Nucharin Songsasen

Lessons from biodiversity - the value of nontraditional species to advance reproductive science, conservation, and human health.

Reproduction is quintessential to species survival. But what is underappreciated for this discipline is the wondrous array of reproductive mechanisms among species— variations as diverse as the morphology of the species themselves (more than 55,000 vertebrate and 1.1 million invertebrate types). We have investigated only a tiny fraction of these species in reproductive science. Besides the need to fill enormous gaps in a scholarly database, this knowledge has value for recovering and genetically managing rare species as well as addressing certain reproductive issues in humans. This article provides examples, first to advise against oversimplifying reproduction and then to show how such knowledge can have practical use for managing whole animals, populations, or even saving an entire species. We also address the expected challenges and opportunities that could lead to creative shifts in philosophy and effective actions to benefit more species as well as a future generation of reproductive scientists. Mol. Reprod. Dev. 77: 397–409, 2010.

Protecting and Extending Fertility for Females of Wild and Endangered Mammals

Sustaining viable populations of any wildlife species requires a combination of adequate habitat protection and a good understanding of environmental and biological factors (including reproductive mechanisms) that ensure species survival. Thousands of species are under threat of extinction due to habitat loss/degradation, over-exploitation, pollution, disease, alien species invasions and urban sprawl. This has served as incentive for intensive management of animal populations, both ex situ (in captivity) and in situ (living in nature). Assisted reproductive technologies developed for addressing human infertility and enhancing livestock production have shown encouraging promise in a few wildlife species. However, species-specific physiological variations and a lack of fundamental knowledge have limited how these tools can be used to help rapidly re-build endangered species numbers. Despite limitations, there is enormous potential in applying human-related fertility preservation strategies to wild animals, especially approaches that could assist managing or ‘rescuing’ the genomes of genetically valuable individuals. Indeed, one of the highest priorities in wildlife ex situ management is sustaining all existing genetic diversity to (1) preserve heterozygosity to avoid inbreeding depression and (2) ensure species integrity and the persistence of genomic adaptability to environmental changes. There are components of the rapidly emerging field of oncofertility in women that are highly compatible with preserving valuable genomes of individuals or populations of threatened wildlife. Strategies associated with ovarian tissue cryopreservation and follicle in vitro culture are especially attractive for protecting and extending fertility for wild females. Given adequate attention and more basic studies, we predict that these approaches could assist in the intensive and practical management of gene diversity in endangered species.

Comparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species

A major challenge to retaining viability of frozen gametes and reproductive tissues is to understand and overcome species-specificities, especially because there is substantial diversity in cryobiological properties and requirements among cell types and tissues. Systematic studies can lead to successful post-thaw recovery, especially after determining: 1) membrane permeability to water and cryoprotectant, 2) cryoprotectant toxicity, 3) tolerance to osmotic changes, and 4) resistance to cooling and freezing temperatures. Although species-dependency ultimately dictates the ability of specific cells and tissues to survive freeze-thawing, there are commonalities between taxa that allow a protocol developed for one species to be useful information for another. This is the reason for performing comparative cryopreservation studies among diverse species. Our laboratory has compared cellular cryotolerance, especially in spermatozoa, in a diverse group of animals-from corals to elephants-for more than 30 yrs. Characterizing the biophysical traits of gametes and tissues is the most efficient way to develop successful storage and recovery protocols, but, such data are only available for a few laboratory, livestock, and fish species, with virtually all others (wild mammals, birds, reptiles, and amphibians) having gone unstudied. Nonetheless, when a rare animal unexpectedly dies, there is no time to understand the fundamentals of biophysics. In these emergencies, it is necessary to rely on experience and the best data from taxonomically-related species. Fortunately, there are some general similarities among most species, which, for example, allow adequate post-thaw viability. Regardless, there is a priority for more information on biophysical traits and freezing tolerance of distinctive biomaterials, especially for oocytes and gonadal tissues, and even for common, domesticated animals. Our colleague, Dr John Critser was a pioneer in cryobiology, earning that moniker because of his advocacy and devotion to understanding the differences (and similarities) among species to better store living genetic material.

In vitro growth and steroidogenesis of dog follicles are influenced by the physical and hormonal microenvironment

The present study examined the influences of the physical and hormonal microenvironment on in vitro growth and steroidogenesis of dog follicles. Follicles were enzymatically isolated and individually encapsulated in 0.5% (w/v; n=17) or 1.5% (n=10) alginate and cultured with 0.5 IU/ml equine chorionic gonadotropin for 192 h. In a separate experiment, follicles were encapsulated in 0.5% alginate and cultured with 0 (n=22), 1 (n=23), 10 (n=20) or 100 (n=21) μg/ml FSH for 240 h. Follicle diameter and steroid production were assessed every 48 h in both studies. Follicles encapsulated in the 0.5% alginate grew faster (P<0.05) than those cultured in the 1.5% concentration. Oestradiol (E(2)) and progesterone (P(4)) increased consistently (P<0.05) over time, and follicles in the 1.5% alginate produced more (P<0.05) P(4) than those in the 0.5% solution. Follicles cultured in the highest FSH concentration (100 μg/ml) increased 100% in size after 240 h compared with 50 to 70% in lower dosages. E(2) concentration remained unchanged over time (P>0.05) across FSH dosages. However, P(4) increased (P<0.05) as culture progressed and with increasing FSH concentration. Results demonstrate that dog follicles cultured in alginate retain structural integrity, grow in size and are hormonally active. Lower alginate and increasing FSH concentrations promote in vitro follicle growth. However, the absence of an E(2) rise in follicles cultured in FSH alone suggests the need for LH supplementation to support theca cell differentiation and granulosa cell function.

Potential factors affecting semen quality in the Asian elephant (Elephas maximus)

BackgroundOne of the major obstacles in using artificial insemination to manage genetics of elephant population in captivity is the large variations in semen quality among ejaculates within the same and among individuals. The objectives of this study were to determine the influences of (1) age (2) seasonality (3) and circulating testosterone (SrTest), triiodothyronine (SrT3) and tetraiodothyronine (SrT4), as well as seminal (4) testosterone (SpTest), zinc (SpZn) and protein (SpTP) on semen quality in the Asian elephantMethodsAnalyses, including motility, viability and morphology were performed in semen samples collected twice monthly from 13 elephant bulls (age range, 10-to 72-years) by manual stimulation between July 2004 and June 2005. Serum samples obtained monthly were assessed for SrTest, SrT3, SrT4, and seminal plasma samples were evaluated for, SpTest, SpZn and SpTP.ResultsThe highest semen quality was observed at age 23 to 43 years. Percentages of progressive motility and viable sperm were lowest at age 51 to 70 years (P < 0.05); on the other hand, sperm concentration was lowest at age 10 to 19 years (P < 0.05). Percentage of sperm with normal morphology was highest at age 23 to 43 years. The levels of SrT3, SrTest, SpTest and SpZn were lowest at age 51 to 70 years, whereas SrT4 was lowest at age 23 to 43 years. Seasonality significantly affected semen characteristics in which percentage of viable sperm and cell concentration were highest during rainy season and lowest during summer months (P < 0.05). However, percentage of sperm with normal morphology was highest in summer and lowest in rainy season (P < 0.05). Seasonality significantly influenced SrTest with elevated concentrations observed in rainy season and winter (P < 0.05).ConclusionThis study indicates that age and seasonality had influence on semen characteristics in the Asian elephant. The knowledge obtained in this study will improve our understanding of the reproductive biology of this species.

Follicular Morphology, Oocyte Diameter and Localisation of Fibroblast Growth Factors in the Domestic Dog Ovary

Remarkably little is known about folliculogenesis in the dog. Objectives were to characterise (1) changes in follicle/oocyte diameter and granulosa cell number and (2) localisation of fibroblast growth factor (FGF)-2 and FGF-7 during dog ovarian follicle development. Fourteen ovarian pairs were excised and processed for histological evaluation. Two to four serial sections/bitch were stained with hematoxylin, and follicle/oocyte diameters and granulosa cell number were determined at each developmental stage. Mean follicle and oocyte size were compared among stages by one-way analysis of variance. Relationships between follicle and oocyte size and granulosa cell number were determined using correlation and regression analysis, respectively. Another eight serial sections/bitch were processed for immunostaining to determine FGF-2 and FGF-7 localisation. Primordial and primary follicles were similar in size, but smaller than the progressively increasing (p < 0.05) diameter of the later stages. Oocyte diameter gradually increased (p < 0.05) among oocytes derived from primordial, primary, secondary and early antral follicles with no difference (p > 0.05) thereafter. Oocyte size and granulosa cell number increased (p < 0.01) with follicular diameter. Except during anoestrus, FGF-2 occurred in oocytes and granulosa cells of primordial to secondary follicles. In adult bitches, FGF-7 was localised in granulosa cells of primary and secondary follicles and also occurred in the theca layer of antral follicles during prooestrus and oestrus. In summary, folliculogenesis in the domestic dog occurs in two phases: pre-antral phase characterised by increasing follicle size in association with oocyte growth and granulosa cell proliferation and antral phase linked with marked granulosa cell proliferation and accumulation of antral cavity fluid. Finally, the temporal localisation pattern of FGF-2 implies its role in follicular activation, whereas FGF-7 activities appear related to later folliculogenesis.

Epidermal Growth Factor (EGF) Sustains In Vitro Primordial Follicle Viability by Enhancing Stromal Cell Proliferation via MAPK and PI3K Pathways in the Prepubertal, but Not Adult, Cat Ovary

ABSTRACT This study examined the influences of epidermal growth factor (EGF) and growth differentiation factor 9 (GDF9) on in vitro viability and activation of primordial follicles in the ovarian tissue of prepubertal (age, <6 mo) versus adult (age, >8 mo) cats. Ovarian cortical slices were cultured in medium containing EGF and/or GDF9 for 14 days. EGF, but not GDF9, improved (P < 0.05) follicle viability in prepubertal donors in a dose-dependent fashion. Neither EGF nor GDF9 enhanced follicle viability in ovarian tissue from adults, and neither factor activated primordial follicles regardless of age group. We then explored how EGF influenced primordial follicles in the prepubertal donors by coincubation with an inhibitor of EGF receptor (AG1478), mitogen-activated protein kinase (MAPK; U0126), or phosphoinositide 3-kinase (PI3K; LY294002). EGF enhanced (P < 0.05) MAPK and AKT phosphorylation, follicle viability, and stromal cell proliferation. These effects were suppressed (P < 0.05) when the tissue was cultured with this growth factor combined with each inhibitor. To identify the underlying influence of age in response to EGF, we assessed cell proliferation and discovered a greater thriving stromal cell population in prepubertal compared to adult tissue. We conclude that EGF plays a significant role in maintaining intraovarian primordial follicle viability (but without promoting activation) in the prepubertal cat. The mechanism of action is via stimulation of MAPK and PI3K signaling pathways that, in turn, promote ovarian cell proliferation. Particularly intriguing is that the ability of cat ovarian cells to multiply in reaction to EGF is age-dependent and highly responsive in prepubertal females.

Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein-Free Medium

Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α-minimum essential medium (α-MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock-out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free α-MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α-MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p < 0.05). In contrast, α-MEM was superior (p < 0.05) to MEM in maintaining dog follicle viability (32.7% vs 8.1%) in protein-free condition at 15 days. Serum was detrimental (p < 0.05) to follicle survival in both species. Knock-out serum replacement supplementation and a protein-free condition supported cat follicle viability, whereas the latter was superior (p < 0.05) to the former for sustaining dog follicle survival. Likewise, dog follicle viability was enhanced (p < 0.05) by the agarose gel compared to the cell culture insert and control groups after 3 and 9 days of culture. For the cat, the agarose gel better (p < 0.05) supported follicle viability compared to the control, but was equivalent to the cell culture insert. Therefore, sustaining primordial follicle survival from intracortical ovarian slices requires a different in vitro microenvironment for the cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange.

Behavioral and physiologic responses to environmental enrichment in the maned wolf (Chrysocyon brachyurus)

The ex situ population of maned wolves is not self-sustaining due to poor reproduction, caused primarily by parental incompetence. Studies have shown that environmental enrichment can promote natural parental behaviors in zoo animals. The objective of this study was to determine the effects of environmental enrichment on behavioral and physiological responses of maned wolves. During an 8-week experimental period, daily behavior observations and fecal sample collection were conducted on four adult wolves (2.2) individually housed in environments without enrichment. After 2 weeks, the wolves were chronologically provided with 2-week intervals of hiding dead mice around the exhibit, no enrichment, and introduction of boomer balls. Responses of the wolves to enrichment were assessed based on activity levels and exploratory rates, as well as the level of corticoid metabolites in fecal samples collected daily throughout the study period. Providing wolves with environmental enrichment significantly increased exploratory behaviors (P<0.05), especially when mice were hidden in the enclosure. Fecal corticoid concentrations were increased during periods of enrichment in males (P<0.05), but not in females. Overall, there were no correlations between behavioral responses to enrichment and fecal corticoid levels. Behavioral results suggest that environmental enrichment elicits positive effects on the behavior of captive maned wolves. There is evidence suggesting that providing animals with ability to forage for food is a more effective enrichment strategy than introducing objects. There is need for a longer term study to determine the impact of environmental enrichment in this species. Zoo Biol 26:331-343, 2007. (c) 2007 Wiley-Liss, Inc.

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.