Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Università degli Studi di Milano

To evaluate the ability of a 3D culture system in improving the nuclear and molecular competence of canine oocytes, barium alginate microcapsules were used for in vitro maturation (IVM) and the expression profile of one selected oocyte-secreted factor, the growth differentiation factor-9 (GDF-9) was analysed. In Experiment I, canine grade I cumulus-oocyte complexes (COCs) were in vitro matured in 3D microcapsules in a controlled atmosphere for 72 hr, and meiosis resumption rates were compared to those of oocytes cultured in traditional 2D microdrops of medium. In Experiment II, a primer pair specific for canine GDF-9 was designed, and preliminary tested in conventional PCR on genomic DNA. Total RNA content was isolated from oocytes at different time intervals (T0-T24-T48-T72) during in vitro 3D culture, and a reverse transcription to cDNA was performed. The expression of target gene was assessed by quantitative Reverse Transcription Real-Time PCR (qRT-PCR), and the obtained amplicons were sequenced to check the specificity of the analysis. Canine COCs resumed meiosis at higher rates in 3D microcapsules than in 2D microdrops (p < 0.05), even though no significant differences in the proportions of oocytes achieving full maturational stages were obtained. A significant dynamic decrease in GDF-9 expression was recorded during culture: after 72 hr of IVM, the GDF-9 transcription significantly dropped (p = 0.018) compared to 24 and 48 hr. In conclusion, in vitro 3D culture represents an efficient system for IVM of canine oocytes, and the expression profile of GDF-9 well reflects temporal dynamics for the acquisition of developmental competence in this species.

Nuclear competence and genetic expression of growth differentiation factor-9 (GDF-9) of canine oocytes in 3D culture.

In the domestic cat, in vitro fertilization started 40 years ago, but an ideal culture system has yet to be achieved. The physiological microenvironments, which interact with oocytes and embryos promoting their competence, have been investigated. However, recreating in vitro follicle- and oviduct-like conditions is challenging and a matter of both chemistry and physics. This review presents an excursus of the experimental investigations focused on the improvement of feline oocytes and embryos culture through the modulation of chemical and physical factors. Medium supplementation with components of follicular and oviductal fluids, or the use of different co-cultures, supports or substrata have been considered. Innovative and sophisticated systems as "organ-on-a-chip" might lead to the creation of artificial follicles and oviducts and they may represent the ideal combination of chemical and physical factors. Will the search ever end?

The never-ending search of an ideal culture system for domestic cat oocytes and embryos.

Attempting to contribute to the development of a more objective morphological evaluation of dog spermatozoa, in this study the indices of multiple sperm defects (multiple abnormalities index [MAI]; teratozoospermic index [TZI]; sperm deformity index [SDI]) were calculated following the World Health Organization (WHO) guidelines. In Experiment I, the concordance of MAI, TZI and SDI with the proportions of morphologically normal spermatozoa (MNS) was evaluated in fresh ejaculated spermatozoa (dogs = 47). In Experiment II, the potential role of indices as prognostic values was assessed in spermatozoa of different origin and treatment (fresh ejaculated: n = 6; fresh epididymal: n = 6; frozen-thawed ejaculated: n = 6) by their correlation with different semen parameters (motility, membrane integrity and acrosome status) and with an in vitro sperm function test. Samples with different proportions of MNS showed different values of SDI, the index that better represented the decline of sperm morphology in both fresh and frozen-thawed samples (Exp. I and II; p < 0.05). No correlations between indices and semen parameters were observed (Exp. II), but when samples were evaluated collectively, negative correlations (SDI-motility, p = 0.01; SDI-acrosome integrity, p = 0.002) were found. Including all the defects of each spermatozoon, SDI might be a useful index during morphological analysis and better discriminates the increase in multiple defects. A more objective morphological evaluation for dog spermatozoa was achieved by the WHO method, and in vitro tests allowed to elucidate the validity of SDI as prognostic indicator of in vitro fertilizing potential.

Morphological indices for canine spermatozoa based on the World Health Organization laboratory manual for human semen.

Cryoinjuries severely affect the competence of vitrified oocytes (VOs) to develop into embryos after warming. The use of culture conditions that provide physical and chemical support and resemble the in vivo microenvironment in which oocytes develop, such as 3D scaffolds and coculture systems, might be useful to improve VOs outcomes. In this study, an enriched culture system of 3D barium alginate microcapsules was employed for the in vitro embryo production of domestic cat VOs. Cryotop vitrified-warmed oocytes were in vitro matured for 24 h in the 3D system with or without fresh cumulus-oocyte complexes (COCs) in coculture, whereas a control group of VOs was cultured in traditional 2D microdrops of medium. After in vitro fertilization, presumptive embryos were cultured in 3D or 2D systems according to the maturation conditions. Vitrified oocytes were able to mature and develop into embryos in 3D microcapsules (17.42 ± 11.83%) as well as in 2D microdrops (14.96 ± 8.80%), but the coculture with companion COCs in 3D resulted in similar proportions of VOs embryo development (18.39 ± 16.67%;  = 1.00), although COCs presence allowed for blastocyst formation (0.95 ± 2.52%). In conclusion, embryos until late developmental stages were obtained from cat VOs, and 3D microcapsules were comparable to 2D microdrops, but improvements in post-warming conditions are still needed.

Developmental Competence of Domestic Cat Vitrified Oocytes in 3D Enriched Culture Conditions.

Follicle-like structures are three-dimensional matrices joint with living cells that allow the in vitro development of female gametes in more physiological conditions. They have been shown to be beneficial to fresh oocytes in different species, and in this study, domestic cat (Felis silvestris catus) granulosa cells were used to create a functional follicle-like structure aimed at supporting the in vitro maturation of conspecific vitrified oocytes, key players of fertility preservation programmes that usually struggle to acquire their full developmental competence after warming. Cat granulosa cells were cultured for up to 6 days in three-dimensional barium alginate microcapsules (i.e. follicle-like structures) or in two-dimensional monolayers, and their steroidogenic ability (estradiol and progesterone secretion) was assessed to confirm their functionality. The same systems were used (on day 2 or 6 of granulosa cells culture) for the in vitro maturation (IVM) of Cryotop vitrified immature cat oocytes and compared with microdrops of IVM medium without cells (control). Granulosa cells were able to maintain their functionality in vitro in both the conditions, even if with a different extent of hormonal secretion along culture (p = .02). Vitrified oocytes resumed meiosis at higher rates when cultured with 2 days old granulosa cells (p = .03), but full maturation rates slightly raised when granulosa cells were cultured longer, albeit without differences with the control group. This study paved the road for the creation of enriched culture systems in the domestic cat, but innovations are strongly needed for vitrified oocytes that deserve better chances to develop in vitro.

Granulosa cells in three-dimensional culture: A follicle-like structure for domestic cat vitrified oocytes.

Germplasm preservation of animals, whether they are valuable domestic breeds or rare species, is the main goal of gamete cryobanking. Dogs and cats act as models for this purpose thanks to the wide availability of biological material which can be employed to experiment protocols that can then be applied to wild animals. This review is focused on spermatozoa, oocytes and gonadal tissues cryobanking in small domestic animals, which is still an unsolved case. Like in a courtroom, evidences of cryoinjuries affecting cellular structures will be presented, penalties as loss of functionality due to cellular alterations will be described, and appeal as strategies to protect gametes from damages or rescue their functionality will be discussed. Differences and similarities between single cell or tissue cryopreservation will be highlighted, together with the rationale for the choice of one type of preservation or another and the fundamental principles which they are based on. The deep analysis of different aspects that still hamper the success of cryopreservation in small animals can help clarify where research is most needed. Therefore, as in a cold case, investigation should remain open in order to hopefully find the solution and make these procedures more and more efficient in the future.

Martina Colombo

Dipartimento di Scienze Veterinarie per la Salute,
la Produzione Animale e la Sicurezza Alimentare 'Carlo Cantoni',
Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare 'Carlo Cantoni'

Università degli Studi di Milano

Minimum Volume Vitrification of Immature Feline Oocytes