Comparative Tensile Properties and Collagen Patterns in Domestic Cat (Felis catus) and Dog (Canis lupus familiaris) Ovarian Cortical Tissues.

The importance of the ovarian extracellular environment and tissue rigidity on follicle survival and development has gained attention in recent years. Our laboratory has anecdotally observed differences in the rigidity of domestic cat and dog ovarian cortical tissues, which have been postulated to underlie the differences in in vitro culture responses between the species, wherein cat ovarian tissues display higher survival in extended incubation. Here, the tensile strengths of cat and dog ovarian cortical tissues were compared via micropipette aspiration. The underlying collagen patterns, including fiber length, thickness, alignment, curvature, branch points and end points, and overall tissue lacunary and high-density matrix (HDM) were quantified via picrosirius red staining and TWOMBLI analysis. Finally, we explored the potential of MMP (-1 and -9) and TIMP1 supplementation in modulating tissue rigidity, collagen structure, and follicle activation in vitro. No differences in stiffness were observed between cat or dog cortical tissues, or pre- versus post-pubertal status. Cat ovarian collagen was characterized by an increased number of branch points, thinner fibers, and lower HDM compared with dog ovarian collagen, and cat tissues exposed to MMP9 in vitro displayed a reduced Young's modulus. Yet, MMP exposure had a minor impact on follicle development in vitro in either species. This study contributes to our growing understanding of the interactions among the physical properties of the ovarian microenvironment, collagen patterns, and follicle development in vitro.

Cryopreservation of African painted dog (Lycaon pictus) ovarian tissue.

Development of techniques for the preservation and use of gonadal tissues are increasingly needed for the genetic management of the endangered African painted dog (Lycaon pictus). Here we evaluated two cryopreservation techniques for ovarian tissue (2 × 2 × 1 mm3 fragments, n = 11 individuals): needle immersed vitrification (NIV), with equilibration in a 7.5% dimethyl sulfoxide (DMSO) and 7.5% ethylene glycol (EG) solution, and vitrification in a 15% DMSO, 15% EG, and 0.5 M sucrose solution, and slow freezing in cryovials with either the equilibration (SF-E) or vitrification (SF-V) solutions. Following warming, tissues were either fixed and embedded for evaluation of density of morphologically normal follicles, semi-quantitative scoring of stromal cell preservation, and apoptotic index (TUNEL stain), and/or flash-frozen for expression of proliferation (PCNA), apoptosis (CASP3, BCL2), or oxidative stress (GPX3, SOD1, SOD2) pathway genes (n = 4). Needle immersed vitrification maintained higher density of morphologically normal follicles compared to the slow freezing protocols applied (p < 0.05), with no significant changes in expression of select genes among treatment groups. A slight increase in apoptotic index was observed in all cryopreservation groups, but only reached significance in SF-E compared with fresh tissue controls (p < 0.05). Future research should be dedicated to developing improved methods for ovarian tissue culture in the species, both as a means to evaluate the efficacy of tissue cryopreservation techniques and for the production of viable oocytes from banked ovarian tissue in the endangered African painted dog.

In vitro development of mechanically and enzymatically isolated cat ovarian follicles.

Isolation of ovarian follicles is a key step in culture systems for large mammalian species to promote the continued growth of follicles beyond the preantral stage in fertility preservation efforts. Still, mechanical isolation methods are user-skill dependent and time-consuming, whereas enzymatic strategies carry increased risk of damaging theca cell layers and the basement membranes. Here, we sought to determine an optimal method to rescue domestic cat (Felis catus) early antral and antral stage follicles from ovarian tissue and to evaluate the influence of isolation strategy on follicle development, survival, and gene expression during 14 days of in vitro culture in alginate hydrogel. Mechanical isolation was compared with 90 min digestion in 0.7 and 1.4 Wünsch units/mL Liberase blendzyme (0.7L and 1.4L, respectively). Mechanical isolation resulted in improved follicle growth and survival, and better antral cavity and theca cell maintenance in vitro, compared with 1.4L (P < 0.05) but displayed higher levels of apoptosis after incubation compared with enzymatically isolated follicles. However, differences in follicle growth and survival were not apparent until 7+ days in vitro. Expressions of CYP19A1, GDF9, LHR, or VEGFA were similar among isolation-strategies. Cultured follicles from all isolation methods displayed reduced STAR expression compared with freshly isolated follicles obtained mechanically or via 0.7L, suggesting that prolonged culture resulted in loss of theca cell presence and/or function. In sum, early antral and antral stage follicle development in vitro is significantly influenced by isolation strategy but not necessarily observable in the absence of extended culture. These results indicate that additional care must be taken in follicle isolation optimizations for genome rescue and fertility preservation efforts.

Canid Reproductive Biology: Norm and Unique Aspects in Strategies and Mechanisms.

The reproductive physiology of canids is unique compared to other mammalian species. Specifically, the reproductive cycle of female canids is characterized by extended periods of proestrus and estrus followed by obligatory diestrus and protracted ovarian inactivity (anestrus). Although canid reproduction follows this general pattern, studies have shown variations in reproductive biology among species and geographic regions. Understanding of these differences is critical to the development of assisted reproductive technologies including estrus induction, gamete rescue, and embryo production techniques for canid conservation efforts. This review summarizes current knowledge of canid reproduction, including estrus cyclicity, seasonality, and seminal traits, with the emphasis on species diversity. The application of reproductive technologies in wild canid conservation will also be discussed.

Cryopreservation of grey wolf (Canis lupus) testicular tissue.

Development of genomic preservation technologies for canids, especially for seasonally breeding species like the grey wolf (Canis lupus), is needed in advance of growing species conservation concerns. Here, we evaluated the efficacy of two cryopreservation protocols - needle immersion vitrification (NIV) and slow freezing (SF) on grey wolf (n = 7) testicular tissue morphology. NIV samples were equilibrated in a 7.5% v/v dimethyl sulfoxide (DMSO or Me2SO) + 7.5% ethylene glycol (EG) solution in minimum essential medium with 20% FBS for 10 min at 4 °C, then exposed to 15% DMSO + 15% EG + 0.5 M sucrose for 10 min at 4 °C before plunging into liquid nitrogen. For slow freezing, we assessed two cryoprotectant (CPA) strategies, DMSO, 15% v/v alone (SF-D) or 7.5% EG + 7.5% DMSO (SF-ED). Following thawing, there were no significant differences in seminiferous tubule area among treatment groups, although all cryopreserved tissues displayed reduced tubule size compared with fresh controls and increased apoptosis, the latter reaching significance for SF-D treated tissues. Slow freezing improved maintenance of testis architecture, with minimal detachment of seminiferous tubule basement membranes post-thaw. Spermatogonia densities were reduced in NIV tissues compared with fresh, with no differences in spermatocyte, spermatid, or Sertoli cell counts, or germ cell marker DDX4+ cell densities among groups. In sum, we conclude that slow freezing better maintained morphology of cryopreserved testicular tissues compared with needle vitrification with 15% each DMSO and EG and 0.5 M sucrose, and that DMSO + EG combination SF supports cell viability. This represents a first step in the development of male gonadal tissue preservation strategies for the grey wolf.

Investigating media that support red wolf (Canis rufus) sperm viability and capacitation in vitro.

The red wolf is a critically endangered canid, with ~250 and ~20 individuals in the ex situ and reintroduced wild populations, respectively. Assisted reproductive technologies such as sperm cryopreservation and in vitro fertilization therefore represent critically-needed tools to manage these populations. However, the motility of post-thaw red wolf sperm rapidly declines during in vitro incubation, hindering the ability to develop these technologies. In this study, we evaluated the influence of several culture media (a modified canine capacitation medium (mCCM), a modified North Carolina State University-23 medium (mNCSU-23), a synthetic oviductal fluid (SOF), a fertilization Tyrode's medium base or Fert-TALP (FERT), and a TRIS-based buffer (TRIS)) on the survival and capacitation of red wolf sperm during extended (18 h) incubation at 38.5°C and 5% CO2. Red wolf sperm motility averaged (±s.e.m.) 73.8 ± 7.1% at the time of collection, and was better maintained over 4 h incubation in mCCM (55.0 ± 9.8%) and mNCSU-23 (54.7 ± 10.4), compared to mSOF (43.8 ± 8.3%), FERT (30 ± 10.5), and TRIS (16.4 ± 4.1%) solutions. Patterns of tyrosine phosphorylation signal, as assessed via immunocytochemistry, indicated induction of capacitation between 2 and 4 h in vitro culture. Tyrosine phosphorylation signal was particularly robust in mCCM and mNCSU-23 incubated sperm, although significant acrosome exocytosis was not observed in response to progesterone supplementation after 3 h incubation in any of the media. In sum, results indicate mCCM and mNCSU-23 are promising base media for the in vitro incubation and capacitation of red wolf sperm, for assisted reproduction applications. LAY SUMMARY: Development of assisted reproductive technologies such as in vitro fertilization and artificial insemination is of high importance to the genetic management of critically endangered species such as the red wolf (Canis rufus). However, these technologies require the ability to maintain sperm viability and function during extended incubation, which has not been successful for the red wolf thus far. In this study, various culture media developed for sperm/egg/embryo culture in large mammalian species were evaluated for their ability to maintain red wolf sperm motility under physiological incubation conditions. Media and conditions previously utilized for domestic dog sperm were found to best support sperm incubation and capacitation (process of becoming competent to fertilize an egg) in the red wolf, representing a key step for future development of assisted reproductive technologies for the species.

Activin promotes growth and antral cavity expansion in the dog ovarian follicle.

Understanding regulators of folliculogenesis remains limited in the domestic dog (Canis familiaris), which challenges our ability to develop in vitro follicle culture systems for canid genome rescue efforts. Here, we investigated the influence of activin on dog follicle development and survival, oocyte quality, and FSH receptor expression in culture. Preantral (150 - ≤230 µm diameter), early antral (231 - ≤330 µm), and antral (>330-550 µm) stage follicles were encapsulated in a fibrin-alginate hydrogel with 0, 100, or 200 ng/ml rhActivin plus 0, 0.1, 1, or 10 µg/ml FSH for 12 or 21 d of in vitro culture. All follicle groups increased in diameter (P < 0.05) with activin acting synergistically with FSH to improve (P < 0.05) growth and antral cavity expansion (to >630 µm) in early antral and antral cohorts. This complementary effect was not linked to changes in FSHR mRNA expression (P > 0.05). Although not influencing (P > 0.05) follicle survival or transzonal projection (TZP) density in shorter term 12 d culture, activin in the presence of 1 ng/ml FSH maintained TZP density from the 12-21 d interval. Activin also increased oocyte diameter and improved nuclear integrity compared to un-supplemented controls. These results indicate that activin acts synergistically with FSH to promote growth and antral cavity expansion of the dog follicle in vitro, information useful to formulating an effective culture microenvironment for this species.

Evaluation of an ovary-on-a-chip in large mammalian models: Species specificity and influence of follicle isolation status.

The ability to grow oocytes from immature ovarian follicles in vitro has significant potential for fertility preservation; yet, it has proved challenging in large mammalian species due to the complex metabolic needs and long-term culture requirements. Currently, follicular incubations are based on a "static" system with manual exchange of medium. Despite the numerous advantages of conventional culturing approaches, recapitulating the native microenvironment and supporting the survival of ovarian follicles from large mammalian species still represent challenges. In this study, we utilized an innovative, dynamic microfluidic system to support the in vitro survival of domestic cat and dog follicles enclosed within the ovarian cortex or isolated from ovarian cortex. Results indicate both species-specific and tissue type-specific differences in response to microfluidic culture. Domestic cat but not dog ovarian cortical tissues maintained viability under flow similar to conventional agarose gel controls. Preantral stage isolated follicles from both species that grew most favourably in conventional alginate bead culture, but overall, there was no influence of culture system on expression of follicle development or oocyte health markers. This system represents an important exploration toward the development of an improved ovarian in vitro culture system of large mammalian species (e.g., cats and dogs), which has potential applications for fertility preservation, reproductive toxicology, and endangered mammal conservation efforts.

Age-associated and deslorelin-induced declines in serum anti-Müllerian hormone concentrations in female cheetahs, Acinonyx jubatus.

Anti-Müllerian hormone (AMH) is widely used in human medicine to non-invasively estimate the size of the ovarian follicle reserve and to predict the ovarian response to gonadotropin stimulation in the context of assisted reproductive technologies (e.g., IVF). These applications of AMH testing have recently expanded to non-human mammals, with production animals, such as cows, goats and sheep being the primary focus of AMH research. However, few investigations have involved exotic species, and in particular carnivores. In this study, we measured AMH concentrations (0.078-3.078ng/mL) in archived serum samples that had been collected from 36 adult female cheetahs across their reproductive lifespan (2-15years of age). Similar to other mammals, AMH concentration in cheetahs declined with age, and its variability among females of the same age was considerable. The rates at which AMH declined over time in individual cheetahs were also highly variable. Five cheetahs had been contracepted with the long-acting GnRH agonist deslorelin for 6-18months prior to sample collection, and their AMH concentrations were relatively low compared to untreated females. In this first study of AMH in an exotic carnivore, the findings demonstrate that the age-associated decline in AMH is highly variable and that deslorelin appears to suppress AMH concentration in serum. Owing to the increased use of assisted reproductive technologies in ex situ populations of threatened and endangered species, such as cheetahs, the present study's findings will need to be taken into consideration if AMH is to be used successfully to optimize breeding management decisions in exotic species.

Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization.

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies-an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4-5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.