Understanding critical roles of warming and reanimation is critical to improve the survival of vitrified testicular tissue in domestic cats. The objective was to study structural and functional properties of testicular tissues from prepubertal domestic cats after standard vitrification followed by two warming protocols (directly at 37°C or with a 5-second pre-exposure to 50°C) and three reanimation time points (immediately, 24 h and 5 days post-warming). In Experiment 1, tissues were evaluated for histo-morphology and mitochondrial activity immediately or 24 h after warming protocols. In Experiment 2, cell viability, DNA fragmentation, and germ cell composition were assessed immediately, 24 h, or 5 days after optimal warming. Preservation of seminiferous tubule structure was better using warming at 50°C for five seconds, and survival of somatic as well as germinal cells was higher compared to direct warming at 37°C for one minute. Short term in vitro culture (for reanimation) also proved that cellular composition and functionality were better preserved when warmed for a short time at 50°C. Collective data showed that short warming at 50°C led to better quality of seminiferous tubule structure and cell composition after vitrification and short-term culture. In addition, data suggest clear directions to further understand and optimize testicular tissue survival after fertility preservation procedures.

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We aimed to compare fresh sperm and sperm cooled to 4ºC that had been recovered from the epididymides of cats using powdered coconut water (ACP-117c) and Tris extenders. Sixty epididymides were divided into 6 groups: 10 fresh epididymides were recovered using Tris (T0h); 10 were kept at 4°C/2h and recovered using Tris (T2h); 10 were kept at 4°C/4h and recovered using Tris (T4h); 10 fresh were recovered using ACP-117c (A0h); 10 were kept at 4°C/2h and recovered using ACP-117c (A2h), and 10 were kept at 4°C/4h and recovered using ACP-117c (A4h). The testis-epididymis complexes (TEC) control were not cooled. The others were cooled at 4°C for 2 or 4h. The epididymis was separated and the sperm was recovered by the modified flotation method. Sperm kinetic parameters were evaluated by a computer-system analysis, and vigor, viability, concentration, membrane function and morphology of the sperm were assessed under a light microscope. The progressive motility with ACP-117c declined after 2h of cooling, but did not differ between fresh and 4h. The vigor and membrane function were higher in A4h than A0h. The vigor at T2h and T4h were decreased compared to T0h. T0h was higher than A0h for vigor and sperm membrane function. However, after 4h of cooling, ACP-117c maintained a higher percentage of living cells. Feline epididymal sperm quality can be maintained to the degree necessary for artificial breeding programs following cooling and ACP-117c may be successfully used to recover cat sperm that have been cooled for up to 4h.

Cryopreservation of testicular tissue enables the maintenance of reproductive capacity in different animal species, and contributes to the formation of gene banks for endangered species. The spermatogonia present in the testes can be grown in vitro and the sperm obtained can be used in artificial breeding programs. This review aimed to describe the main techniques of testicular cryopreservation, the main cryoprotectants used, as well as the progress made in different animal species thus far. In the last decade, significant progress has been made in obtaining viable and functional germ cells from testicular tissue. However, more research is needed to better establish protocols that can be used in clinical practice with various species.

Systematic cryo-banking of semen and testicular tissues is critical to preserve the genetic value of recently deceased or neutered black-footed ferrets (BFFs). Specifically, recovering or producing mature sperm cells from vitrified-warmed issues offers additional options in assisted reproduction. This could, in turn, enhance the genetic management of this rare and endangered species over multiple generations. The objective of the study was to evaluate structural properties, DNA fragmentation, cell viability, and germ cell composition in vitrified testicular tissues from BFFs directly after warming or after warming plus a short in vitro culture period. Tissue biopsies from five adult BFFs were either kept fresh or vitrified with a standard protocol (using dimethylsulphoxide (DMSO) and glycerol) and warmed at 50 °C for 5 s. Some of the warmed samples were then cultured in vitro for 24 h. Fresh, warmed, and warmed/cultured tissues were analyzed using different indicators: histology of seminiferous tubules, intact Sertoli cells (vimentin labeling), DNA integrity, cell viability, germ cell composition (Oct4 and Boule labeling). Percentages of intact seminiferous tubules decreased after vitrification/warming and returned to the level of fresh samples after culture. While percentages of cells labeled with vimentin, with intact DNA integrity, or proportions of viable cells were affected by vitrification/warming, they all reached similar or better levels than the fresh tissue after culture. Proportions of cells labeled with Boule antibodies also improved during in vitro culture post-warming. We demonstrated for the first time that BFF testes subjected to vitrification, rapid warming, and short in vitro culture were viable and maintained the ability to resume germ cell progression. Cryopreserved testicular tissues could potentially contribute to new strategies to enhance BFF assisted reproduction as well as conservation efforts.

Testicular vitrification is an alternative to preserve the genetic material of pre‐pubertal animals. However, there are few studies on post‐vitrification warming. Hence, the aim was to compare the influence of different warming temperatures on vitrified testicular fragments from pre‐pubertal cats. The testicles were fragmented and divided into a control group (non‐vitrified) and vitrified, using an association between dimethylsulphoxide and glycerol. The vitrified fragments were warmed at 50, 55 and 60°C/5 s. Morphological and morphometric evaluations were carried out using classical histology. Afterwards, the mitochondrial activity was evaluated using Rhodamine 123. The data were expressed in mean and standard error. The differences were considered significant when p < .05. In the histomorphological analysis, the testicular fragment presented seminiferous tubules with poorly developed germinal epithelium, compatible with pre‐pubertal animals. The group warmed at 50°C presented similar to the control regarding the maintenance of the integrity of the tubules and cells, without stromal rupture and lamina propria alteration, as well as regarding the maintenance of the junctions between the cells. The group warmed at 55°C showed reduction of the cell junctions, and the one warmed at 60°C had increased detachment of the basement membrane (p < .05). The warming caused a reduction in the tubular diameter inversely proportional and progressive to the increase in temperature, with the highest diameter in the control group and the lowest in the 60°C group. The control group showed a lower incidence of Rhodamine 123, followed in ascending order of the warmings at 55 and 60°C. The higher mitochondrial activity was obtained with 50°C, showing an increase of the metabolic cell function at this temperature. It was concluded that the testicular fragment of pre‐pubertal cats presents a better preserved morphology, morphometry and viability when warmed at 50°C.