. Here, we generated knowledge about the reproductive endocrinology of the hoary fox (Lycalopex vetulus), a small canid (2.5-4 kg) endemic to open areas of the Brazilian Cerrado and listed in the Brazilian National List of Endangered species. Specifically, we utilized non-invasive hormone monitoring methods to assess oestrogen and progestagen metabolites from eight female hoary foxes housed in five zoological institutions in the state of São Paulo-Brazil. We observed the elevations of oestrogen and progestagen metabolites between July and September in six of the eight females. No significant evidence of ovarian activity was observed during other months. Two females, who shared the same enclosure, did not show a pattern of reproductive cyclicity. Based on these characteristics, we concluded that captive hoary foxes are seasonal monoestric, with the beginning of the oestrus cycle occurring mainly in July followed by 2 months of the luteal phase when conception does not occur. We suggest the dosage of faecal metabolites of estradiol and progesterone could be used to differentiate the reproductive period from a non-reproductive period in Lycalopex vetulus females, providing relevant information about their reproductive biology that may contribute to species conservation and management strategies, such as increased ex situ reproductive success.

Effect of oxygen tension on in vitro viability and development of dog follicles enclosed within the ovarian cortex.

Oxygen concentration has been shown to influence in vitro viability and growth of ovarian follicles. The present study examined the effect of oxygen tension on in vitro development of dog follicles enclosed within the ovarian cortex. Ovaries were obtained from domestic dogs (age, 8 months to 2 years), and cortical fragments were recovered. The cortices were then incubated on 1.5% (w/v) agarose gel blocks within a 4-well culture plate containing Eagle Minimum Essential Medium (MEM). Ovarian follicles within the tissues were processed for histology and assessed for follicle density, viability and diameter immediately after collection (Control) or after 2 or 5 days of in vitro incubation. Apoptotic cells were assessed using TUNEL assay. Comparisons of follicular viability and diameter were performed using analysis of variance followed by Tukey's test (p < 0.05). Comparisons of follicle density and apoptosis among treatments were conducted using Non-parametric Kruskal-Wallis test followed by Friedman's test (p < 0.05). No difference (p > 0.05) in follicle density was observed among groups at Day 2 of in vitro culture. However, the density of follicles within cortices cultured in 20% oxygen for 5 days significantly reduced compared to the Control and those incubated in 5% concentration. The viability of cultured follicles in all treatments decreased (p < 0.05) compared to the Control after 2 days incubation, and this value further reduced (p < 0.05) in 20% oxygen group at Day 5. There were no differences in the percentages of apoptotic follicles between the two treatment groups (p > 0.05). Nevertheless, after 5 days of culture, the percentage of TUNEL-positive follicles increased significantly (p < 0.05) in cortices incubated in 20% oxygen environment. In conclusion, our findings demonstrated that 5% oxygen level was superior to 20% concentration in sustaining in vitro viability of dog follicles enclosed within the ovarian cortex.

Physiological impacts of housing maned wolves (Chrysocyon brachyurus) with female relatives or unrelated males.

The maned wolf is a threatened canid species native to South America. Previous studies have suggested the species exhibits induced ovulation. In captive breeding facilities, reproductive success is low while rates of neonatal mortality are high. Females that are not recommended for breeding are frequently housed together. However there has never been a systematic study of the reproductive consequences of co-housing females. This study was conducted for three purposes, to: (1) corroborate the presence of induced ovulation, (2) determine whether elevated cortisol is implicated in neonatal pup mortality, and (3) evaluate the endocrine correlates of group housed females. Using fecal hormone monitoring for estrogen, progesterone, and cortisol, 43 cycles from 33 female maned wolves were studied from 2002 to 2015. Females were categorized by their reproductive status: pregnant and successfully raised pups (PR; n = 11), pregnant with neonatal pup demise within 3 days (PL; n = 7), housed with a male but no signs of breeding or pregnancy (PP; n = 10), housed singly (S; n = 8), or housed with related females (F; n = 7). Estrogen and progestagen remained at baseline for all females not housed with a male (S, F), while elevations consistent with ovulation were seen in females housed with a male (PP, PL, PR). Compared to PR females, PL individuals showed similar cortisol levels throughout the cycle and slightly lower progesterone levels during gestation. As for the effect of co-housing related females, F females showed estrogen and progesterone levels lower even than S females while cortisol levels were elevated compared to all other groups. These findings support the previous evidence of induced ovulation in the maned wolf. Although elevated cortisol does not seem to be implicated in pup loss, a non-significant trend towards lower progesterone during gestation could be implicated. Future studies should assess depressed progesterone levels as a correlate to neonatal pup mortality. Female maned wolves housed with related females experience suppressed reproductive hormones and elevated adrenal hormones. Therefore, a more systematic study of hormonal and behavioral correlates to co-housing with related females is warranted.

Challenges in wildlife conservation: present and future perspectives / Desafios na conservação de vida selvagem: perspectivas presentes e futuras

The global environment is under increasing pressure from expanding human activities and climate changes. The change in global environment, including increase environmental temperature, marine pollution, ocean acidification among others, has resulted in significant loss in biodiversity. To dates, the International Union for Conservation of Nature (IUCN) has estimated that 41% of amphibians, 33% of corals, 26% of mammals and 13% of birds are threatened by extinction (International Union for Conservation of Nature - IUCN, 2015). Imminent extinction of wild species is often caused by multiple factors and may not always be due to failure of animals to breed. Nevertheless, reproductive sciences play critical roles in wildlife conservation, especially captive breeding program. A clear example of how reproductive biology contributes to species recovery program is the case of black footed ferret (Howard et al., 2003; Santymire et al., 2014), endemic to North America. In 1980s, the species underwent severe population decline with only 18 individuals remained in the wild which were brought into captivity. To-date, >150 ferret kits have been produced by artificial insemination (AI), including offspring produced from frozen founder sperm stored for as long as 20 years. Since the inception of the captive breeding program, 8,000 black footed ferrets have been produced, half of which have been reintroduced into 20 sites in eight US States, Canada and Mexico.

Current status and future perspective of canine and feline ovarian tissue culture / Status atual e perspectiva futura de cultura de tecido ovariano canino e felino

The culture of ovarian follicles is an important tool for understanding of the mechanisms controlling follicle development and differentiation of its oocyte. The benefit from recovering developmentally competent oocytes from small, immature follicles (primordial, primary, secondary and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in female cancer survivors. To-date, live offspring from cultured primordial follicles has occurred only in the mouse. Progress in larger, more complex species has been limited because these animal models have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. In this presentation, we highlight current status of this topic for domestic carnivores (i.e., dogs and cats) as well as future research priorities.

A cultura de folículos ovarianos é uma ferramenta importante para o entendimento dos mecanismos que controlam o desenvolvimento de folículos e a diferenciação de oócitos. O benefício da recuperação de oócitos competentes em termos de desenvolvimento de folículos pequenos e imaturas (primordiais, primários, secundários e antral precoce) também seria significativo, variando do resgate de genoma de espécies em extinção à preservação da fertilidade em sobreviventes de câncer femininos. Até o momento, descendentes de folículos primordiais cultivados ocorreram somente em ratos. O progresso em espécies maiores e mais complexas tem sido limitado porque estes modelos de animais tem durações de foliculogênese mais longa, requerendo maior tempo de cultura para gerar folículos e oócitos completamente crescidos. Nesta apresentação ressaltamos o status atual deste tópico para carnívoros domésticos (i.e. cães e gatos), além de prioridades futuras de pesquisa.

Current status and future perspective of canine and feline ovarian tissue culture / Status atual e perspectiva futura de cultura de tecido ovariano canino e felino

The culture of ovarian follicles is an important tool for understanding of the mechanisms controlling follicle development and differentiation of its oocyte. The benefit from recovering developmentally competent oocytes from small, immature follicles (primordial, primary, secondary and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in female cancer survivors. To-date, live offspring from cultured primordial follicles has occurred only in the mouse. Progress in larger, more complex species has been limited because these animal models have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. In this presentation, we highlight current status of this topic for domestic carnivores (i.e., dogs and cats) as well as future research priorities.(AU)

A cultura de folículos ovarianos é uma ferramenta importante para o entendimento dos mecanismos que controlam o desenvolvimento de folículos e a diferenciação de oócitos. O benefício da recuperação de oócitos competentes em termos de desenvolvimento de folículos pequenos e imaturas (primordiais, primários, secundários e antral precoce) também seria significativo, variando do resgate de genoma de espécies em extinção à preservação da fertilidade em sobreviventes de câncer femininos. Até o momento, descendentes de folículos primordiais cultivados ocorreram somente em ratos. O progresso em espécies maiores e mais complexas tem sido limitado porque estes modelos de animais tem durações de foliculogênese mais longa, requerendo maior tempo de cultura para gerar folículos e oócitos completamente crescidos. Nesta apresentação ressaltamos o status atual deste tópico para carnívoros domésticos (i.e. cães e gatos), além de prioridades futuras de pesquisa.(AU)

Challenges in wildlife conservation: present and future perspectives / Desafios na conservação de vida selvagem: perspectivas presentes e futuras

The global environment is under increasing pressure from expanding human activities and climate changes. The change in global environment, including increase environmental temperature, marine pollution, ocean acidification among others, has resulted in significant loss in biodiversity. To dates, the International Union for Conservation of Nature (IUCN) has estimated that 41% of amphibians, 33% of corals, 26% of mammals and 13% of birds are threatened by extinction (International Union for Conservation of Nature - IUCN, 2015). Imminent extinction of wild species is often caused by multiple factors and may not always be due to failure of animals to breed. Nevertheless, reproductive sciences play critical roles in wildlife conservation, especially captive breeding program. A clear example of how reproductive biology contributes to species recovery program is the case of black footed ferret (Howard et al., 2003; Santymire et al., 2014), endemic to North America. In 1980s, the species underwent severe population decline with only 18 individuals remained in the wild which were brought into captivity. To-date, >150 ferret kits have been produced by artificial insemination (AI), including offspring produced from frozen founder sperm stored for as long as 20 years. Since the inception of the captive breeding program, 8,000 black footed ferrets have been produced, half of which have been reintroduced into 20 sites in eight US States, Canada and Mexico.(AU)

Adrenal activity in maned wolves is higher on farmlands and park boundaries than within protected areas.

In this study we measured excreted fecal corticoid metabolites (FCM) in maned wolves (Chrysocyon brachyurus) living within a protected reserve, on farmlands or in a boundary zone between the two habitats, and determined the impacts of season and reproductive status on adrenal activity. Feces were collected within a national park (n=191 samples), a park boundary zone (n=39) and on nearby farmlands (n=27), processed and analyzed by enzyme immunoassay. FCM amounts from samples collected on farmlands were higher (P<0.05) than in those collected inside the reserve and from the boundary zone. In relation to seasonality, FCM were elevated (P<0.05) in spring (September-November) when wolf pairs were raising young. We then divided the samples collected during breeding season (March-August) into cycling females and male/non-cycling females based on fecal progesterone: fecal testosterone ratio. FCM concentrations of the former collected inside the park were higher than (P<0.05) than the latter group. However, there were no differences in FCM levels between the two groups for samples collected in the boundary zone and on farmlands. Furthermore, FCM concentrations of male/non-cycling females samples collected on farmlands were 2- to 5-fold higher (P<0.05) than in counterparts collected inside the park. The consistently high FCM concentrations in samples collected on farmlands indicate that, in addition to seasonality, gender and reproductive status, anthropogenic pressures also contribute to elevating adrenal steroid for individuals living in altered habitat.