Effects of different subcutaneous sites on heterotopic autotransplantation of canine ovarian tissue

Ovarian tissue transplantation makes it possible to restore fertility; however, the success of this technique depends on the transplant region used. Therefore, this study aimed to evaluate the effect of two subcutaneous regions on canine ovarian transplantation, pinna (Pi) and neck (Ne), for 7 and 15 days. Ovaries collected by ovariosalpingohysterectomy were fragmented using a punch device. Fresh fragments were fixed, and the others were immediately grafted onto the animal itself in the Pi and Ne regions for 7 and 15 days. Recovered fragments were evaluated for histology (morphology, development and stromal density), picrosirius (collagen fibers), and immunohistochemistry (fibrosis and cell proliferation). The results showed that follicular normality rates were lower in Pi-7 (78%) vs. control (90%) and Pi-15 (86%), similar in Ne-7 (92%) and superior in Ne-15 (97%) compared to the control, with the effect of the region Ne (94%) superior (P < 0.05) to Pi (82%). Stromal density reduced in both regions vs. control but was similar within 15 days. Fragments from both regions showed higher fibronectin labeling and deposition of type I and lower type III collagen fibers (P < 0.05) vs. control. Proliferation rates in Ne-7 were higher (P < 0.05) than in control, and Pi-15 was higher (P < 0.05) than Ne-15. In conclusion, the pinna may be a region with greater potential than the neck after a 15-day autotransplantation of canine ovarian tissue.


Introduction
The wild canid population has been constantly affected due to anthropic actions, like deforestation, which can lead to invasion of urban environments, roadkill, and, consequently, the death of these individuals (Orozco et al. 2014).Thus, some canid species have been classified as near threatened (Maned wolf -Chrysocyon brachyurus) or threatened (Ethiopian wolf-Canis simensis), or critically endangered (Red wolf -Canis rufus), according to the red list (IUCN 2023).Therefore, different reproductive strategies are being developed to preserve the fertility of both wild species that are at risk of extinction, as well as domestic animals of high genetic value, commercial or sentimental, using animal Abbreviations HVSBC-UECE Veterinary Hospital Prof. Sylvio Barbosa Cardoso of the State University of Ceará MEM-HEPES Minimum Essential Medium-buffers OSH Ovariosalpingohysterectomy models (Abe et al. 2011;Johnson et al. 2014;Paz 2015;Lee et al. 2018), that can even configure applications for human reproduction (Scalercio et al. 2015;Donfack et al. 2018;Costa et al. 2020;Souza et al. 2020).
Transplantation of ovarian tissue, associated with cryopreservation, has become an increasingly important tool in studies focused on folliculogenesis and reproductive biology ( (Terazono et al. 2012a;Campos-Junior et al. 2016), enabling fertility restoration (Bordes et al. 2005;Donnez et al. 2014;Tammiste et al. 2019;Hoekman et al. 2020).Among the types of transplants, autotransplantation has been showing highlights for not requiring immunosuppressive therapy, immune regulators, or knockout mice and for presenting less possibility of rejection (Costa et al. 2020).In addition, allotransplantation or xenotransplantation requires expensive techniques and specialized and equipped laboratories.Thus, autotransplantation can be considered an alternative for preserving the fertility of canid species, especially for those who have undergone oncological treatment, as in the case of transmissible venereal tumor (TVT) common in domestic dogs (Ganguly et al. 2016).Heterotopic implantation sites have been considered in autotransplants (Lee et al. 2004).The subcutaneous site has proven to be a suitable environment because it is easy to monitor and access, is low invasive (Souza et al. 2020), and does not require general anesthesia (Donfack et al. 2017), in addition to facilitating graft recovery.These sites are considered preferred sites for collecting oocytes to be used in in vitro fertilization (Costa et al. 2020).In subcutaneous sites, the neck region has been used to obtain favorable follicular survival rates and functional resumption of ovarian tissue after autotransplantation (Leonel et al. 2018;Vilela et al. 2019;Costa et al. 2020;Souza et al. 2020).However, despite the good results obtained, the type of region used can also hinder the success of subcutaneous autotransplantation due to hypoxia or ischemia/reperfusion injury, to which the tissue can be subjected (Donfack et al. 2017).This way, other subcutaneous regions must be considered.Recently, Terren et al. (2022) performed autotransplantation of cryopreserved whole ovaries from mice, between the outer layer of skin and the cartilage of the ear, with recovery up to 21 days and obtained an increase in vascularization and cell proliferation in the ovary.These results led the authors to conclude that the ear transplant model may be suitable for ovarian tissue transplantation and helpful in testing pharmaceutical strategies.
Regarding the quality of ovarian tissue after autotransplantation, preantral follicles morphology and stromal density have been frequently used (Pinto et al. 2020;Souza et al. 2020).Furthermore, the expression of proliferation markers, such as PCNA (proliferating-cell nuclear antigen), has played an essential role in the functionality of ovarian tissue after transplantation (Hariya et al. 2016).Excessive deposition of extracellular matrix components can also determine the quality of the graft and is considered indicative of tissue fibrosis, one of the main obstacles in transplant systems (Scalercio et al. 2015;Souza et al. 2020).Among the factors involved in fibrosis formation, the synthesis of extracellular matrix proteins can be highlighted, like collagen type I, type III, and fibronectin (Barnes and Gorin 2011), which are responsible for remodeling the tissue matrix, mainly after cell loss (Scalercio et al. 2015;Zhou et al. 2017;Souza et al. 2020).
Based on that, the present study aimed to evaluate, for the first time in female dogs, the effect of the pinna and neck regions in the subcutaneous site on autotransplantation of ovarian tissue for 7 and 15 days, about the normality of preantral follicles, development, and stromal density, as well as tissue proliferation and fibrosis, using the domestic dog as an experimental model.

Ethical aspects
This study was approved and carried out under the Ethics in Animal Use Committee guidelines of the State University of Ceará (3,349,245/2018).

Chemicals
Unless otherwise indicated, the chemical reagents used in this study are from Sigma Chemical Co.(St.Louis, MO, USA).

Origin of animals and collection of ovarian tissue
Eight adult female dogs (3-6 years old), mixed breed, at different stages of estrous (two females in anestrus and the others in diestrus), belonging to the Zoonosis Surveillance Unit (UVZ -Fortaleza, Ceará, Brazil), were submitted to ovariosalpingohysterectomy (OSH) at Veterinary Hospital Prof. Sylvio Barbosa Cardoso of the State University of Ceará (HVSBC-UECE).After OSH, the ovarian bursa was removed, and the ovaries were washed once in 70% alcohol and twice in the Minimum Essential Medium (MEM-HEPES) supplemented with 100 µg/mL of penicillin and 100 µg/mL of streptomycin.Then, the ovaries were sectioned with the auxiliary of a 2 mm punch device (Punch Keyes for 2 mm Skin Biopsy -ABC Surgical Instruments, São Paulo, Brazil) to obtain fragments of sizes 2 × 2 mm, according to the modified protocol by Wiedemann et al. (2012).

Experimental design
Fifteen fragments (n = 15) were recovered by each pair of ovaries from eight females and randomly distributed in different experimental conditions (Fig. 1).Three fragments (n = 3) were fixed immediately and classified as fresh control (FC).Then, the remaining fragments (n = 12) were autotransplanted subcutaneously in the following regions (Fig. 1A and B): external side of the pinna for 7 days (Pi-7; n = 3) and 15 days (Pi-15; n = 3); lateral neck for 7 days (Ne-7; n = 3) and 15 days (Ne-15; n = 3).This procedure was applied to all eight females, obtaining a total of 120 fragments.After the transplantation time had elapsed, all fragments were submitted to histological, histochemical (picrosirius), and immunohistochemistry evaluation.

Autotransplantation and recovery of fragments
Autotransplantation was performed according to an adaptation of the anesthetic and surgical protocol described by Terazono et al. (2012a).Briefly, the bitches were induced with intramuscular anesthesia of 0.3 mg/kg of methadone hydrochloride (10 mg/mL; Mytedom, São Paulo, Brazil) and intravenous midazolam hydrochloride of 0.5 mg/kg (5 mg; Dormire, São Paulo, Brazil) associated with 2.0 mg/ kg of ketamine hydrochloride (10%; Ketamine; São Paulo, Brazil), and 4 mg/kg of propofol (10 mg/mL; Propovan, São Paulo, Brazil).Furthermore, inhalation of 3% isoflurane (Isoflurane, São Paulo, Brazil) and oxygen maintained anesthesia.Thus, after performing OSH, ovarian fragments were obtained from the ovarian cortex using the punch device.Then, for autotransplantation, a subcutaneous cut was made in the dorsal region of the neck or on the convex side of the -hematoxylin.The preantral follicles were analyzed under an optical microscope (Nikon E200; Tokyo, Japan) with 400х magnification and classified as morphologically normal when they contained oocytes, cytoplasm, and intact granulosa cells; or degenerated when they presented cytoplasmic retraction, presence of a pyknotic nucleus, and disorganization of granulosa cells (Silva et al. 2002).Morphology evaluations were performed only in follicles with a visible nucleus.Furthermore, the preantral follicles were classified as primordial (oocyte surrounded by one layer of flattened granulosa cells) and based on the stage of development as follows: transitional (one layer of flattened and cuboidal granulosa cells), primary (one layer of cuboidal granulosa cells around the oocyte), and secondary (oocyte surrounded by two or more layers of cuboidal granulosa cells) follicles (Souza et al. 2020).

Density of stromal cells
For cell density of ovarian stroma, 10% of the histological sections of each ovary fragment were analyzed, recording the average of cells from four random quadrants (50 × 50 μm = 2,500 µm 2 ), as described by Alves et al. (2016).A single operator performed all evaluations.

Picrosirius
According to Scalercio et al. (2015), the density of collagen-rich deposits was analyzed.The sections of the ovarian fragments of all experimental conditions were processed according to classic histology and stained using the standard picrosirius red staining protocol (0.1%) (# 365548-5G) with a saturated picric acid solution (1.2%), for 1 h, at room temperature.For the evaluation, four sections were photographed under polarization microscopy (Nikon E200; Tokyo, Japan) at 400× magnification, coupled with an image capture system (Nikon, Coolpix 4500).Collagen fibers were determined using the different polarizing colors of collagen I and III types, yellowish-orange and green birefringence, respectively (Junqueira et al. 1978).Image analysis was performed by measuring the red threshold, green and blue to obtain the percentages of the red and green colors, expressed in pixels (ImageJ software, version 1.45; National Institutes of Health, USA) (Pinto et al. 2020).

Immunohistochemistry
Ovarian fragments from all experimental conditions were fixed in 4% paraformaldehyde (PAF) at room temperature for 2 h, dehydrated, clarified, included in paraffin, and sectioned at 5 μm.The sections were mounted on positively charged slides and processed for immunohistochemistry for pinna; the fragments were fixed using surgical 6 − 0 nylon.Next, the suture of the subcutaneous cut was performed with 3 − 0 nylon thread.
After seven days of transplant, three grafts were recovered from each region (pinna and neck).The animals received 0.3 mg/kg of methadone hydrochloride intramuscularly and intravenously, combining 0.5 mg/kg of midazolam hydrochloride and 5.0 mg/kg of ketamine hydrochloride.Furthermore, local anesthesia was applied using 2 mL of lidocaine hydrochloride (2%; Xylestesin, São Paulo, Brazil).Posteriorly, at the end of the 15 days, the same animals were euthanized to remove the other grafts in the respective regions.Thus, 0.3 mg/kg of methadone hydrochloride was administered intramuscularly and 25 mg/mL of thiopental intravenous (50 mg/mL; Thiopentax, São Paulo, Brazil) until the loss of consciousness.Then, 1 mL/kg of potassium chloride was applied intravenously (10%; Potassium chloride, São Paulo, Brazil) following the standard protocol of the HVSBC-UECE.Euthanasia is a required procedure for animals from the UVZ.

Macroscopy of the grafts
The recovery rate was determined, and the macroscopic appearance of the recovered fragments was qualitatively described using a scoring system (see Table 1), following the criteria: (I) morphology, considering if the graft is necrotic, intact (similar to the original fragment) or swollen; (II) adhesion when the graft is only secured by the suture (absent tissue adhesion) or adhered to host tissue; (III) extent of bleeding at the time of graft removal.The data were presented as median.Furthermore, the presence or absence of suture in the graft was observed.A single individual performed all evaluations.

Histological analysis
Fresh and autotransplanted ovarian fragments were fixed in Davidson's solution (Formalin 22.2%, alcohol 33.4%, glacial acetic acid 11%, and tap water) for 12 h at room temperature (Brito et al. 2021).Posteriorly, they were dehydrated, clarified, included in paraffin, and sectioned at 5 μm.A total of 7736 Sect.(30 sections/slides; 51 slides/treatment) were obtained and stained with periodic acid-Schiff error of the mean (± SEM), and differences were considered significant when P < 0.05.

Recovery rate and macroscopic aspects of the graft
Recovery rates in Pi-7 regions, Ne-7 and Ne-15, were 100% (24/24); only the Pi-15 region obtained 95,8% (23/24).The median of the scores referring to the macroscopic criteria of the recovered grafts are shown (Table 2).Most grafts recovered showed intact morphology, similar to the original fragment, except the Pi-15 region, which presented three grafts (3/23) moderately swollen and whitish.The fragments with a longer transplant time (15 days) visibly showed greater adherence regarding adherence to host tissue.Most fragments did not show bleeding, except for the 15-day grafts from the Ne region, which had few bleeding points.Regarding the presence of sutures, all grafts kept their original suture.

Morphology and development of preantral follicles
A total of 3502 preantral follicles were analyzed in the present study.Figure 2 A shows the percentage of morphologically normal preantral follicles.Representative images are shown (Fig. 2B-F).As observed, despite the percentage of morphologically normal follicles having decreased in Pi-7 (78.5% ± 5.6; P < 0.05), when compared to FC (90.8% ± 0.8), the Pi-15 (86.6% ± 7.6) and Ne-7 (92.8% ± 1.3) regions did not show significant difference with FC.However, curiously, in 15 days of autotransplantation, the Ne region showed an increase (97.0% ± 1.2; P < 0.05) in the percentage of normal follicles compared to FC.When treatments were compared, it was observed that the percentage of normal follicles did not differ (P > 0.05) between days in both regions.However, Ne-7 had a percentage of normal follicles significantly higher than Pi-7.In contrast, this percentage was similar between Pi-15 and Ne-15 (P > 0.05).
In addition, our data also showed that the percentages of developing preantral follicles (transitional, primary, and secondary) were significantly lower in both regions after 7 and 15 days of autotransplantation compared to the control.

Stromal cell density
Figure 3 A shows the density of stromal cells in each treatment, and their representative images are shown in Fig. 3B-F.Stromal density was significantly reduced in all transplants when compared to FC.In addition, between treatments, it was observed that autotransplantation for 15 days also PCNA and fibronectin proteins.For antigenic recovery, the slides were incubated in a recovery buffer at 98 °C for 20 min (protein-specific pH; Dako, Santa Clara, CA, USA).The blocking of endogenous peroxidase activity was performed using 10% hydrogen peroxide in methanol.Then, incubation was performed with rabbit polyclonal to PCNA (1:1000; AB2426, Abcam Inc., Cambridge, MA, 461 USA) or rabbit polyclonal to fibronectin (1:50; AB2413, Abcam Inc., Cambridge, MA, 461 USA) primary antibody for 30 min.Posteriorly, the slides were incubated for 30 min with the biotinylated goat anti-rabbit IgG secondary antibody (1:200; AB9049, Abcam Inc., Cambridge, MA, 461 USA).Finally, the slides were incubated for 30 min with the avidin-biotin enzyme complex (ABC; Vector laboratories, 465 Burlingame, CA, USA) to react with chromogen 3,3'-diaminobenzidine (DAB, ACB030; ScyTek Laboratories Inc., West Logan, USA) for 5 min.Hematoxylin and 0.5% ammonia solution were used as counterstain.The negative control was performed by omitting the primary antibody.For positive control, they used mouse spleen tissue for PCNA and fibronectin.The evaluation of the PCNA was performed on the stromal cells; in an area of 400 µm 2 , the proliferation of stromal cells and the percentage of stromal cells positive for PCNA were determined (Hariya et al. 2016).However, the evaluation of tissue fibrosis was determined using the modified H-score method, described by Wiweko et al. (2019): The color intensity was obtained by the intensity scale (weak = 1, medium = 2, strong = 3).

Statistical analysis
The statistical analyzes were performed using Sigma Plot (version 11.0 Systat Software Inc., USA).When appropriate, normality (Shapiro-Wilk) and homogeneity of variance tests (Levene's) were performed.One-way ANOVA, followed by post-hoc test, was used to verify differences between the fresh control and the other treatments.Twoway ANOVA was used to evaluate the main effects (factors) of the transplant region (Pi vs. Ne) and days post-transplant (7 vs. 15).All data were presented as the mean and standard

PCNA (stromal cell proliferation)
According to Fig. 5A, no significant difference with FC was observed in the Pi region on both days of autotransplantation (7 and 15).However, although the Ne region showed an increased cell proliferation within seven days (P < 0.05), there was a significant reduction in 15 days of autotransplantation compared to FC.This reduction in 15 days was observed in both regions (P < 0.05) when days 7 and 15 were compared among themselves, although being more pronounced in Ne.In contrast, when different regions were compared, it can be observed that although Pi-7 had lower cell proliferation than Ne-7 (P < 0.05), Pi-15 has shown to be significantly superior to Ne-15.Representative images are shown in Fig. 5 (C-H).

Fibronectin
Figure 5B shows that fibronectin labeling was significantly higher in all autotransplant treatments compared with FC.Conversely, between treatments, it has been observed that a significant reduction in stromal cell density in both regions; however, although Pi-7 had a higher stromal density (P < 0.05) than Ne-7, both regions (Pi and Ne) were similar (P > 0.05) in 15 days of autotransplantation.

Quantification of collagen fibers type I and III
Picrosirius staining revealed that all autotransplants had high levels of type I collagen fibers (Fig. 4A; P < 0.05) and a reduction in type III collagen (Fig. 4B; P < 0.05) compared to FC.Furthermore, when comparing the treatments, it was observed that the Pi region maintained similar levels of type I collagen between days 7 and 15 (P > 0.05).In contrast, Ne-15 promoted a higher deposit of the fiber type I than Ne-7 (P < 0.05).Unlike that, the type III collagen level showed a significant reduction in 15 days of autotransplantation in both regions, compared to 7-day autotransplantation.No difference in collagen I and III levels was observed when the Pi and Ne regions were compared on the same day.Representative images of picrosirius coloration in the respective treatments are shown in Fig. 4 (C-Q).

Discussion
Ovarian tissue transplantation may be a promising technique for resuming the reproductive potential of domestic species with high genetic value or endangered wild species ((Terazono et al. 2012a;Leonel et al. 2018).Still, among the types of transplants, autotransplantation has stood out for preventing graft rejection and not requiring immunosuppressive therapy (Costa et al. 2020).However, the graft region has been considered essential in maintaining and functional resumption of ovarian tissue in transplantation (Cao and Li, Thus, using the domestic dog as a model, the present study investigated for the first time the effect of the pinna the Pi region did not show any difference between days (P > 0.05), unlike the Ne region, which showed the highest percentage of labeling in Ne-15 (P < 0.05).However, the comparison between different regions showed that Pi-7 was significantly higher than Ne-7.Nevertheless, in 15 days of autotransplantation, the Pi region had a lower level of fibronectin labeling than the Ne region (P < 0.05).Representative images are shown in Fig. 5I-N.
Fig. 3 Cell density/2500µm 2 between treatments in canine ovarian tissue autotransplantation (A).Representative images of fresh control stroma (B) and pinna autotransplants for 7/15 days (C/D) and neck for 7/15 days (E/F).Black arrows indicate a decrease in stromal cells.Bar 40 μm.* indicates significant differences with FC; a,b indicate differences between days in the same region; A,B indicate sameday differences between different regions control in 15 days of autotransplantation.Studies have suggested that transplant sites with greater vascularization have better rates of morphologically normal follicles (Youm et al. 2015;Donfack et al. 2018;Pinto et al. 2020) by allowing better support of nutrients and essential factors for survival maintenance.This fact may explain our findings in the neck region, although the ear region needed more transplant days for better support and maintenance, despite being a region less vascularized than the neck.However, we believe that the higher percentage of normal follicles found in Ne-15, compared to control, may be due to follicular heterogeneity, i.e., variation in follicular density in the same fragment often found in different regions of the mammalian ovary cortex Despite the results obtained in morphological normality, our data showed that autotransplantation in both regions did not show follicular development after days 7 and 15, in addition to presenting a reduction in preantral follicles in development (transitional, primary, and secondary).It is known that developing follicles are more susceptible than quiescent preantral follicles (Webb et al. 2016).Furthermore, significant reductions in follicular density are reported after autotransplantation of ovarian tissue in the subcutaneous region of the neck for seven days in mares (Souza et al. 2020) and the dorsal subcutaneous region for and neck region on heterotopic autotransplantation of ovarian tissue for 7 and 15 days.
Our macroscopic findings showed that most of the recovered grafts were morphologically intact.Furthermore, during the 15 days of transplantation, grafts showed greater adherence in both regions studied (pinna and neck).However, the Ne-15 stood out for having presented bleeding on withdrawal.Pinto et al. (2020) also observed good adhesion and bleeding upon graft removal, in the subcutaneous site in the neck region, in 15 days of goat ovarian tissue allotransplantation.The subcutaneous implantation site has been widely used for the grafting of ovarian tissue from different species (Oktay et al. 2011;Scalercio et al. 2015;Youm et al. 2015;Pinto et al. 2020;Souza et al. 2020) for being a place of easy access and monitoring with little invasiveness (Souza et al. 2020).However, this is the first study using a subcutaneous site in the pinna region in female dogs.The subcutaneous site in the neck region is known to be more anatomically irrigated than in the pinna region (Evans 1971;Degner et al. 2004); this fact may justify the bleeding observed in the neck region after removal in 15 days of autotransplantation.
The present study also showed that the neck region maintained rates of morphologically normal preantral follicles similar to and higher than the control in 7 and 15 days of autotransplantation, respectively.However, the pinna region showed a percentage similar to the neck region and the  (2004) demonstrated that stromal cells are more vulnerable to ischemia than ovarian follicles.Thus, it is assumed that the damage to the graft in the first days of avascular transplantation and the vulnerability of stromal cells may have contributed to reducing these cells.Furthermore, the neck region, being more vascularized, can facilitate a series of cellular processes, such as the production of inflammatory cytokines, which can intensify tissue damage (Kalogeris et al. 2012;Slegtenhorst et al. 2014).
Regarding matrix proteins, our data also showed that autotransplantation caused an increase in type I collagen and fibronectin, but a reduction in collagen type III in the two regions studied when compared with the control.Furthermore, an intriguing finding, high rates of type I collagen and fibronectin were observed in autotransplants only in the neck region (15 days).On the contrary, between 7 and 15 days of autotransplantation, type III collagen rates were reduced by almost half in both regions.Unlike our findings, Souza et al. (2020) observed that ovarian fragments from mares treated with VEGF (vascular endothelial growth seven days in monkeys (Scalercio et al. 2015).In contrast, Khoram et al. (2011) demonstrated that 75-76% of follicles survived ischemia after isotransplantation of canine hemi-ovaries in the muscle layer region of the stomach wall for 60 days.Despite the promising results of this study, the methodology used in the use of hemi-ovaries differs from our study, because we use ovarian fragments of sizes difference may be related both to the use of the whole ovary in the study, which may present more coordinated functional responses than small fragments of the same organ in longterm transplant (Terazono et al. 2012b;Gavish et al. 2014;Lew 2019), as well as, to injuries and oxidative stress from ischemia-reperfusion, that are severe in transplanted ovarian fragments (Damous et al. 2015;Cacciottola et al. 2018), as already discussed earlier.However, despite that, the ear region showed a better response than the neck region.
In conclusion, this study showed that the pinna could be a potential region for autotransplantation of canine ovarian tissue because it manages to keep the survival and the stability of the fragment during the 15 days of autotransplantation.We observed this mainly by maintaining morphologically normal follicles and by promoting less extracellular matrix remodeling.However, in both regions, there was no follicular development, and the density of stromal cells was reduced.Furthermore, although the neck showed a more significant proliferative potential of stromal cells in seven days, this region presented loss of stroma and proliferation within 15 days of autotransplantation, adding to a more significant deposition of tissue fibers when compared to the pinna.It is essential to highlight that this is the first comparative study of canine ovarian tissue autotransplantation between the pinna region and the neck.However, more research is needed to obtain better survival rates and follicular development.Like this, future proposals using antioxidant factors as a pre-treatment may promote adequate support in canine ovarian tissue autotransplantation.This study can contribute to pharmacological research and applications of autotransplantation in animal reproduction centers or zoos to preserve the fertility of precious domestic animals or endangered canine species, respectively.factor) before autotransplantation subcutaneous obtained a reduction in type I collagen and an increase in type III collagen when compared to the non-transplanted control.The ovarian cortex comprises connective tissue cells and collagen fibers (Bochner et al. 2015).The events surrounding ovarian fibrosis are very complex, but they usually involve a proliferation of fibroblasts and excessive deposition of extracellular matrix, characterizing fibrosis (Zhou et al. 2017).Several factors are involved in the formation of fibrosis, such as transforming growth factor-beta (TGF-β) released by paracrine and autocrine pathways, which induces the differentiation of myofibroblasts and, consequently, the synthesis of matrix proteins, like collagen type I, type III, and fibronectin (Barnes et al. 2011).Fibronectin is responsible for remodeling the matrix, and type I collagen is closely related to this protein.Thus, fibronectin deposition led to an increase in type I collagen fibers in extracellular matrix remodeling (Shi et al. 2010).This remodeling can be part of a healing process between 8 and 16 days after inflammation (Broughton et al. 2006).In the initial phase of this process, there is a high percentage of type III collagen, which is gradually replaced by type I collagen (Lindsey et al. 2015).Therefore, and according to our findings, we believe that the neck region promoted a higher inflammatory reaction than the pinna, showing a significant increase in fibrotic tissue after 15 days of autotransplantation, confirmed by the increase in type I collagen and fibronectin and the reduction in type III collagen, as well as the loss of stromal cells in the region, as discussed earlier.
The analysis of stromal cell proliferation showed that the 7-day autotransplantation in the neck region had a higher number of PCNA-positive cells, but this number significantly reduced with 15 days of grafting compared with the control.Scalercio et al. (2015) also obtained an increase in stromal cell proliferation with seven days of subcutaneous autotransplantation of monkey ovarian tissue.However, this reduction in the proliferative potential in the region, with the passage of transplantation time, may have occurred due to the tissue damage that the region developed, such as the loss of stromal population since the first days of transplantation, more tissue fibrosis, provoked by the attempt to reconstruct the damage.Such facts may have affected or inhibited factors responsible for some of the signals that promote proliferation (Vogler et al. 2003).In addition, our findings also show that the pinna region was similar to the control throughout transplantation, although, despite the decrease in cell proliferation within 15 days of transplantation, this rate was higher than the neck region.Unlike our findings, Terren et al. (2022) observed an increase in cell proliferation (by immunostaining with Ki67) and a decrease in fibrosis at 21 days compared to 3 days of cryopreserved whole ovary autotransplantation into the ear of mice.We believe that this

Fig. 4
Fig. 4 Quantification of type I (A) and type III (B) collagen in canine ovarian autotransplantation.Representative images of picrosirius staining, highlighting collagen types I (C-G), III (H-L), and the merge (M-Q) of the respective treatments.Bar 40 μm.* indicates significant differences with FC; a,b indicate differences between days in the same region; A,B indicate same-day differences between different regions

Fig. 5
Fig. 5 Percentages (± SEM) of PCNA (A) and fibronectin (B) immunostaining in canine ovarian tissue autotransplantation.Representative images of PCNA (C-H) and fibronectin (I-N) marking in the negative (-), positive (+) control, and in the respective treatments.Bar 40 μm.* indicates significant differences with FC; a,b indicate differences between days in the same region; A,B indicate same-day differences between different regions

Table 1
Macroscopic criteria of canine ovarian tissue after transplanta-

Table 2
Median of the scores referring to the macroscopic criteria