Culture of vitrified bovine ovarian tissue on agarose gel inserts maintains follicle integrity.

In brief: Ovarian tissue cryopreservation and culture provide an option for fertility preservation without tissue grafting, but need optimization. This study reveals that vitrified bovine ovarian tissue can be cultured on agarose gel and maintain follicle morphology, low activation, and low apoptosis. Abstract: Ovarian tissue preservation is hitherto a promising fertility insurance option for precious animals. Ovarian tissue vitrification and culture combined approach would eliminate the need of transplanting ovarian tissue to obtain mature oocytes. We aimed at optimizing vitrification and in vitro culture conditions for improved bovine ovarian tissue viability. Ovaries obtained from the slaughterhouse were punched into fragments and divided into three groups. Group 1 (fresh) was divided into two and immediately placed in two-culture systems (culture inserts and agarose inserts). Group 2 was vitrified, warmed, and placed in the two-culture systems, while group 3 was only equilibrated and then placed in the two-culture systems. All cultures were maintained for 6 days and spent media were collected on alternate days for cytokine (interleukin 1ß and interleukin 6) evaluation. Fragments were fixed for morphology assessment and immunohistochemistry. Higher percentages (P < 0.05) of grade 1 (morphologically intact) follicles were observed in fragments on agarose compared to those on culture inserts on days 2 and 4 of the culture. Conversely, we found higher (P < 0.05) shifts of primordial follicles to transitional follicles in fragments on culture inserts vis-à-vis agarose inserts which was consistent with a higher proportion of Ki-67 and MCM-7 and activated caspase-3-positive follicles. In conclusion, in vitro culture of bovine ovarian tissue on agarose inserts maintained follicle morphology, low follicle activation, and low apoptosis compared to culture inserts.

Ovary cold storage and shipment affect oocyte yield and cleavage rate of cat immature vitrified oocytes.

In feline species, cooled transport of ovaries can be employed without detrimental effects to retrieve immature oocytes intended for in vitro embryo production purposes. Indeed, this is the most common way to collect gametes from gonads of wild, valuable animals after they die or are castrated far from specialized laboratories. However, fresh retrieved gametes are generally used, and their cryosensitivity is not known. This study employed ovariectomy-derived domestic cat gonads as a model for wild felids, and aimed to compare the yield and developmental competence of Cryotop-vitrified oocytes (VOs) collected and cryopreserved right after ovary excision (In loco-VOs) or after 24 h cooled transport of ovaries (Shipped-VOs). The number of collected oocytes was higher in In loco-VOs than in Shipped-VOs (mean ± SD: 8 ± 3.36 vs 5.6 ± 3.1, p = 0.05). In vitro embryo production resulted in similar maturation (35% for both vitrified groups, p = 1) and fertilization rates (In loco-VOs: 29.1%; Shipped-VOs: 22.2%; p = 0.295), but showed a difference in cleavage (In loco-VOs: 25.6%; Shipped-VOs: 14.5%; p = 0.0495). No differences were found in further embryo development. Taken together, results suggested that delayed oocyte vitrification after cooled transport of organs was feasible and allowed embryo development. However, the number of collected oocytes and the cleavage rate of matured oocytes were higher when oocyte vitrification was performed without delay after ovary excision, and this should be considered in gamete conservation programs for endangered felids.

Advanced ultrasound techniques in small animal reproduction imaging.

Ultrasonography is the imaging technology of choice for the evaluation of the reproduction system and of pregnancy in both humans and animals. Over the past 10 years, there have been significant technological improvements of the equipment, while new technologies have been developed. Doppler, contrast-enhanced ultrasonography, elastography, and 3D/4D ultrasonography are advanced ultrasound techniques that have been designed as methods to increase the diagnostic sensitivity of two-dimensional (b-mode) ultrasound, and not as stand-alone tests. The basic physics as well as the advantages and limitations of these advanced ultrasound methods are briefly described. In the reproductive diagnostics of small animals, these techniques have gained an increased popularity as proved by the increased publication of several reports that are also briefly summarized in this review. Clinical applicability is to date limited because of a lack of research on the diagnostic value in concrete situations. Future research projects should focus also on standardization of the used techniques, on determination of thresholds to discriminate between healthy or diseases or fertile versus infertile and on the predictive value of advanced ultrasound findings. Continuing development and optimization of different ultrasound techniques as well as the increase of related scientific interest and worldwide research promises that the clinical interest on the use of advanced ultrasound techniques will increase in future.

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

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.

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

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.

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

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?

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

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.

FGF2 and EGF Are Required for Self-Renewal and Organoid Formation of Canine Normal and Tumor Breast Stem Cells.

Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self-renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi-lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self-renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570-584, 2017. © 2016 Wiley Periodicals, Inc.

Melatonin reduces oxidative stress and improves follicular morphology in feline (Felis catus) vitrified ovarian tissue.

Ovarian tissue vitrification is associated with multiple events that promote accumulation of ROS (reactive oxygen species) which culminate in follicular apoptosis. Thus, this study was aimed at evaluating the role of melatonin in vitrification and culture of feline (Felis catus) ovarian tissue. In phase 1, domestic cat ovaries were fragmented into equal circular pieces of 1.5 mm diameter by 1 mm thickness and divided into four groups (fresh control and 3 treatments). The treatments were exposed to vitrification solutions supplemented with melatonin at 0 M, 10-9 M, and 10-7 M, then vitrified-warmed, histologically evaluated and assayed for ROS. Consequently, phase 2 experiment was designed wherein ovarian fragments were divided into two groups. One group was exposed to vitrification solution without melatonin and the other with 10-7 M melatonin supplementation, then vitrified-warmed and cultured for ten days with fresh ovarian fragments as control prior to assessment for histology, immunohistochemistry (Ki-67, MCM-7 and caspase-3) and ROS. Concentration of ROS was lower (p = 0.0009) in 10-7 M supplemented group in addition to higher proportion of grade 1 follicles. After culture, proportions of intact and activated follicles were higher (p < 0.05) in melatonin supplemented group evidenced by higher expression of Ki-67 and MCM-7. Follicular apoptosis was lower in melatonin supplemented group. In conclusion, melatonin at 10-7 M concentration preserved follicular morphological integrity while reducing ROS concentration in vitrified-warmed feline ovarian tissue. It has also promoted the follicular viability and activation with reduced apoptosis during in vitro culture of vitrified-warmed feline ovarian tissue.

Effect of Fixatives and Fixation Period on Morphology and Immunohistochemistry of Feline Ovarian Tissue.

Fixatives and fixation protocol have a profound effect on both the morphology and epitope sensitivity of ovarian tissue, which hampers accurate ovarian tissue evaluation. We aimed to establish the most suitable fixation protocol for feline (Felis catus) ovarian tissue. Fragments (1.5 mm diameter) were punched from 1 mm-thick feline ovarian tissue, divided into three groups then fixed with three different fixatives (Bouin, neutral buffered formalin [NBF] and form acetic acid [new compound fixative formulation for ovarian tissue composed of 5% acetic acid in NBF]) for five fixation periods. Subsequently, fragments were processed and evaluated for the morphology and intensity of immunohistochemical signals against three antigens (Ki-67, MCM-7 and activated caspase-3). Proportions of grade 1 or morphologically intact follicles were significantly lower in NBF when compared with Bouin and form acetic acid fixatives. However, Bouin fixative had the lowest mean DAB intensity (p < 0.05) in all three antigen targets, while NBF had the highest (p < 0.05) in Ki-67 and caspase-3, but in MCM-7, it was no different from form acetic acid. In conclusion, form acetic acid maintained ovarian tissue architecture with excellent follicular morphology in the same manner as Bouin fixative, and it also maintained reasonable DAB signals similar to NBF, thus providing a better alternative for feline ovarian tissue studies.

Vitrification of feline ovarian tissue: Comparison of protocols based on equilibration time and temperature.

Global contraction of biodiversity pushed most members of Felidae into threatened or endangered list except the domestic cat (Felis catus) thence preferred as the best model for conservation studies. One of the emerging conservation strategies is vitrification of ovarian tissue which is field-friendly but not yet standardized. Thus, our main goal was to establish a suitable vitrification protocol for feline ovarian tissue in field condition. Feline ovarian tissue fragments were punched with biopsy punch (1.5 mm diameter) and divided into 4 groups. Group 1 was fresh control (Fr), while the other three were exposed to 3 vitrification protocols (VIT_CT, VIT_RT1 and VIT_RT2). VIT_CT involved two step equilibrations in solutions containing dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 10 min each at 4 °C. VIT_RT1 involved three step equilibration in solutions containing DMSO, EG, polyvinylpyrrolidone and sucrose for 14 min in total at room temperature, while in VIT_RT2 all conditions remained the same as in VIT_RT1 except equilibration timing which was reduced by half. After vitrification and warming, fragments were morphologically evaluated and then cultured for six days. Subsequently, follicular morphology, cellular proliferation (expression of Ki-67, MCM-7) and apoptosis (expression of caspase-3) were evaluated, and data obtained were analysed using generalised linear mixed model and chi square tests. Proportions of intact follicles were higher in Fr (P = 0.0001) and VIT_RT2 (P = 0.0383) in comparison to the other protocols both post warming and after the six-day culture. Generally, most follicles remained at primordial state which was confirmed by the low expression of Ki-67, MCM-7 markers. In conclusion, VIT_RT2 protocol, which has lower equilibration time at room temperature has proven superior thus recommended for vitrification of feline ovarian tissue.

Microenvironment factors promoting the quality of vitrified cat oocytes.

In oocyte cryopreservation programs, vitrification has overthrown conventional slow freezing both in veterinary and human medicine. In animals, its feasibility in field conditions makes it the preferred technique for the safeguard of genetic resources from zoo or wild animals, including threatened felids, for which the domestic cat is an excellent model. However, many cellular injuries, such as cytoskeleton, mitochondria and meiotic spindle alterations, DNA damage, zona pellucida hardening and cumulus cell loss, might occur following vitrification. After warming, although the exact mechanisms are still unclear, degeneration is a frequent outcome for cat vitrified oocytes. For immature (germinal vesicle) gametes, in vitro maturation after warming is a challenge, and cleavage after fertilization barely reaches 15-30%, while for mature (metaphase II) cryopreserved gametes it can get to 30-50%. Anyway, the progression to late embryos stages is often impaired, and improvements are needed. Standard cryopreservation protocol and the use of conventional in vitro culture systems after warming may not be enough for vitrified oocytes to recover and demonstrate their full developmental potential. Physical or chemical factors applied to oocytes undergoing vitrification, as an enrichment to the vitrification step, or to the culture microenvironment, could create more favorable conditions and promote vitrified oocyte survival and development. From the use of three-dimensional culture systems to the regulation of metabolic activities and cellular pathways, this review aims to explore all the possibilities employed so far, including the studies performed by our own lab, and the future perspectives, to present the most effective strategies for cat oocyte vitrification and the best time for their application (i.e., before, during, or after vitrification-warming).

In vivo embryo development in bitches inseminated laparoscopically after ovulation time estimated based on a single progesterone determination.

Logistic and economical limitations are often the causes of dog owners not accurately monitoring the estrous cycle and the optimal insemination time. The aim of this study was to evaluate in vivo early embryonic development in bitches, after the analysis of sequential vaginal cytologies associated to single progesterone measurement and single laparoscopic insemination with high quality semen (fresh and with high spermatozoa concentration) or low-quality semen (frozen/thawed and with low spermatozoa concentration) at 48 h post- ovulation time predicted on a single progesterone measurement. Ten bitches were inseminated with 250 x 106 fresh spermatozoa (80% motility), and ten with 80 x 106 frozen/thawed spermatozoa (60% motility) in the cranial part of each uterine horn. Seven days later, ovariohysterectomy was performed and the oviducts and uterine horns and body were flushed to recover embryos and unfertilized oocytes. In 80% of the bitches inseminated with fresh and 50% of bitches inseminated with frozen/thawed semen, embryos at 2 to 8 cells stage were recovered mostly from the, oviducts. This study indicates that pregnancies can be obtained with a single laparoscopic intrauterine insemination after single serum progesterone measurement, although with a low number of embryos. This result should be taken into account in case economic or logistic restrictions that affect the possibility of owners to plan an accurate monitoring of the optimal breeding time using fresh and frozen semen.

Canine Spermatozoa-Predictability of Cryotolerance.

Markers of freezability allow the selection of ejaculates of good freezability. So far, most investigations were conducted in boars, bulls, rams and horses, with high economic interests triggering the efforts. The progress in dogs is comparably slow. A critical evaluation of the methods requires consideration of practicability, with most labs not even possessing a computer assisted sperm analyser (CASA); furthermore, small canine ejaculates mostly do not allow the use of large semen volumes. In dogs, modern markers of freezability no longer assess single membrane constituents or seminal plasma components but comprise tests of cell functionality and adaptability, energy metabolism, cluster analyses of kinetic and morphometric parameters, as well as DNA intactness. Identification of the most efficient combination of tests seems useful. At present, examination by CASA combined with cluster analysis of kinetic subgroups, JC-1 staining and COMET assay or staining with toluidine blue seem most appropriate; however, cell volumetry and other functional tests deserve better attention. A better understanding of spermatozoa energy metabolism might reveal new markers. This review focuses on the requirements and markers of freezability of canine semen, highlighting potential future candidates.

Ultra-Rapid Freezing Preserves Morphofunctional Integrity and Fertilizing Ability of Epididymal Cat Spermatozoa.

Vitrification and ultra-rapid freezing, which are more commonly used for oocytes and embryos, have recently been applied to spermatozoa in an attempt to make semen cryopreservation in field conditions easier compared to conventional freezing. It is well-known that in case of unexpected death of rare and wild animals, preserving epididymal spermatozoa from isolated testicles represents a great chance of salvaging male germplasm for future use in assisted reproductive technologies. The aim of this study was to evaluate the morphofunctional integrity of cat epididymal spermatozoa ultra-rapid frozen in pellets or straws with two different extenders [E1 (Tris buffer with 20% egg yolk and 0.25 M sucrose) or E2 (Ham's F10 with 1% bovine serum albumin and 0.4 M sucrose)] and to test whether spermatozoa preserved by the best combination were able to fertilize oocytes and produce embryos in vitro by intracytoplasmic sperm injection (ICSI) of in vitro matured cat oocytes. The results showed that E1 and E2 in straw or pellet were comparable (at warming, about 30% normal morphology, 45% intact membranes, and 20% intact acrosomes), except for post-warming motility that was better maintained along time by E1 pellet (21.7 ± 7.4% at warming and 3.6 ± 2.9% after 6 h). Such spermatozoa could fertilize conspecific oocytes and support embryonic development (cleavage 35.5%) as well as frozen control spermatozoa (cleavage 54.29%, p = 0.22). In conclusion, cat epididymal spermatozoa better maintained their morphofunctional features after ultra-rapid freezing with E1 and could successfully produce embryos in vitro after ICSI. This underscores their usefulness as cryobanked material for fertility and biodiversity preservation purposes.

Freezability of Dog Semen after Collection in Field Conditions and Cooled Transport.

Dog semen freezing is gaining popularity, but it has to be performed in equipped facilities, which can be far from the place where the stud dog lives. The aim of this study was to evaluate whether freezing dog semen after 24 or 48 h of cooled transport to an equipped laboratory was possible when semen collection was performed in the field such as in local breeding kennels. Single ejaculates from different dogs (mixed breeds and ages) were collected. In Experiment I, 10 ejaculates were conventionally frozen using the Uppsala method or frozen after 24 or 48 h of storage in a Styrofoam transport box cooled by icepacks. In Experiment II, 10 ejaculates were used to assess the influence of two extenders (Uppsala chilling extender or freezing extender 1) used for semen dilution during the 24 or 48 h storage. Motility, morphology, membrane, and acrosome integrity were analyzed as well as spermatozoa zona-binding ability. No significant differences were observed among the frozen groups, regardless of freezing time (Experiment I) or extender (Experiment II). Motility at thawing, however, decreased in absolute value at 48 h. Freezing of freshly collected semen is the gold standard, but the results obtained in this study prompt the application of freezing after cooled transport for the long-term preservation of dog semen, especially if the transport can be organized in 24 h.

Spermatozoa Survival in Egg Yolk-Based and Soybean-Based Extenders at Ambient and Chilling Temperature in Domestic Turkeys (Meleagris gallopavo).

Populations of many galliform species have declined mainly due to habitat loss and over-hunting, notably the Congo peacock, which has been classified as a vulnerable species by the International Union for Conservation of Nature (IUCN). The domestic turkey, being a species of least concern, which has been reported to be closely related to peacocks, could serve as a model for the optimization of assisted reproductive technologies for the Congo peacock. This study was aimed at developing a suitable turkey semen extender for artificial insemination in field conditions. Semen was collected using the dorso-abdominal massage technique from seven turkey toms and analyzed. Ejaculates with >70% motility and >80% live spermatozoa were pooled and divided into four aliquots (four treatments). Each of the four treatments was extended in a soybean-based extender or an egg yolk-based extender, with or without L-ascorbic acid. Two liquid preservation protocols (ambient temperature (35 °C) and chilled (4 °C)) were employed, and quality parameters including motility, viability and morphology were evaluated. The results show that the two extenders were similar with regard to semen quality parameters, and L-ascorbic acid supplementation of the turkey semen extenders improved semen quality during liquid storage.

Fighting Like Cats and Dogs: Challenges in Domestic Carnivore Oocyte Development and Promises of Innovative Culture Systems.

In vitro embryo production in cats and dogs still presents some challenges, and it needs to be optimized to transfer efficient protocols to related wild, endangered species. While the chemical composition of culture media has been the focus of several studies, the importance of culture substrates for oocyte and embryo culture has often been neglected. Traditional in vitro systems, i.e., two-dimensional cultures, do not resemble the physiological environments where cells develop, and they may cause morphological and functional alterations to oocytes and embryos. More modern three-dimensional and microfluidic culture system better mimic the structure and the stimuli found in in vivo conditions, and they could better support the development of oocytes and embryos in vitro, as well as the maintenance of more physiological behaviors. This review describes the different culture systems tested for domestic carnivore reproductive cells along the years, and it summarizes their effects on cultured cells with the purpose of analyzing innovative options to improve in vitro embryo production outcomes.

Canine and Feline Epididymal Semen-A Plentiful Source of Gametes.

Canine and feline epididymal semen provide an additional source of gametes to preserve the genetics of valuable breeding dogs and tomcats, especially for those that fail to ejaculate, need castration as a therapy or die unexpectedly. Moreover, since it is quite common to perform castration of non-breeding dogs and cats, the development of a gene bank of epididymal semen collected after castration would greatly contribute to increase the genetic diversity in dogs and cats. Collection and cryopreservation of epididymal semen necessitates a full understanding of the function of the epididymis and of the characteristics of epididymal spermatozoa as opposed to ejaculated semen. During collection of epididymal semen, specific factors may have a negative effect on epididymal semen quality and freezability. Accordingly, the elimination of these triggers could enhance epididymal semen freezability and consequently positively influence post-thaw semen quality and outcome for different ARTs.

Cold case: Small animal gametes cryobanking.

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.