Oviductal extracellular vesicles interact with the spermatozoon's head and mid-piece and improves its motility and fertilizing ability in the domestic cat.

Fertilization and early embryo development are regulated by a unique maternal-gamete/embryo cross-talk within the oviduct. Recent studies have shown that extracellular vesicles (EVs) within the oviduct play important roles in mediating this developmental process. Here, we examined the influence of oviductal EVs on sperm function in the domestic cat. We demonstrated that (1) EVs are enriched in proteins related to energy metabolism, membrane modification, and reproductive function; (2) EVs bound and fused with the membranes of the acrosome and mid piece; and (3) incubating sperm with EVs improved motility, fertilizing capacity of cat spermatozoa and prevented acrosomal exocytosis in vitro. These findings indicated that oviductal EVs mediate sperm function and fertilization in the cat and provides new insights to improve sperm cryopreservation and in vitro fertilization in the domestic and wild felids and human.

The Domestic Dog Embryo: In Vitro Fertilization, Culture, and Transfer.

Advances in embryo technologies in the domestic dog have made significant strides in the past decade. This progress has been spurred by interests in taking advantage of the dog as a biomedical research model for human and companion animal medicine, developing assisted reproductive technologies to manage genetic diversity in endangered canids maintained ex situ, and improving breeding in rare or working breeds of dogs. Here, we focus on recent advancements and techniques for collection of in vivo-matured oocytes, in vitro fertilization (IVF), in vitro culture of early (≤8-cell) and advanced stage (≥16-cell) embryos, and embryo transfer.

Equine chorionic gonadotropin induces in vitro follicular growth from the multi-layered secondary developmental stage in cats.

Equine chorionic gonadotropin (eCG) has been commonly used to induce estrus in several felid species. However, the mechanisms by which this gonadotropin regulates cat folliculogenesis are still unclear. We investigated the responsiveness of cat ovarian follicles at different developmental stages to various eCG concentrations supplemented in vitro. Follicles were mechanically isolated from the ovaries of 22 cats and categorized into three developmental stages based on their morphology and diameter: 1) two-layered secondary follicle (SF), 100-150 µm (n = 139); 2) multi-layered SF, 150-300 µm (n = 154); and 3) early antral follicle (AF), ≥300-500 µm (n = 123). The follicles were then encapsulated in 0.5% (w/v) sodium alginate and cultured for 12 days in Minimum Essential Medium supplemented with 0, 0.05, 0.1 or 0.5 IU/mL eCG. Follicle and oocyte diameters were assessed every 3 days. On Day 12, mRNA expression levels of FHSR, LHCGR, GDF9, BMP15, CYP17A1, CYP19A1 and STAR were analyzed using real-time PCR. After being cultured for 12 days, follicle growth and mRNA expression of two-layered SF were not influenced by eCG at all concentrations (P > 0.05). However, the concentration of eCG at 0.05 IU/mL stimulated follicular growth and gene expressions in the multi-layered SF and early AF (P < 0.05). Correspondingly, the diameter of oocytes in the multi-layered SF and early AF treated with 0.05 IU/mL eCG was unchanged. Considering the mRNA expression, the level of STAR was enhanced in the early AF (P < 0.05) and tended to increase in the multi-layered SF (P = 0.08) cultured in 0.05 IU/mL eCG, whereas the expression of other genes was not affected. In sum, the responsiveness of cat follicles to eCG is apparent from the multi-layered SF stage onward. The eCG supplementation at 0.05 IU/mL appeared to be optimal for the follicle culture in the domestic cats.

Responsiveness of intraovarian dog follicles in vitro to epidermal growth factor and vascular endothelial growth factor depends on ovarian donor age.

We investigated the influence of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) on in vitro follicle development within ovarian cortices recovered from pre-pubertal (≤6 months) versus peri-pubertal dogs (≥10 months). Ovarian cortices were cultured for 3 or 7 days in EGF (0 or 10 ng/ml) and VEGF (0, 0.1 or 1 ng/ml) and subjected to histological and apoptosis analyses. Fresh cortices from the same dogs served as "non-cultured controls" (NCC) and were evaluated similarly. The response of ovarian follicles to growth factors differed between pre-pubertal versus peri-pubertal tissues. For pre-pubertal dogs, percentage of structurally normal follicles in cortices cultured for 3 days in low VEGF (0.1 ng/ml) and EGF alone was comparable to that of the NCC. Follicle density declined in all cultured groups even after 3 days. Primary follicle diameter in all cortices cultured for 7 days, except in low VEGF, was smaller than that of the NCC, and percentage apoptotic follicles sharply increased in all treatment groups compared to the NCC. For peri-pubertal donors, percentages of structurally normal follicles decreased in all culture treatments at 3 and 7 days of incubation compared to the NCC. However, more normal follicles were found in cortices cultured in low VEGF and the two VEGF and EGF combinations than in the absence of growth factors or with EGF alone. Culture reduced the density of developing follicles, but follicle diameter was similar to that of the NCC. TUNEL analysis revealed that high-VEGF (1 ng/ml) treatment protected follicles against apoptosis, with the proportion of apoptotic follicles at Day 7 being comparable to that of the NCC. The findings demonstrate that the response of ovarian cortices to growth factor supplementation varied between pre-pubertal versus peri-pubertal donors.

Short-term hypertonic exposure enhances in vitro follicle growth and meiotic competence of enclosed oocytes while modestly affecting mRNA expression of aquaporin and steroidogenic genes in the domestic cat model.

Using the domestic cat as a non-rodent, larger animal model, the objective was to determine the impact of a brief incubation in a hypertonic microenvironment on (1) ovarian follicle and oocyte growth in vitro, (2) developmental capacity of the resident oocyte, and (3) expression of aquaporin (AQP) genes in parallel with genes involved in regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 0.5% alginate were allocated to one of three treatment groups: 1) culture in standard medium at 290 mOsm for 15 d (Control); 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for 15 d (Hypertonic-1h); or 3) incubation in 350 mOsm medium for 24 h followed by incubation in standard medium for additional 14 d (Hypertonic-24h). After measuring follicle and oocyte diameters on Day 15, in vitro-grown oocytes were incubated for 24 h before assessing nuclear status. In Study 2: secondary or early antral follicles were subjected to one of the three treatments: 1) culture in standard medium at 290 mOsm for 48 h; 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for additional 47 h; or 3) incubation in 350 mOsm medium for 24 h followed by culture in standard medium for additional 24 h. At the end of the culture period, all follicles were assessed for mRNA level of Cyp17a1, Cyp19a1, Star, Aqp1, 3, 5, 7 and 8 as well as Fshr using qPCR. Freshly collected follicles also were subjected to gene expression analysis and served as the 'Non-cultured control'. Hypertonic-24h follicles grew larger (P < 0.05) than the control, whereas those in Hypertonic-1h group exhibited intermediate growth, especially when the culture started at the early antral stage. Oocytes in the Hypertonic-24h group were larger and resumed meiosis at a higher rate than in the other treatments. In vitro culture affected (P < 0.05) mRNA expression of Cyp19a1, Star, Aqp1, and Aqp7 in both the secondary and early antral stage while Fshr was only affected in the former compared to the non-cultured control. Pre-incubating follicles in 350 mOsm medium for 24 h enhanced (P < 0.05) Star and Aqp7 while decreasing (P < 0.05) Aqp1 expression compared to the control in secondary follicles, but not in the early antral stage. In summary, short-term hypertonic exposure promoted cat follicle development in vitro (including the meiotic competence of the enclosed oocyte) possibly through a mechanism that does not involve water transport genes.

Endocrine and paracrine controls of canine follicular development and function.

Canid reproduction is unique among other mammals in that females experience long and variable periods of ovarian inactivity. While the domestic dog exhibits a non-seasonal, largely sporadic monoestrus occurring once or twice a year, most wild canids, such as the gray wolf (Canis lupus) and red wolf (Canis rufus), are seasonal breeders with onset apparently dependent on species, latitudinal location and/or variety of environment factors. Neuroendocrine controls of ovarian functions have been mostly studied in the dog, but less so in their wild counterparts, due to difficulties in regular blood sampling. Yet, development of non-invasive hormone monitoring has advanced the understanding of reproductive cycle in wild canids. Recent advances in in vitro follicle culture technology also have begun to provide insights into paracrine controls of canid ovarian folliculogenesis. This review highlights current knowledge on canid reproduction with emphasis on endocrine and paracrine controls of follicular development. We also discuss future research priorities, including advancing the understanding of anoestrous termination and role of paracrine factors in canine folliculogenesis.

Dynamic changes in mitochondrial DNA, distribution and activity within cat oocytes during folliculogenesis.

Mitochondria play fundamental roles during oocyte development. The accumulation and spatial redistribution of these energy-producing organelles have been linked to the developmental competence of mammalian oocytes. Here, we assessed the copy number, distribution and activity of mitochondria within cat oocytes during folliculogenesis. In Experiment 1, oocytes were recovered from primordial (n = 152), primary (112), secondary (95), early (131), small (118), antral (86) and advanced antral (5) stages follicles, and mitochondria DNA extracted and quantified using qPCR. In Experiment 2, oocytes from pre-antral (n = 44), early antral (n = 66), small antral (n = 59), antral (n = 41) and advanced antral (n = 21) follicles were isolated and stained with CMXRos MitoTracker dye to assess mitochondrial distribution pattern and activity levels. Oocyte's mitochondria DNA (mtDNA) copy numbers gradually increased as folliculogenesis progressed, with a significant shift at the small antral stage (0.5 to <1 mm in diameter). The location of mitochondria gradually shifted from a homogeneous distribution throughout the cytoplasm in pre-antral oocytes to a pericortical concentration in the advanced antral stage. Quantification of CMXRos fluorescent intensity revealed a progressive increase in mitochondrial activity in oocytes from the pre-antral to the large antral follicles. Taken together, these findings demonstrated that cat oocytes undergo dynamic changes in mitochondrial copy number, distribution and activity during folliculogenesis. These significant modifications to this crucial cytoplasmic organelle are likely associated with the acquisition of developmental competency by cat oocytes.

Time within reproductive season, but not age or inbreeding coefficient, affects seminal and sperm quality in the whooping crane (Grus americana).

All living whooping cranes (Grus americana) are descended from 16 or fewer birds that remained alive in the early 1940s, a bottleneck that puts the species at potential risk for inbreeding depression. Although AI is commonly used in the management of the captive population of this species, little is known about seminal traits or factors affecting sperm quality in the whooping crane. In the present study, semen samples were collected from 29 adult males (age 3-27 years) during the early (March), mid (April) and late (May) breeding season over 2 consecutive years. The effects of donor age, time within reproductive season and level of inbreeding on seminal characteristics were analysed using regression and information-theoretic model selection. Only time within reproductive season significantly affected seminal traits, with total numbers of spermatozoa and proportions of pleiomorphisms increasing across the season. We conclude that, even with a highly restricted number of founders, there is no discernible influence of inbreeding (at the levels described) on sperm output or quality. Furthermore, although there is variance in seminal quality, the whooping crane produces significant numbers of motile spermatozoa throughout the breeding season, similar to values reported for the greater sandhill crane (Grus canadensis tabida).

Expression pattern of matrix metalloproteinases changes during folliculogenesis in the cat ovary.

Matrix metalloproteinase (MMP) has been implicated as having roles in ovarian folliculogenesis. Here, we determined the expression pattern of six MMPs (MMP1, MMP2, MMP3, MMP7, MMP9 and MMP13) and their endogenous tissue inhibitor, TIMP1, during cat follicle growth. Different developmental stage follicles were mechanically isolated and gene expression analysed by real-time qPCR while MMP1, 2, 9 and 13 localization was determined by immunohistochemistry. With the exception of MMP13, the amount of MMP mRNA was lowest in primordial follicles and increased thereafter. Peak levels were detected in early antral follicles for MMP1 (72.2-fold increase above primordial follicle amount), MMP2 (10-fold), MMP3 (57-fold) and MMP9 (2.8-fold). MMP7 transcripts increased 2-fold by the primary follicle stage and then plateaued. MMP13 mRNA peaked in primary follicles (2.5-fold) and was lower in more advanced counterparts. TIMP1 sharply increased (6-fold) in secondary follicles and gradually declined in the later stages. MMP1 and MMP9 expression were expressed in the granulosa cells of all follicle stages. MMP2 was immunoreactive in early and antral follicles, especially at granulosa cells adjacent to the antral cavity. By contrast, the MMP13 was weakly detected in primary follicles onward. In summary, there are distinctive and consistent changes in MMPs and TIMP1 expression during follicle development, suggesting that these enzymes play one or more roles in cat folliculogenesis. In particular, high mRNA and protein expression levels of MMP1 and MMP2, especially at the antral stage, indicate that these enzymes likely are involved in antrum formation and expansion.

Anti-Müllerian Hormone in the Domestic Dog during the Anestrus to Oestrous Transition.

The reproductive cycle of the domestic dog features a long period of relative ovarian inactivity or anestrus. The mechanism of anestrous termination/oestrous resumption is not yet fully understood, which presents a challenge to the development of oestrous induction protocols. In this study, we assess the possibility that anti-Müllerian hormone (AMH) might play a role in this transition by characterizing its patterns of expression in the circulation during the transition from anestrus to oestrous and in all stages of ovarian follicular growth. Serum samples from five beagles (2.0-4.5 years) were collected three times per week at least 30 days prior to the onset of oestrous and assessed for AMH concentrations. Serum AMH concentration increased significantly during the transition from anestrus to proestrus and then declined back to the anestrous baseline beginning on day -4 before the luteinizing hormone surge, which was determined by changes in serum progesterone concentrations. Cortical sections of ovaries from females undergoing routine ovariohysterectomy (aged 8 months-5 years, n = 4) were evaluated for AMH by immunohistochemistry. Pre-antral and small antral follicles were most strongly immunoreactive for AMH. These data suggest that the increase in the number of antral follicles is associated with the rise in serum AMH as the anestrous period comes to an end. The rise in AMH might be useful in predicting the onset of oestrus and therefore assist with the optimization of oestrous induction protocols and possibly other assisted reproductive technologies.

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.

The domestic dog and cat as models for understanding the regulation of ovarian follicle development in vitro.

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.

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.

Activity pattern, reproductive behaviors and gonadal hormones in the raccoon dog (Nyctereutes procyonoides).

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.

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.

Oocyte quality and estradiol supplementation affect in vitro maturation success in the white-tailed deer (Odocoileus virginianus).

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.

Advances in reproductive science for wild carnivore conservation.

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.

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.