N Songsasen

Conservation Biology Institute, Corvallis, Oregon, United States

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Publications (47)90.71 Total impact

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    ABSTRACT: Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are known to play key roles in the remodelling of extracellular matrix during ovarian folliculogenesis, especially during the final stage of follicle development. To date, little is known about the significance of MMPs and TIMPs during preantral follicle development. This study determined the expression of MMPs and TIMP-1 during various stages of cat folliculogenesis, largely for the purpose of securing information useful to improving in vitro follicle culture. Primordial (~10 follicles/cat), primary (~5 follicles/cat), secondary (~9 follicles/cat), early antral (~9 follicles/cat), and antral (~4 follicles/cat) follicles were physically isolated from ovaries recovered from 15 cats (5 months to 3 years old during follicular stage) undergoing ovariohysterectomy and assessed for expression of MMP-1, -2, -3, -7, -9, and -13 as well as TIMP-1 using real-time quantitative polymerase chain reaction (q-PCR; 2-4 replicates/follicle stage). Additional ovaries were obtained from three prepubertal (6 months old) and three adult (1 year old) cats and ovarian pieces were fixed in Bouin's solution and assessed for MMP-2 and -13 localization using immunohistochemistry. MMP expression data were analysed using the Kruskull-Wallis one-way ANOVA. Follicles from all stages of development expressed MMPs and TIMP-1. Specifically, expression of MMP-2 increased (P<0.05) as folliculogenesis progressed (10-fold increases from primordial to early-antral and antral stage). There were no differences (P>0.05) in the expression of other MMPs among follicular classes. For TIMP-1, there was a tendency (P=0.07) for increased expression after antrum formation (early antral and antral stages). Immunohistochemistry analysis revealed that MMP-2 was expressed in both the oocyte and somatic cells of all follicular stages in prepubertal cats. However, MMP-2 expression was limited to granulosa and theca cells of antral follicles in adult females. MMP-13 was expressed in the granulosa and theca cells of primary, secondary, and antral stage follicles, and there were no differences (P>0.05) in localization patterns for this protein between prepubertal and adult females. In summary, the study is the first to report the expression of MMPs as well as TIMP-1 in isolated cat follicles. The difference in MMP-2 expression between prepubertal and adult cats suggests that there may be age-specific requirements for in vitro follicle growth. We are keenly interested in this information for underpinning the development of new in vitro microenvironments for growing immature cat follicles. We suspect that such information will be crucial for understanding how to promote the remodelling of the extracellular matrix by creating degradable biomaterials containing MMP-sensitive peptides to allow optimal follicle expansion.
    Reproduction Fertility and Development 12/2014; 27(1):183. · 2.58 Impact Factor
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    ABSTRACT: Contents 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.
    Reproduction in Domestic Animals 12/2012; 47(s6). · 1.39 Impact Factor
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    ABSTRACT: Contents The culture of ovarian follicles is an important tool for understanding the mechanisms controlling follicle development and differentiation of the oocyte. The benefit of recovering meiotically and developmentally competent oocytes from early stage follicles (primordial, primary, pre-antral and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in women facing cancer treatments. This research field is at an early stage of scientific discovery. To-date, live offspring from cultured primordial follicles that produced fertilizable oocytes has occurred only in the mouse. Progress in other more complex species has been limited because larger animals have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. We believe the dog and cat are excellent models for understanding more about folliculogenesis in vitro. This review highlights what is known about this topic for these two species as well as future priorities. We have discovered that it is more challenging to maintain viability of primordial follicles within ovarian tissues in vitro in the dog than the cat. Nonetheless, it is possible to grow both isolated cat and dog pre-antral follicles in culture. Although the follicles of both species have the capacity to increase in size and produce steroids, only cat oocytes appear morphologically normal. The reason for this striking difference between these two species is an area of high research priority. While much more fundamental data are required, we envision advanced technology that will allow harvesting oocytes from the vast, unused follicle stores sequestered within carnivore ovaries. These gametes have utility for reproducing genetically valuable dogs and cats that are ‘companions’ or biomedical models for investigating human disorders as well as for salvaging the genomes of rare canid and felid species that die before contributing to genetic management programs.
    Reproduction in Domestic Animals 12/2012; 47(s6). · 1.39 Impact Factor
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    ABSTRACT: 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.
    Theriogenology 06/2012; 78(8):1666-81. · 2.08 Impact Factor
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    N. Songsasen
    Meiosis - Molecular Mechanisms and Cytogenetic Diversity, 02/2012; , ISBN: 978-953-51-0118-5
  • N Songsasen, C Guzy, D E Wildt
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    ABSTRACT: A previous study from our laboratory has demonstrated that preantral follicles from the dog that are cultured in alginate are able to grow and produce steroid hormones (Songsasen et al. 2011 Reproduction 142, 113-122). Here we investigated the influence of using a combination of alginate and a degradable biomaterial, fibrin, on dog follicle development in vitro. We hypothesised that the alginate and fibrin gel matrix would be superior to alginate alone because the former has dynamic mechanical properties that permit more expansive follicle development than the inert alginate-only system. Secondary follicles (128-220μm in diameter) were collected from the ovaries of 4 prepubertal dogs (<6 months of age) and encapsulated in 0.5% alginate (n=26) or 0.5% alginate+12.5mgmL(-1) of fibrin (n=22). Follicles were cultured for 12 days at 38.5°C in 100μL of α-minimal essential medium+2mM of glutamine+5.5μgmL(-1) of insulin+5.5μgmL(-1) of transferrin+6.7ngmL(-1) of selenium+10μgmL(-1) of FSH and 1ngmL(-1) of LH+3mgmL(-1) of polyvinyl alcohol. Follicle diameter was monitored and half of the medium exchanged every 48h. Follicle survival was assessed based on ability to increase in size, as well as on oocyte and granulosa cell morphology. Comparisons of follicle growth rate for each culture day between the 2 treatments were conducted using Student's t-test and among culture days within the same group using ANOVA followed by a Holm-Sidak multiple comparison. Follicle survival was compared using a chi-square test. In both groups, follicles maintained the 3-dimensional structure and increased (P<0.05) in size as culture period progressed. However, follicles encapsulated in alginate+fibrin grew larger (P<0.05) than those in alginate alone. Specifically, follicles in alginate+fibrin were doubled in size by 12 days compared with a 60% increase for alginate alone. There were no differences (P>0.05) in follicle survival between the 2 groups (27.0 and 38.1% for alginate and alginate-fibrin, respectively). Results demonstrate that a dynamic alginate-fibrin matrix enhances in vitro follicle growth. We suspect that the mechanism involved is related to facilitating expansion capacity. Specifically, it is likely that nondegradable alginate offers physical, but eventually restrictive, support to encapsulated cells. By contrast, in the gel combination, the fibrin degrades due to cell-secreted proteases that, in turn, permit more robust follicle expansion. Low follicle survival (<40%) in both treatments emphasises the need for more studies to identify influential endocrine/paracrine factors that enhance follicle growth and production of competent oocytes.
    Reproduction Fertility and Development 12/2011; 24(1):173. · 2.58 Impact Factor
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    N Songsasen, T K Woodruff, D E Wildt
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    ABSTRACT: 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.
    Reproduction 04/2011; 142(1):113-22. · 3.56 Impact Factor
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    ABSTRACT: The objectives of this study were to (1) assess year-round behaviors and activity patterns of captive raccoon dogs (Nyctereutes procyonoides) and (2) characterize the species' reproductive endocrinology. Behaviors and activity patterns of 12 (5.7) animals were recorded over a 1-year period. During that time, fecal samples were collected 2-7 times/week from 16 (7.9) individuals (six of these were included in the behavioral study) for the analysis of testosterone, progesterone and estrogen metabolite concentrations. Activity pattern and excretion of gonadal steroids followed a seasonal pattern. Specifically, dogs were cathemeral in summer, and primarily nocturnal in winter. In the males, testosterone concentrations were at baseline from April through September, began to rise in October and reached peak concentrations in February (P<0.05). In the females, elevated estrogen (P<0.05) was observed in March followed by an increase in progestagen concentrations from March through May (P<0.05) in both pregnant and pseudopregnant animals. Gender significantly influenced monthly testosterone/estrogen ratio (P<0.01); values were higher in males than in females throughout the year with overall percentage of overlapping values between males and females being 28%. In summary, this study characterized cirannual fluctuations in behaviors and gonadal steroid metabolites in the raccoon dog maintained in captivity. Because there is no obvious sexual dimorphism, the differences in testosterone/estrogen ratio may be useful for gender differentiation (72% accuracy), especially among individuals living in the wild.
    Zoo Biology 03/2011; 30(2):134-48. · 1.14 Impact Factor
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    ABSTRACT: Induction of ovarian activity and ovulation is a valuable tool for the genetic management of ex situ populations of wildlife. Deslorelin, a gonadotropin releasing hormone (GnRH)-agonist, has been used earlier to induce oestrus in the grey wolf (Canis lupus). The objective of the present study was to determine the efficacy of Deslorelin (2.1mg, Ovuplant(®), Peptech Animal Health, Australia) for manipulating ovarian activity of the maned wolf, a species speculated to be an induced ovulator. Eight female (4-12 years old) maned wolves were (i) paired with a male (n=3) or (ii) housed alone (n=5). Of the 8 females, 1 (1 in a pair and 1 singleton) were implanted with Deslorelin under the vulvar mucosa for 12 days. The remaining 6 received implants in the subcutaneous layer of the ear for 7 days. Fresh fecal samples were collected 5 to 7 days/week for 1 month before Deslorelin treatment through 6 weeks after implant withdrawal. Reproductive steroid metabolites were extracted from the fecal samples and quantified using a validated enzyme immunoassay. Baseline progestagen concentrations for each individual were calculated by an iterative process, whereby high values exceeding the mean plus one standard deviation were excluded. Comparisons of oestrogen and progestagen concentrations among pre-, peri-, and post-Deslorelin implant periods were performed using analysis of variance. Site and duration of the GnRH agonist treatment had no effect (P>0.05) on subsequent ovarian responses. In paired females, oestrogen metabolites reached the highest (P<0.05) concentration during Deslorelin (i.e. peri-) treatment (441.7±20.2ngg(-1) of feces) compared to pre- (174.9±16.7) and post- (177.8±9.1) treatment. Progesterone metabolites rose (P<0.05) above the baseline (indicative of ovulation) starting on Day 10 after the onset of Deslorelin implantation and remained elevated throughout the study (pre-, 11645±4798; peri-, 31521±6444; post-, 55843±2924ngg(-1) of feces). In singleton females, oestrogen metabolites increased (P<0.05) immediately after implantation (from pre-, 184.2±45.3 to peri-, 334.2±29.8ngg(-1) of feces) and then declined (post-, 192.3±12.4). Progestagen metabolites exhibited a similar pattern with a rise (P<0.05) during Deslorelin treatment (from pre-, 3870±1336 to peri-, 10546±880ngg(-1) of feces) followed by a decline after implant withdrawal (post-, 6171±366), indicating that ovulation did not occur. These results suggest that Deslorelin can induce ovarian activity in the maned wolf. However, administration of an ovulatory agent after Deslorelin treatment may be required in females managed in the absence of a male, further supporting the assertion that this species is an induced ovulator.
    Reproduction Fertility and Development 01/2011; 23(1):112-113. · 2.58 Impact Factor
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    ABSTRACT: The whooping crane is one of the most critically endangered species in North America. The species underwent a severe genetic bottleneck with only 16 individuals remaining in the wild as of 1942. Captive breeding began in 1966 and continues to produce chicks for release in order to establish new wild populations. However, captive birds experience poor reproduction with approximately 40% of eggs being infertile. Males have been known to reach sexual maturity at 5 years of age and continue to reproduce almost as long as the duration of their adult life (i.e. 40 years). Understanding factors affecting seminal quality may assist in identifying and correcting causes of suboptimal reproduction. Our objectives were to determine the influence of age and reproductive seasonality on seminal quality. We hypothesised that seminal quality variations among whooping cranes and ejaculates within a given individual over time were due to bird age and stage of breeding season. In 2010, twenty-nine whooping cranes of 5 age groups housed at Patuxent Wildlife Research Center (Laurel, MD, USA) were studied: ≤5 years (n=3); 6-10 years (n=7); 11-15 years (n=7); 16-20 years (n=4); >20 years old (n=8). Semen was collected using a manual manipulation technique at 3 stages of the breeding season: early (March, n=29) mid (April, n=24), and late (May, n=14). Samples were evaluated for seminal volume and sperm concentration, motility, and morphology, with data evaluated by analysis of variance. Bird age had no influence on seminal quality, whereas stage of breeding season affected seminal volume and the proportion of sperm with normal morphology (95% confidence interval). Specifically, samples collected during Mid breeding season had the highest volume (mean±SEM; early: 42.0±8.0μL; mid: 66.0±15.2μL; late: 39.7±17.8μL), but lowest proportions of structurally normal sperm (early: 78.4±3.7%: mid: 61.5±3.2%; late: 69.7±3.4%). There was a significant difference (P=0.06) in sperm concentration among stages of the breeding season (early: 66.3±18.8×10(6) spermmL(-1); mid: 179.2±46.2×10(6) spermmL(-1); late: 91.4±47.8×10(6) spermmL(-1)). Sperm motility was unaffected by season (early: 36.4±3.5%; mid: 45.9±4.1%; late: 48.0±4.9%). In summary, there is a peak in seminal quality that corresponds with higher volume and more sperm during the mid stage of the season, although with higher instances of structural abnormalities. Despite the small founder base for this species, males in this population produce sperm with no variation in seminal quality across a wide variation in age.
    Reproduction Fertility and Development 01/2011; 23(1):214. · 2.58 Impact Factor
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    N. SONGSASEN, M. D. RODDEN
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    ABSTRACT: The Association of Zoos and Aquariums Maned Wolf Species Survival Plan (MWSSP) was established almost 25 years ago. The goals of the MWSSP are to (1) maintain a viable self-sustaining captive population in North America, (2) enhance health and well-being of individuals living in North American zoos and (3) promote conservation of this species through education and field-conservation initiatives. Since its inception, the MWSSP and member institutions have supported studies on nutrition, medical management, behaviour and reproductive biology, and published a husbandry manual, which serves as a guide for captive management of Maned wolves Chrysocyon brachyurus in North and South American zoos. Furthermore, the MWSSP has provided funding for field studies aimed at identifying potential threats to wild populations in range countries, including Brazil, Bolivia and Argentina, as well as for the first Population and Habitat Viability Assessment Workshop for this species. Finally, the MWSSP has played an active role in promoting education and outreach efforts in both the United States and range countries. In this paper, we review and discuss the roles of the MWSSP in ex situ and in situ conservation of the Near Threatened Maned wolf.
    International Zoo Yearbook 12/2009; 44(1):136 - 148.
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    ABSTRACT: White-tailed deer oocyte biology is not well documented. The objective of this study was to determine (1) the influence of estradiol (E(2)) supplementation on meiotic resumption and the ability to "rescue" poorer quality (lower grade) oocytes and (2) the kinetics of oocyte nuclear maturation in vitro in the white-tailed deer. In Experiment 1, immature oocytes harvested during hunting-culling operations were cultured for 24h in the presence or absence of E(2). Incubation in 1mug/mL E(2) promoted nuclear maturation (to telophase I, TI; or to metaphase II, MII) in a higher proportion of Grade 1 oocytes ( approximately 77%; P<0.05) compared with that in Grade 2 or Grade 3 counterparts ( approximately 51%). For Grades 2 and 3 oocytes, there was no advantage (P>0.05) for E(2) supplementation in reaching TI/MII. In Experiment 2, Grade 1 oocytes were cultured in the presence of E(2) and nuclear status evaluated at 0, 3, 6, 12, and 24h of in vitro incubation. At 0h,>70% of oocytes already had undergone germinal vesicle breakdown. After 12h, approximately 70% of oocytes had reached metaphase I of nuclear maturation, with approximately 75% achieving TI/MII by 24h in vitro. In summary, adding E(2) to an in vitro maturation (IVM) culture system for white-tailed deer was advantageous, but only for the highest quality oocytes, with approximately 75% achieving nuclear maturation. In contrast, E(2) supplement did not benefit lower-grade oocytes, half of which will reach MII, with the other half failing. Under the described culture conditions, good-quality white-tailed deer oocytes achieve nuclear maturation over a time duration comparable with that reported in other ungulates.
    Theriogenology 10/2009; 73(1):112-9. · 2.08 Impact Factor
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    ABSTRACT: 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.
    Reproduction in Domestic Animals 07/2009; 44 Suppl 2:65-70. · 1.39 Impact Factor
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    ABSTRACT: Knowledge about reproduction is critical for predicting the viability of wildlife populations in nature and for managing breeding programmes in captivity. Intensive species-based studies are the priority, because reproductive mechanisms are extraordinarily diverse, even within the same taxonomic family. Carnivores deserve more attention as such species are highly vulnerable to environmental change and human persecution. The present review provides contemporary illustrations of how reproductive science is contributing to understand unique reproductive mechanisms that are both of fundamental and applied interest. In the case of the endangered African wild dog (Lycaon pictus) free-living in South Africa, non-invasive faecal corticosteroid assessments have yielded new insights about the impact of animal relocation and reintroduction on adaptive responses, reproductive fitness and survival. For the maned wolf (Chrysocyon brachyurus), advances have been made in characterizing and comparing reproductive traits in free-ranging vs captive individuals. For the cheetah (Acinonyx jubatus), recent studies have focused on the cryosensitivity of sperm and the ability to develop a field-friendly sperm cryo-method. The by-product has been a large-scale frozen repository of sperm from wild-caught cheetahs useful for infusing new genes into ex situ populations. Finally, rigorous, multi-disciplinary and cross-institutional reproductive studies of the black-footed ferret (Mustela nigripes), including the use of artificial insemination, have contributed to the remarkable recovery and restoration of this species, once on the brink of extinction. In summary, advances in reproductive science are not necessarily related to 'assisted breeding'. However, understanding the unique ways of carnivore reproduction greatly contributes to species management and conservation.
    Reproduction in Domestic Animals 07/2009; 44 Suppl 2:47-52. · 1.39 Impact Factor
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    ABSTRACT: To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.
    Cryo letters 04/2009; 30(3):190-201. · 0.84 Impact Factor
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    ABSTRACT: There has been growing interest in the specific impacts of anthropogenic factors on the health of wildlife. This study examined hematology and serum chemistry status of a prominent carnivore, the maned wolf (Chrysocyon brachyurus), living in, on the boundaries to, or on adjacent farmlands to the Serra da Canastra National Park, Brazil. Twenty-eighty wolves were captured, and values were compared 1) between subadults (n=8 animals) and adults (n=20 animals), 2) males (n=12 animals) and females (n=16 animals), and 3) among wolves living inside the park (n=11), near the park border (n=11 animals), and in neighboring farming areas (n=6 animals). Age, gender, and wolf locations influenced (P<0.05) hematology and serum biochemistry values. Specifically, adults had lower (P<0.05) circulating phosphorus than subadults. Males had lower (P<0.05) serum glucose, creatinine phosphokinase, and cholesterol and higher (P<0.05) potassium than females. Erythrocyte count and serum cholinesterase were lower (P<0.05) in wolves living within the park compared with near the park border or on farmlands. Mean corpuscular volume was lower (P<0.05) in wolves living near the park border than those ranging within the park and on farmlands. Aspartate transaminase and chloride were higher (P<0.05) in wolves living inside the park compared with those ranging near the park border. Creatinine phosphokinase was lower (P<0.05) in wolves living on farmland compared with the other two locations. These results clearly reveal a relationship between age and gender on hematology and serum biochemistry values in free-living maned wolves. More importantly, certain traits indicative of health are potentially compromised in wolves living in areas under anthropogenic pressure. These data lay a foundation for examining the influence of farming and local domestic species on disease susceptibility and fitness in the maned wolf.
    Journal of wildlife diseases 02/2009; 45(1):81-90. · 1.27 Impact Factor
  • Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2009; 21(1).
  • Reproduction Fertility and Development 01/2009; 21(1). · 2.58 Impact Factor
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    ABSTRACT: The size of donor follicles influences meiotic maturation of oocytes, including those of the domestic dog (Songsasen and Wildt 2005 Mol. Reprod. Dev. 72, 113–119). Maturation promoting factor (MPF, CDK1) and mitogen activating protein kinase (MAPK) play a pivotal role in regulating meiosis in several species (Abrieu et al. 2001 J. Cell Sci. 114, 257–267). Accordingly, we determined (1) MPF and MAPK activities, and (2) mRNA expression of cell cycle genes, including CDK1, CCNB1, and CDC25 in oocytes obtained from small (<1 mm diameter), medium (1 to 2 mm) and large (>2 mm) follicles. In Study 1, ovarian oocytes were classified into three groups (based on these sizes) and then cultured (38.5°C in 5% CO2) for 0, 24, or 48 h in TCM-199 (+25 µm β-mercaptoethanol, 10 ng mL–1 epidermal growth factor, 0.25 mm pyruvate, 2.0 mm glutamine, and 0.1% polyvinyl alcohol). Oocytes were denuded, assessed for nuclear status, and stored individually at –80°C until MPF and MAPK activities were assayed using a double kinase assay. Kinase activities of in vitro-matured (IVM) oocytes were expressed as a ratio of MPF and MAPK to that in metaphase I (MI) oocytes flushed from the oviducts of estrous bitches. In Study 2, oocytes (n = 20/follicular size class) were immediately frozen at –80°C; RNA was extracted, reverse transcribed, and subjected to quantitative real-time PCR analysis. Expression levels of each transcript were normalized to levels of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Statistical analyses were performed using ANOVA followed by the Holm-Sidak test for multiple comparisons. Both MPF and MAPK activities varied among follicular size classes. Kinase activities increased in oocytes from large follicles upon meiotic resumption, with germinal vesicle (GV) oocytes expressing the lowest levels compared to their metaphase II (MII) counterpart (GV: 25.9 ± 5.2% v. MII: 211.5 ± 19.5% and 36.4 ± 9.1% v. 150.7 ± 22.9% for MPF and MAPK, respectively; P < 0.01). For medium follicles, MPF increased (P < 0.01) as oocytes progressed from GV (23.3 ± 6.3%) to MII (243.4 ± 55.5%). However, MAPK levels remained constant until the MI stage, and then increased (P < 0.01) in MII oocytes. For small follicles, MPF increased (P < 0.05) at the MI stage and then remained constant until meiosis was completed, whereas MAPK activities remained constant after GVBD. Kinase activity levels were not different (P < 0.05) between MI and MII oocytes harvested from the three follicular classes. Interestingly, follicular size had no effect (P > 0.05) on expression levels of cell cycle transcripts. These findings suggest that the compromised developmental competence of dog oocytes from small follicles likely is related to the oocytes' inability to regulate MAPK activity during meiotic resumption.
    Reproduction Fertility and Development - REPROD FERT DEVELOP. 01/2008; 20(1).
  • N Songsasen, R E Spindler, D E Wildt
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    ABSTRACT: Supplementation of energy substrates to culture medium is essential for resumption and completion of meiosis in vitro for many mammalian species. Objectives were to study the dog oocyte, specifically the influences of pyruvate and glutamine on maturation and the utilization of these two substrates at various developmental stages and incubation times. Ovarian oocytes (n=681) were obtained from spayed bitches and cultured for 48 hr in TCM 199 medium containing various concentrations of pyruvate (0-2.5 mM) and glutamine (0-4 mM) before being assessed for nuclear status. For analyzing metabolic activity, 259 dog oocytes were cultured for 0, 12, 24, 36, or 48 hr, assessed for pyruvate and glutamine metabolism using the hanging drop method and then evaluated for nuclear status. Neither pyruvate nor glutamine had influence (P > 0.05) on oocyte maturation in vitro (IVM). However, both culture interval and meiotic status influenced pyruvate uptake (P < 0.05). Specifically, pyruvate uptake declined as the oocyte progressed from the germinal vesicle (GV) to metaphase II (MII) stage. Glutamine oxidation decreased as culture duration progressed (P < 0.05). In summary, pyruvate or glutamine is not required to promote successful IVM of dog oocytes. But, both substrates are being metabolized, and in patterns different to the domestic cat, another carnivore species. Pyruvate played an important role earlier in the maturational process, and less glutamine was oxidized as the oocyte neared nuclear maturation. These variations emphasize the importance of defining species specificities in carnivores before expecting consistently successful IVM/IVF.
    Molecular Reproduction and Development 08/2007; 74(7):870-7. · 2.81 Impact Factor

Publication Stats

951 Citations
90.71 Total Impact Points

Institutions

  • 2011–2012
    • Conservation Biology Institute
      Corvallis, Oregon, United States
    • University of Leipzig
      • Institut für Tierhygiene und Öffentliches Veterinärwesen
      Leipzig, Saxony, Germany
  • 2006–2009
    • Smithsonian Institution
      • • Center for Species Survival
      • • Department of Reproductive Sciences
      Washington, Washington, D.C., United States
  • 2002–2003
    • University of New Orleans
      • Department of Biological Sciences
      New Orleans, LA, United States
  • 1999–2001
    • Department of Livestock Development
      • Department of Livestock Development
      Bangkok, Bangkok, Thailand
  • 1995–1998
    • University of Guelph
      • • Department of Biomedical Sciences
      • • Department of Population Medicine
      Guelph, Ontario, Canada