Influence of intravaginal 4-vinylcyclohexene diepoxide on ovarian follicles and estrous cycle in rats

Influence of intravaginal 4-vinylcyclohexene diepoxide on ovarian follicles and estrous cycle in rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of intravaginal 4-vinylcyclohexene diepoxide on ovarian follicles and estrous cycle in rats Endreo Alan Pail dos Santos, Etiele Maldonado Gomes, Sandra Elisa Haas, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4649606/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background For the population control of stray dogs and cats, alternative methods to surgical castration are being studied and developed, such as chemosterilization. In this context, substances that lead to irreversible degeneration of the tissue administered directly to the gonads are being investigated, with most studies focusing on males due to the ease of access to the testicles. In females, ovarian degeneration is reported with the use of a chemical compound called 4-vinylcyclohexene diepoxide, a selective ovotoxic agent, used in experimental studies in rats to mimic menopause. However, it requires fifteen consecutive days of intraperitoneal application. Envisioning its use for the population control of dogs and cats, a less invasive and effective route is necessary. Thus, the aim of this study was to evaluate the effect of 4-vinylcyclohexene diepoxide on the ovaries and its chemosterilizing potential by intravaginal gel inoculation in rats. Twenty Wistar female rats were used, randomly divided into two groups (n = 10/group): control and treatment. The control group received 0.2 mL of chitosan-based gel, and the treatment group received the same gel containing 4-vinylcyclohexene diepoxide (160 mg/kg) for fifteen days. The rats were evaluated daily by vaginal cytology until euthanasia. The animals were euthanized at two time points (n = 5/group): Sixteen (M1) and thirty (M2) days after the start of treatment. The ovaries were collected, prepared, and evaluated by optical microscopy for counting of primordial and primary follicles. Results In the group treated with 4-vinylcyclohexene diepoxide, a prolongation of the diestrus phase (M1 and M2), as well as a prolongation of the estrus phase (M2), was noted after inoculations of the intravaginal gel. Even so, in the treated group, there was an increase in the number of estrous cycles compared to the control group (M1 and M2). In the follicular count, a higher median number of atretic primordial follicles was noted in the group treated in M2. Follicular evaluation in M1 and M2 revealed a higher proportion of healthy primordial follicles in the control group, as well as an increase in the proportion of atretic primordial follicles in the treated group. Conclusions It was possible to observe evidence of ovotoxicity of chitosan gel containing 4-vinylcyclohexene diepoxide, applied intravaginally, due to the increase in the population and proportion of atretic primordial follicles and interference in the estrous cycle. Chemosterilization female chemical castration follicular atresia ovotoxicity Figures Figure 1 Figure 2 Figure 3 Background The overpopulation of stray dogs and cats is a problem in different parts of the world, causing direct and indirect disruptions to public health, animal welfare and threats to wildlife [ 1 – 4 ]. As a form of population control for these animals, surgical sterilization is still the most applied, humanly accepted, and recommended option [ 5 – 7 ]. Despite this, surgical castration is still not completely viable in some locations, due to the difficulty of mass applicability and challenging access for low-income populations [ 8 – 11 ]. In this context, alternative methods to surgical sterilization aiming at reproductive control are being studied and developed, such as chemosterilization [ 2 , 5 , 6 ]. As it is considered low-cost, does not require much care after application and can be used on a large scale, it is believed that chemosterilization is an alternative to surgical sterilization, effective for castration/population control programs for stray animals [ 5 ]. However, most studies were carried out with substances that require application directly to the gonads, therefore, they are in male animals, due to the testicles being easily accessible [ 5 , 13 , 14 , 15 ]. For population control, however, there is a need for reproductive control methods to be applied to both sexes [ 6 , 16 , 17 ]. In this context, a chemical compound known as 4-vinylcyclohexene diepoxide (4-VCD) has been used experimentally in rodents to induce ovarian failure and mimic menopause [ 18 , 19 ]. This compound selectively promotes ovotoxicity, inducing apoptosis of ovarian pre-antral follicles (primordial and primary) [ 20 , 21 ]. However, the effective route of use to produce this effect is intraperitoneal (IP), which is invasive and prone to complications [ 22 ]. Envisioning its use in dogs and cats for population control, a less invasive, effective, and safe route is necessary. Therefore, the objective of this study is to evaluate the effect of an intravaginal gel based on chitosan containing 4-VCD on the ovarian follicles and estrous cycle of Wistar rats. Methods Animals Twenty adult Wistar rats ( Rattus norvergicus albinus ) were used. The rats were acclimatized for a period of fifteen days and kept in boxes appropriate to the species (5 animals/box), with food and water ad libitum , in a controlled temperature environment (22 ± 2°C) and a light-dark lighting cycle (12 hours each). During the acclimatization period, the animals were conditioned to the manipulation and collection of vaginal cytology. The study was approved by the Ethics Committee on the Use of Animals in Research at the Federal University of Pampa – UNIPAMPA under protocol 020/2018. The study was carried out in accordance with ARRIVE guidelines and regulations. Study design The animals were equally and randomly divided into four experimental groups, according to the treatment received: control (CG) and treatment with 4-VCD gel; and moment of euthanasia: sixteen (M1) and thirty (M2) days after the beginning of treatment (Fig. 1 ). Prior to the study, the appropriate vaginal volume for intravaginal inoculation of chitosan-based gel was investigated so that there was no extravasation, which was established as 0.2 mL (data not shown). Daily, the rats were subjected to intravaginal inoculation at the same time in the morning with up to 0.2 mL of gel for 15 days. Animals in the control group received 0.2 mL of chitosan-based gel without any active ingredient and animals in the treatment group received 0.2 mL of chitosan-based gel at a dose of 160 mg/kg of 4-VCD (~ 32 mg/rat = 0.2 ml of gel) (Fig. 1 ). Intravaginal gel The 2.5% (w/v) chitosan gel was prepared by manually mixing, with a mortar and pestle, chitosan and distilled water acidified with 1% lactic acid. The homogenate was left to rest for approximately 12 hours, under refrigeration. For the treatment group, the chemical compound 4-VCD was slowly incorporated, with a mortar and pestle, into the base gel, resulting in a final concentration of 160 mg/g of 4-VCD. Estrous cycle The rats were subjected to estrous cycle assessment daily from the first day of acclimatization. As an exclusion criterion, rats that had irregular estrous cycles prior to treatment were not used in the study. To this end, daily vaginal washes were performed at the same time, prior to inoculation of the gel. Washings were carried out by placing and retrieving 10 µL of intravaginal saline solution and immediate cytological evaluation using optical microscopy (40x) according to the protocol and classification established by Goldman et al. [ 23 ] and Marcondes et al. [ 24 ]. The stages of the estrous cycle were classified as proestrus, estrus and diestrus (1 and 2) based on the predominance of cell types [ 23 ]. For each group, the number of days in each phase of the estrous cycle of each rat were counted and added, for later determination of the average. Data were compared between CG and 4-VCD in M1 and M2. The number of estrous cycles of each rat/group were added and the average number of cycles/group was determined and compared between the CG and 4-VCD at both times of the study (M1 and M2). The estrous cycle was classified according to its length according to Goldman et al. [ 23 ], and consecutive episodes of ≥ 4 days of diestrus and ≥ 3 days of estrus were considered abnormal/irregular cycles. Ovarian follicle count After euthanasia, the collected ovaries were fixed in 10% formalin, processed, and embedded in paraffin. The slides were mounted in 3.5 µm thick sections with 40 µm intervals between each, avoiding multiple counts of the same follicle [ 25 ]. The first three slides of each ovary were evaluated. On each slide, the preantral follicles containing nuclei in the oocyte were classified as primordial (oocytes surrounded by a single layer of flattened granulosa cells) and primary (oocytes surrounded by a single layer of cuboidal cells) [ 25 – 27 ]. Follicular quality was assessed according to Kao et al. [ 20 ] and Springer et al. [ 27 ], for classification into atretic or healthy follicles. All slides were analyzed with an optical microscope (40 and 100x) by three evaluators in consensus, blindly. The number of healthy and atretic primordial and primary follicles were counted and added together in each rat/group, establishing the average for each group. Therefore, GC and 4-VCD were compared in M1 and M2. Euthanasia To collect the ovaries, euthanasia was performed at two different times: M1 on the 16th day of the experiment, 12 hours after the last inoculation of the intravaginal gel, with five rats from the GC and five from the 4-VCD group; and M2, on the 30th day of the experiment, that is, 15 days after the end of treatment, containing the rest of the animals. Euthanasia was performed with an overdose of propofol intraperitoneally and, after anesthesia, IV potassium chloride was injected. After euthanasia, the ovaries were collected for follicular counting. Statistical analysis The data obtained from the different stages of each phase, the number of estrous cycles, number of healthy and atretic primordial and primary follicles were statistically analyzed using GraphPad Prism version 8.0.1 (Graphpad Software, San Diego, USA) and expressed as median, lower and upper quartile. Comparisons were made between GC and 4-VCD according to the time of euthanasia (M1 and M2). Differences between GC and 4-VCD were considered significant when p < 0.05. As data did not follow a normal distribution, they were analyzed using the non-parametric Mann-Whitney test. For each group, at each time point (M1 and M2), the total number of follicles was computed, the proportion (percentage) for each follicular phase was determined and proportions were calculated using Minitab Inc. (Software, version 18.1). For the proportion test, the 95% confidence interval was considered, and significant differences were considered when P < 0.05. Results Effects of 4-VCD on estrous cycle analysis In M1, the 4-VCD group showed shorter proestrus phases and longer diestrus phases (P < 0.05) compared to the CG (Table 1 ). The number of estrous cycles was lower in the 4-VCD group when compared to the CG (P < 0.05) (Table 1 ). Of the rats in the 4-VCD group in M1, all showed an abnormal estrous cycle, with prolongation of the diestrus phase (data not shown). Table 1 – Number of days in each phase of the estrous cycle and number of estrous cycles. Moment Group Proestrus Estrous Diestrus N of estrous cycle M1 GC 5/4–7 5/3–6 5/4,5–5,5 3/2–3 4-VCD 1/0.5–2.5* 3/1.5-4 11/9.5–11* 1/0–2* P value 0,0119* 0,1105 0,0112* 0,0290* M2 GC 7 /5.5–8.5 10/7.5–11.5 12/11-16.5 4/3-6.5 4-VCD 3/2.5–3.5* 8/5.5–10 20/16–22* 2/2–3* P value 0,0079* 0,2463 0,0362* 0,0459* Comparison of the number of days in each phase of the estrous cycle and the number of estrous cycles in the control (CG) and treated (4-VCD) groups at two different times (M1 = day 16; M2 = day 30). Values are expressed as median/lower and upper quartile, n = 5. * P < 0.05. Statistical significance was determined using the Mann-Whitney test. In M2, there was a decrease in the proestrus phase and an increase in the number of days in diestrus in the 4-VCD group when compared to the CG (P < 0.05) (Table 1 ). Furthermore, the 4-VCD group presented a significantly (P < 0.05) lower number of estrous cycles when compared to the CG (Table 1 ). One rat in the GC presented an abnormal estrous cycle, with prolonged diestrus (during the treatment period). In the 4-VCD group, 100% of the rats had an estrous cycle classified as abnormal, with a prolongation of the diestrus phase during treatment, and four had prolonged estrus in the 15 days after treatment (data not shown). Ovarian follicle count In M1, regarding the global count of healthy and atretic primordial and primary follicles (Fig. 2 ), there was no statistical difference (P > 0.05) between the CG and the 4-VCD group (Table 2 ). However, a significant difference (P < 0.05) was detected between CG and 4-VCD group regarding the proportions of healthy and atretic primordial follicles (Table 3 ). At this time, a higher proportion of healthy primordial follicles was noted in the CG when compared to the 4-VCD group (P = 0.004; 21% of healthy primordial follicles in the CG and 9% of healthy primordial follicles in the 4-VCD group). At the same time, a higher proportion of atretic primordial follicles was noted in the 4-VCD group compared to the CG (P = 0.039; 35% of atretic primordial follicles in the GC group and 46% of atretic primordial follicles in the 4-VCD group). Regarding the comparison between the proportion of healthy and atretic primary follicles, no significant difference (P > 0.05) was detected between the groups (Table 3 ). Table 2 – Global count of primordial and primary follicles (healthy and atretic) in the treated and control groups. Moments Group Primordial Primary Atretic (primordial) Atretic (primary) M1 GC 6/2.5–8.5 1/0–3 8/4.5–14.5 7/6.5–15.5 4-VCD 2/0.5–4.5 1/0–2 12/6.5–16 8/4.5–17 P value 0,1161 1,0000 0,4633 0,8340 M2 GC 3/3–8 1/0–2 8/7-8.5 14/4.5–20.5 4-VCD 2/0.5-5 0/0–1 12/10.5–18* 17/7-21.5 P value 0,3398 0,3447 0,0112* 0,6905 Comparison of the global count of healthy and atretic primordial and primary follicles in the control (CG) and treated (4-VCD) groups at two different moments (M1 = day 16; M2 = day 30). Values are expressed as median/lower and upper quartile, n = 5. * p < 0.05. Statistical significance was determined using the Mann-Whitney test. Table 3 Proportion of healthy and atretic primordial and primary follicles in the treated and control groups. M1 M2 GC 4-VCD GC 4-VCD Primordial (healthy) 28/132 12/125 25/133 13/158 % 21% 9% 19% 8% P value 0,004* 0,004* Primordial (atretic) 46/132 57/125 39/133 69/158 % 35% 46% 29% 44% P value 0,039* 0,005* Primary (healthy) 7/132 5/125 5/133 2/158 % 5% 4% 4% 1% P value 0,31 0,092 Primary (atretic) 51/132 51/125 64/133 74/158 % 39% 41% 48% 47% P value 0,362 0,587 Comparison of the proportion of healthy and atretic primordial and primary follicles in the control (CG) and treated (4-VCD) groups at two different moments (M1 = day 16; M2 = day 30). Values are expressed as number of follicles phase/total follicles counted per group at each time point and as a percentage, n = 5. * P < 0.05. Statistical significance was determined using the proportion test. In M2, the number of healthy primordial follicles, atretic primaries and healthy follicles showed no statistical difference (P > 0.05) comparing CG and 4-VCD group. It was noted that the number of atretic primordial follicles was higher in the 4-VCD group compared to the CG, with a statistically significant difference (P < 0.05) (Table 2 ). It was observed that the CG had a higher proportion of healthy primordial follicles (P < 0.05) when compared to the 4- VCD group (P = 0.004; 19% of healthy primordial follicles CG and 8% of healthy primordial follicles in group 4- VCD) (Table 3 ). At the same time, the proportion of atretic primordial follicles was higher in the 4-VCD group compared to the CG (P = 0.005; 29% of atretic primordial follicles in the CG and 44% of atretic primordial follicles in the 4-VCD group) (Table 3 ). Regarding the proportion of healthy and atretic primary follicles, no statistically significant difference (P > 0.05) was detected when comparing the groups (Table 3 ). Other findings Only one rat in the 4-VCD group presented, on the 15th day of intravaginal gel inoculation, the presence of serosanguineous exudate (Fig. 3 A) in the vulva, without further clinical or behavioral changes. On the 16th day of the experiment, the day of euthanasia in M1, the same rat presented bloody exudate (Fig. 3 B). It is worth noting that the exudate had no odor. This rat was euthanized with the other rats from the same group in M1. After euthanasia, during macroscopic abdominal exploration, the presence of structures with a cystic appearance was noted in the body and right uterine horn (Fig. 2 C). The body and right uterine horn were edematous, with congested vessels and a hemorrhagic appearance. Among the other rats in both groups of the experiment, none showed noteworthy changes. Discussion The use of 4-VCD intraperitoneally in rodents promotes selective ovotoxicity, follicular depletion, premature ovarian failure, and changes in the estrous cycle [ 20 , 28 , 29 ]. The beginning of ovotoxicity was evidenced after ten days of treatment with 4-VCD intraperitoneally [ 27 ], detected by the increase in the population of atretic primordial follicles and, from the 12th day of treatment, a significant decrease is detected. in the counting of healthy primordial and primary follicles [ 20 , 27 ]. In the present study, when evaluating the ovotoxicity of 4-VCD through a less invasive route than the intraperitoneal route, it was noted that the use of intravaginal 4-VCD, using chitosan gel as a vehicle, promoted toxicity only to the primordial follicles and affected the estrous cycle of treated rats. However, there was no evidence of sterility during the study period, suggesting that the ovotoxic action of 4-VCD via the intravaginal route is less intense than intraperitoneal route. The findings are, however, promising, considering chemosterilization and support future studies. Firstly, chitosan is a natural polymer and was chosen for the development of the gel, as it presents good biocompatibility, biodegradability, and easy absorption [ 30 ]. For inoculation, the intravaginal gel containing 4-VCD based on chitosan was easily expelled and applied, with an administration volume of 0.2mL, showing good muco-adhesive characteristics and local retention. These characteristics enhance its use since the chitosan-based gel (positive charge) performs electrostatic interactions with the vaginal mucus (negative charge), thus allowing good mucus adhesion and local permanence, guaranteeing drug release [ 31 , 32 ]. Besides, the gel has antibacterial properties [ 33 ]. Furthermore, 24 hours after applying the compound, it was possible to perform vaginal washes without gel residue or interference in the vaginal cytological analysis. In view of this, the chitosan-based gel appeared to be a suitable means of delivering 4-VCD intravaginally. Regarding observations related to the estrous cycle, it is known that rats treated with compounds that are harmful to the reproductive system, may present periods of estrus or diestrus lengthened in each cycle (≥ 4 days of diestrus and ≥ 3 days of estrus), causing long-lasting estrous cycles [ 23 ]. In the present study, the evaluation of the estrous cycle showed that the rats in the 4-VCD group at both moments (M1 and M2) presented a cycle classified as abnormal, with a greater number of days in diestrus. As a result, a prolongation of the estrous cycle and a lower number of cycles were noted in relation to the GC. These findings corroborate the findings of studies that used 4-VCD in doses of 40 and 80 mg/kg intraperitoneally in rats that presented irregularities in the estrous cycle at the beginning of treatment, characterized by the prolongation of days in the diestrus phase and thus, also a lower number of estrous cycles in the given period [ 28 , 34 , 35 ]. These changes begin to be observed in the first 30 days of treatment, starting from the third estrous cycle [ 28 , 34 ]. In the long term, Mayer et al. [ 29 ] observed that the cyclicity is interrupted 360 days after the start of treatment with 4-VCD, after the exhaustion of antral follicles. In the present study, due to the short evaluation period, it was not possible to determine whether the same evolution would occur with the use of 4-VCD in intravaginal gel, even so, interference with cyclicity was noted, justifying future studies that include long-term evaluation. Furthermore, it was noted that one of the rats in the 4-VCD group (1/10) showed signs of inflammation in the uterus and vagina in M1, noted macroscopically during data collection. It is known that insults to the vaginal mucosa can trigger a local inflammatory response, and leukocytes can be found in vaginal washes up to 48 hours after the event, thus mimicking the diestrus phase and confusing the assessment of vaginal cytology for up to 14 days [ 23 , 36 ]. Clear signs of vaginal inflammation were not evident in the other animals in the treated group (9/10), and the possible interference of the gel containing 4-VCD in the local inflammatory response was not completely ruled out and could have been inferred in the analyzes of the estrous cycle in M1. The 4-VCD has corrosive characteristics that cause insults to inoculating tissues; cases of inflammation of mucous membranes, skin, muscles, and other organs have already been observed [ 22 , 39 , 41 – 44 ]. However, to confirm this hypothesis, studies that include histopathological evaluation of the involved tissues are necessary for further conclusions. However, it is important to highlight that 80% of the rats in the 4-VCD group maintained irregular cycles in M2 after the gel inoculations were suspended, characterized by periods of prolonged estrus and a smaller number of estrous cycles. Interestingly, the irregularities in the estrous cycle detected at the beginning of 4-VCD treatment appear to be related to the onset of 4-VCD ovotoxicity to small preantral follicles. This is because the prolongation of estrous cycle phases occurs in parallel with the decrease in the population of healthy follicles and the increase in the number of atretic follicles [ 20 – 28 , 34 , 35 ]. It is known that follicular damage caused by 4-VCD in rats begins after 10 days of intraperitoneal administration at a dose of 80 mg/kg, confirmed by the degradation of oocyte DNA, without there being yet a significant loss in the number of primordial and primary follicles [ 27 ]; and with 12 to 15 days of intraperitoneal treatment with 4-VCD at the aforementioned dose, a significant decrease in the global count of primordial and primary follicles is detected [ 20 , 27 ]. However, in the present study it was observed that one day after the end of intravaginal treatment with 4-VCD (M1) it was not possible to detect a significant difference in the number of healthy or atretic follicles between the groups. But, at this moment, the proportion of healthy primordial follicles was higher in the CG, as well as the proportion of atretic primordial follicles was higher in the 4-VCD group. Thus, these findings corroborate those of other articles [ 20 , 29 ], in which 4-VCD was able to promote a decrease in the population of healthy follicles, increasing follicular atresia. Although the results indicate an ovotoxic action of 4-VCD in M1, the data suggest that through the intravaginal route the ovotoxicity of the compound does not occur with the same intensity or speed as observed through the intraperitoneal route [ 20 , 27 , 28 ]. Differently from what was observed in M1, in M2 a significant increase in the number of atretic primordial follicles was noted in the 4-VCD group. However, no difference was observed in the number of healthy primordial and primary follicles, nor in the number of atretic primary follicles. These findings are in accordance with literature data on the onset of ovotoxicity induced by 4-VCD. According to Kao et al. [ 20 ], the initial phase of follicular damage induced by the compound is noted by an increase in the population of atretic follicles, followed by a decrease in the number of healthy follicles. Therefore, it is believed that, for there to be a significant decrease in the number of healthy follicles, a greater dosage and/or time of exposure to the compound would be necessary. Nevertheless, Hinds et al. [ 45 ] when using 4-VCD orally at different doses (150–450 mg/kg) for five days in mice, they did not notice ovotoxicity, attributing the inefficiency of the compound to the short exposure time and doses considered low for this route. In parallel, a study conducted by Abolaji et al. [ 46 ], when using 4-VCD in rats orally, at doses of 100, 250 and 500 mg/kg, ovotoxicity of 4-VCD was demonstrated, but exposure to the compound lasted 28 days. Reiterating the findings in M1, in M2 it was found that in the 4-VCD group, a significantly higher proportion of atretic primordial follicles and a lower proportion of healthy primordial follicles was observed in relation to the CG, demonstrating the ovotoxic action of the compound via the intravaginal route with just 15 days of exposure. It is known that 4-VCD has an ovotoxic nature through different routes and doses [ 20 , 22 , 27 , 28 , 47 ] and when associated with other chemical compounds [ 48 , 49 ]. However, the route of inoculation can influence its mechanism of action [ 45 , 50 ]. According to the findings of the present study, the intravaginal route for administration of 4-VCD showed signs of ovotoxicity, but there was no complete damage to the primordial follicles in the groups evaluated. It is known that for chemosterilization to occur and fertility to be compromised, the entire population of primordial follicles must be affected [ 20 , 29 , 51 ]. As growing and antral follicles are recruited from primordial and primary follicles, their population will only be affected when the small preantral follicles begin to decrease significantly [ 20 , 29 ]. According to Hinds et al. [ 52 ], up to 30 repeated doses of 4-VCD orally are necessary for it to affect the fertility of rats. In the study conducted by Reis et al. [ 19 ], the administration of 4-VCD at a dose of 160 mg/kg for 15 consecutive days subcutaneously in rats significantly decreased the population of healthy antral follicles. However, ovotoxicity induced by 4-VCD to primordial follicles, at a dose of 80 mg/kg intraperitoneally, occurs at a constant rate, in response to daily/repeated dosing, between 10 and 30 days, and is not a single event [ 20 , 27 , 28 ]. Because of this, it is believed that longer exposure (more than 15 days) to 4-VCD intravaginally is necessary for ovarian atresia and, thus, sterility to occur. Future studies should be carried out to determine whether a longer exposure time and/or a higher dose of 4-VCD via the intravaginal route will be able to promote infertility in rats. However, the present study shows important evidence that this pathway has the potential to be better studied. Conclusions It was noted that the administration of intravaginal gel composed of 4-VCD at a dose of 160 mg/kg for 15 consecutive days promoted toxicity to the primordial follicles, as well as affecting the estrous cycle of treated rats. The studied gel applied intravaginally showed promise and caused atresia of primordial follicles, encouraging future studies. Declarations Authors’ contributions EAPS and EMG carried out the experimental protocol, evaluation and data collection of the estrous cycle and assisted in the collection of biological samples from rats, together with FWSC and MLAM. ACFR and SH prepared the intravaginal gels (control and treatment). EAPS, EMG and MET performed the processing and histological analysis of ovarian tissues (classification and follicular count). All authors contributed to writing the manuscript. All authors read and approved the final version of the manuscript. Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. Competing interests The authors declare that they have no competing interests. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Consent for publication Not applicable. Ethics approval This study was approved by the Committee on the Ethics of Animal Use of the Federal University of Pampa (No. 020/2018). All methods were carried out in accordance with relevant guidelines and regulations. The study was carried out in compliance with ARRIVE guidelines. 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Evaluation of single intratesticular injection of calcium chloride for nonsurgical sterilization in adult albino rats. Contraception. 2006;73:289–300. https://doi.org/10.1016/j.contraception.2005.07.011 . Cavalieri J. Chemical sterilisation of animals: A review of the use of zinc-and CaCl2 based solutions in male and female animals and factors likely to improve responses to treatment. Anim Reprod Sci. 2017;181:1–8. https://doi.org/10.1016/j.anireprosci.2017.03.010 . Leoci R, Aiudi G, Cicirelli V, Brent L, Iaria C, Lacalandra GM. Effects of intratesticular vs intraepididymal calcium chloride sterilant on testicular morphology and fertility in dogs. Theriogenology. 2019;127:153–60. https://doi.org/10.1016/j.theriogenology.2019.01.006 . Paksoy Z, Kandemi̇r FM, Gokhan N, Ozkaraca M. The effects of 4-vinylcyclohexene diepoxide on the testes of dogs. Vet Arh. 2018;88:807–22. https://doi.org/10.24099/vet.arhiv.0101 . Brown G. Advances in reproductive control technology. 13th Annu Conf National Urban Animal Management, Caloundra, Queensland. 2003;107-9. Massei G. Fertility control in dogs. In: Macpherson CN, Meslin FX, Wandeler AI, editors. Dogs, zoonoses and public health. New York: CABI Publishing; 2013. pp. 259–70. Hoyer PB, Devine PJ, Hu X, Thompson KE, Sipes IG. Ovarian toxicity of 4-vinylcyclohexene diepoxide: a mechanistic model. Toxicol Pathol. 2001;29:91–9. https://doi.org/10.1080/019262301301418892 . Reis FM, Pestana-Oliveira N, Leite CM, Lima FB, Brandão ML, Graeff FG, et al. Hormonal changes and increased anxiety-like behavior in a perimenopause-animal model induced by 4-vinylcyclohexene diepoxide (VCD) in female rats. Psychoneuroendocrinology. 2014;49:130–40. https://doi.org/10.1016/j.psyneuen.2014.06.019 . Kao SW, Sipes IG, Hoyer PB. Early effects of ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats and mice. Reprod Toxicol. 1999;13:67–75. https://doi.org/10.1016/S0890-6238(98)00061-6 . Van Kempen TA, Milner TA, Waters EM. Accelerated ovarian failure: a novel, chemically induced animal model of menopause. Brain Res. 2011;1379:176–87. https://doi.org/10.1016/j.brainres.2010.12.064 . Muhammad FS, Goode AK, Kock ND, Arifin EA, Cline JM, Adams MR, et al. Effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague–Dawley rats: ovarian, clinical, and pathologic outcomes. Comp Med. 2009;59:46–59. Goldman JM, Murr AS, Cooper RL. The rodent estrous cycle: characterization of vaginal cytology and its utility in toxicological studies. Birth Defects Res B. 2007;80:84–97. https://doi.org/10.1002/bdrb.20106 . Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol. 2002;62:609–14. https://doi.org/10.1590/S1519-69842002000400008 . Bernal AB, Vickers MH, Hampton MB, Poynton RA, Sloboda DM. Maternal undernutrition significantly impacts ovarian follicle number and increases ovarian oxidative stress in adult rat offspring. PLoS ONE. 2010;5:e15558. https://doi.org/10.1371/journal.pone.0015558 . Paixao L, Velez LM, Santos BR, Tusset C, Lecke SB, Motta AB, et al. Early ovarian follicular development in prepubertal Wistar rats acutely exposed to androgens. J Dev Orig Health Dis. 2016;7:384–90. https://doi.org/10.1017/S2040174416000222 . Springer LN, McAsey ME, Flaws JA, Tilly JL, Sipes IG, Hoyer PB. Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats. Toxicol Appl Pharmacol. 1996;139:394–401. https://doi.org/10.1006/taap.1996.0180 . Flaws JA, Doerr JK, Sipes IG, Hoyer PB. Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide. Reprod Toxicol. 1994;8:509–14. https://doi.org/10.1016/0890-6238(94)90033-7 . Mayer LP, Pearsall NA, Christian PJ, Devine PJ, Payne CM, McCuskey MK, et al. Long-term effects of ovarian follicular depletion in rats by 4-vinylcyclohexene diepoxide. Reprod Toxicol. 2002;16:775–81. https://doi.org/10.1016/S0890-6238(02)00048-5 . Valenta C. The use of mucoadhesive polymers in vaginal delivery. Adv Drug Deliv Rev. 2005;57:1692–712. https://doi.org/10.1016/j.addr.2005.07.004 . Andersen T, Bleher S, Flaten GE, Tho I, Mattsson S, Škalko-Basnet N. Chitosan in mucoadhesive drug delivery: Focus on local vaginal therapy. Mar Drugs. 2015;13:222–36. https://doi.org/10.3390/md13010222 . Şenyiğit ZA, Karavana SY, Eraç B, Gürsel Ö, Limoncu MH, Baloğlu E. Evaluation of chitosan based vaginal bioadhesive gel formulations for antifungal drugs. Acta Pharm. 2014;64:139–56. https://doi.org/10.2478/acph-2014-0013 . Ludwig A. The use of mucoadhesive polymers in ocular drug delivery. Adv Drug Deliv Rev. 2005;57:1595–639. https://doi.org/10.1016/j.addr.2005.07.005 . Lee JH, Lee M, Ahn C, Kang HY, Tran DN, Jeung EB. Parabens accelerate ovarian dysfunction in a 4-vinylcyclohexene diepoxide-induced ovarian failure model. Int J Environ Res Public Health. 2017;14:161. https://doi.org/10.3390/ijerph14020161 . Tran DN, Jung EM, Yoo YM, Ahn C, Kang HY, Choi KC, et al. Depletion of follicles accelerated by combined exposure to phthalates and 4-vinylcyclohexene diepoxide, leading to premature ovarian failure in rats. Reprod Toxicol. 2018;80:60–7. https://doi.org/10.1016/j.reprotox.2018.06.071 . McLean AC, Valenzuela N, Fai S, Bennett SA. Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification. J Vis Exp. 2012;15:e4389. https://doi.org/10.3791/4389 . Sarwal A, Singh G, Singh S, Singh K, Sinha VR. Novel and effectual delivery of an antifungal agent for the treatment of persistent vulvovaginal candidiasis. J Pharm Investig. 2019;49:135–47. https://doi.org/10.1007/s40005-018-0395-3 . Yano J, Lilly E, Barousse M, Fidel PL Jr. Epithelial cell-derived S100 calcium-binding proteins as key mediators in the hallmark acute neutrophil response during Candida vaginitis. Infect Immun. 2010;78:5126–37. https://doi.org/10.1128/iai.00388-10 . Appt SE, Kaplan JR, Clarkson TB, Cline JM, Christian PJ, Hoyer PB. Destruction of primordial ovarian follicles in adult cynomolgus macaques after exposure to 4-vinylcyclohexene diepoxide: a nonhuman primate model of the menopausal transition. Fertil Steril. 2006;86:1210–6. https://doi.org/10.1016/j.fertnstert.2006.05.004 . Appt SE. Peer review and reflection continues forever. Fertil Steril. 2008;89:1034–5. https://doi.org/10.1016/j.fertnstert.2008.02.108 . Chhabra RS, Elwell MR, Peters AN. Toxicity of 4-vinyl-1-cyclohexene diepoxide after 13 weeks of dermal or oral exposure in rats and mice. Fundam Appl Toxicol. 1990;14:745–51. https://doi.org/10.1016/0272-0590(90)90299-Y . Dyer CA, Raymond-Whish S, Schmuki S, Fisher T, Pyzyna B, Bennett A, et al. Accelerated follicle depletion in vitro and in vivo in Sprague-Dawley rats using the combination of 4-vinylcyclohexene diepoxide and triptolide. J Zoo Wildl Med. 2013;S9–17. https://doi.org/10.1638/1042-7260-44.4S.S9 . Maronpot RR. Ovarian toxicity and carcinogenicity in eight recent National Toxicology Program studies. Environ Health Perspect. 1987;73:125–30. https://doi.org/10.1289/ehp.877312 . Weil CS, Condra N, Haun C, Striegel JA. Experimental carcinogenicity and acute toxicity of representative epoxides. Am Industr Hyg Assoc J. 1963;24:305–25. https://doi.org/10.1080/00028896309343226 . Hinds LA, Henry S, Sharma S, Leung L, Dyer C, Mayer L. Effects of oral uptake of the chemosterilant 4-vinylcyclohexene diepoxide in wild house mice, Mus domesticus. Proc. 26thVertebr. Pest Conf. 2014;380 – 85. https://doi.org/10.5070/V426110589 . Abolaji AO, Adedara IA, Abajingin AO, Fatunmibi OJ, Ladipo EO, Farombi EO. Evidence of oxidative damage and reproductive dysfunction accompanying 4-vinylcyclohexene diepoxide exposure in female Wistar rats. Reprod Toxicol. 2016;66:10–9. https://doi.org/10.1016/j.reprotox.2016.09.009 . Carolino RO, Barros PT, Kalil B, Anselmo-Franci J. Endocrine profile of the VCD-induced perimenopausal model rat. PLoS ONE. 2019;14:e0226874. https://doi.org/10.1371/journal.pone.0226874 . Dyer C, Mayer L. Sprague Dawley female rat consumption of a liquid bait containing vinylcyclohexene diepoxide and triptolide leads to subfertility. Proc. 26thVertebr. Pest Conf. 2014;386 – 90. https://doi.org/10.5070/V426110430 . Pyzyna B, Whish S, Dyer CA, Mayer LP, Witmer G, Moulton R. Free ranging wild-caught Norway rats have reduced fecundity after consuming liquid oral fertility bait containing 4-vinylcyclohexene diepoxide and triptolide. Proc. 27thVertebr. Pest Conf. 2016;314 – 16. https://doi.org/10.5070/V427110405 . Burd AM. In vivo and in vitro studies of 4-vinylcyclohexene diepoxide in wild-caught female brushtail possums (Trichosurus vulpecula) and Norway rats (Rattus norvegicus) and its potential as a fertility control agent (Doctoral thesis, Doctor of Philosophy, Department of Agricultural Sciences, Faculty of Agriculture and Life Sciences, Lincoln University). 2014. Witmer GW, Raymond-Whish S, Moulton RS, Pyzyna BR, Calloway EM, Dyer CA, et al. Compromised fertility in free feeding of wild-caught Norway rats (Rattus norvegicus) with a liquid bait containing 4-vinylcyclohexene diepoxide and triptolide. J Zoo Wildl Med. 2017;48:80–90. https://doi.org/10.1638/2015-0250.1 . Hinds LA, Tran TT, Blome AK. Effect of Different Periods of Treatment with 4-vinylcyclohexene Diepoxide on Fertility of Female Rats. J Zoo Wildl Med. 2013;44(4s). https://doi.org/10.1638/1042-7260-44.4S.141 . http://www.bioone.org/doi/full/10.1638/1042-7260-44.4S.141 . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4649606","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":323930974,"identity":"7d3de384-ed75-4f15-b3b6-250728083375","order_by":0,"name":"Endreo Alan Pail dos Santos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYHACAxAhByIOPCBFizFYSwIpWhIbQCRRWvilm7d9+FBxJ31+2OGHQFvs5HQbCGiRnHOseOaMM89yN95OMwBqSTY2O0DIVTdyjJl52w7nbpydANJyIHEbIS32IC1/2w6nG85O/0CcFgMJoBbGtsMJ8tI5RNoicSOtmLHnzDPDDdI5BQcSDIjwC/+M5M0MPyruyMvPTt8MDDo7OYJaoOAAgwFYpQFxyiFa5BuIVz0KRsEoGAUjDAAAww1IsbtGACYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2376-0681","institution":"Universidade Federal do Pampa","correspondingAuthor":true,"prefix":"","firstName":"Endreo","middleName":"Alan Pail dos","lastName":"Santos","suffix":""},{"id":323930975,"identity":"6b9c2bb4-1209-4d81-b3e6-6c888155953f","order_by":1,"name":"Etiele Maldonado Gomes","email":"","orcid":"","institution":"Universidade Federal do Pampa","correspondingAuthor":false,"prefix":"","firstName":"Etiele","middleName":"Maldonado","lastName":"Gomes","suffix":""},{"id":323930976,"identity":"6411225e-9702-4ffc-a4cb-89c946c935cb","order_by":2,"name":"Sandra Elisa Haas","email":"","orcid":"","institution":"Universidade Federal do Pampa","correspondingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"Elisa","lastName":"Haas","suffix":""},{"id":323930977,"identity":"ddf75e8f-867a-40f3-a156-78d5e81a6c79","order_by":3,"name":"Maria Elisa Trost","email":"","orcid":"","institution":"Universidade Federal do Pampa","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Elisa","lastName":"Trost","suffix":""},{"id":323930978,"identity":"c1cd80ba-b0a8-4f2d-89e0-94b46daa8fec","order_by":4,"name":"Francielli Weber Santos Cibin","email":"","orcid":"","institution":"Universidade Federal do Pampa","correspondingAuthor":false,"prefix":"","firstName":"Francielli","middleName":"Weber Santos","lastName":"Cibin","suffix":""},{"id":323930979,"identity":"5ce47005-572d-4f39-a691-8f609021c278","order_by":5,"name":"Ana Claudia Funguetto Ribeiro","email":"","orcid":"","institution":"Universidade Federal do Pampa","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Claudia Funguetto","lastName":"Ribeiro","suffix":""},{"id":323930980,"identity":"3538c39d-0b35-4c61-be82-71f3fd9ccf3c","order_by":6,"name":"Maria Ligia de Arruda Mestieri","email":"","orcid":"","institution":"Universidade Federal do Pampa","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Ligia de Arruda","lastName":"Mestieri","suffix":""}],"badges":[],"createdAt":"2024-06-27 15:33:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4649606/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4649606/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62138936,"identity":"09f0ee39-c061-4dc2-9dc3-297c3b5a2cba","added_by":"auto","created_at":"2024-08-09 16:50:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23881,"visible":true,"origin":"","legend":"\u003cp\u003estudy flowchart (treatment, moments of euthanasia, study design, analyses).\u003c/p\u003e","description":"","filename":"figure1pzG7SB8C.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4649606/v1/e8bc590b2ce9180002885f0d.jpg"},{"id":62138938,"identity":"4a35141a-488f-433e-915b-a39182211567","added_by":"auto","created_at":"2024-08-09 16:50:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":224343,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of ovary sections (40x magnification) stained with hematoxylin and eosin. A) Sample from the control group, in M1. Note small healthy primordial follicles grouped in the cortical region of the ovarian tissue (arrow). Note the arrangement of granulosa cells with a flat shape (arrowhead) surrounding the oocyte. Nuclear contour of the oocyte well defined and circular in shape (*). B) Sample from the 4-VCD group in M1. Atretic primordial follicle, note shrinkage of the oocyte and granulosa cells with loss of contact with the ovarian stroma (arrow). Furthermore, the contracted oocyte nucleus and irregular shape can be seen (*). C) Sample from the control group in M1. Note healthy primary follicle (arrow), surrounded by a cuboidal-shaped layer of granulosa cells. The follicle has a uniform arrangement of granulosa cells, as well as a circular-shaped nucleus and regular contours (*). D) Sample from the 4-VCD group in M1. Note atretic primary follicle (arrow), with oocyte cytoplasm with a granular appearance and loss of focal contact between granulosa cells and oocyte (arrowhead). Note irregular nuclear contour with shrinkage (*).\u003c/p\u003e","description":"","filename":"figure2YPYxzj8X.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4649606/v1/888f62fd7ed9a895120d00c2.jpg"},{"id":62140048,"identity":"b06c8d45-0b8e-4aa1-814f-b443eda2a3ab","added_by":"auto","created_at":"2024-08-09 16:58:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":123424,"visible":true,"origin":"","legend":"\u003cp\u003eNecropsy and tissue collection in M1. A, B and C treatment, D and E control. A) Rat presenting serosanguineous exudate on the vulva and perineal region (arrow), on the 15th day of intravaginal gel inoculation; B) Rat presenting bloody discharge from the vulva for the second consecutive day, 16th day of experiment (M1); C) Body and right uterine horn of a rat at the time of euthanasia and macroscopic exploration in M1, showing structures with cystic aspects (arrows), congested vessels, as well as tissue with edematous regions; D) Rat in M1, moments before euthanasia. Presenting perineal region free of exudates and with a healthy appearance (arrow); E) Macroscopically healthy uterine body and horns of a control group rat in M1.\u003c/p\u003e","description":"","filename":"figure34mUAX2Fe.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4649606/v1/cbe2c6a683b7d048cbbdf9b2.jpg"},{"id":65694869,"identity":"0f05f300-0d85-4ce4-b97c-d5591d9e6902","added_by":"auto","created_at":"2024-10-01 11:04:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":971943,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4649606/v1/0f27fda1-f1ac-498b-a359-a1a415c27cc9.pdf"}],"financialInterests":"","formattedTitle":"Influence of intravaginal 4-vinylcyclohexene diepoxide on ovarian follicles and estrous cycle in rats","fulltext":[{"header":"Background","content":"\u003cp\u003eThe overpopulation of stray dogs and cats is a problem in different parts of the world, causing direct and indirect disruptions to public health, animal welfare and threats to wildlife [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As a form of population control for these animals, surgical sterilization is still the most applied, humanly accepted, and recommended option [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite this, surgical castration is still not completely viable in some locations, due to the difficulty of mass applicability and challenging access for low-income populations [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this context, alternative methods to surgical sterilization aiming at reproductive control are being studied and developed, such as chemosterilization [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs it is considered low-cost, does not require much care after application and can be used on a large scale, it is believed that chemosterilization is an alternative to surgical sterilization, effective for castration/population control programs for stray animals [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, most studies were carried out with substances that require application directly to the gonads, therefore, they are in male animals, due to the testicles being easily accessible [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For population control, however, there is a need for reproductive control methods to be applied to both sexes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, a chemical compound known as 4-vinylcyclohexene diepoxide (4-VCD) has been used experimentally in rodents to induce ovarian failure and mimic menopause [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This compound selectively promotes ovotoxicity, inducing apoptosis of ovarian pre-antral follicles (primordial and primary) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the effective route of use to produce this effect is intraperitoneal (IP), which is invasive and prone to complications [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Envisioning its use in dogs and cats for population control, a less invasive, effective, and safe route is necessary. Therefore, the objective of this study is to evaluate the effect of an intravaginal gel based on chitosan containing 4-VCD on the ovarian follicles and estrous cycle of Wistar rats.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eTwenty adult Wistar rats (\u003cem\u003eRattus norvergicus albinus\u003c/em\u003e) were used. The rats were acclimatized for a period of fifteen days and kept in boxes appropriate to the species (5 animals/box), with food and water \u003cem\u003ead libitum\u003c/em\u003e, in a controlled temperature environment (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and a light-dark lighting cycle (12 hours each). During the acclimatization period, the animals were conditioned to the manipulation and collection of vaginal cytology. The study was approved by the Ethics Committee on the Use of Animals in Research at the Federal University of Pampa \u0026ndash; UNIPAMPA under protocol 020/2018. The study was carried out in accordance with ARRIVE guidelines and regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003e The animals were equally and randomly divided into four experimental groups, according to the treatment received: control (CG) and treatment with 4-VCD gel; and moment of euthanasia: sixteen (M1) and thirty (M2) days after the beginning of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Prior to the study, the appropriate vaginal volume for intravaginal inoculation of chitosan-based gel was investigated so that there was no extravasation, which was established as 0.2 mL (data not shown). Daily, the rats were subjected to intravaginal inoculation at the same time in the morning with up to 0.2 mL of gel for 15 days. Animals in the control group received 0.2 mL of chitosan-based gel without any active ingredient and animals in the treatment group received 0.2 mL of chitosan-based gel at a dose of 160 mg/kg of 4-VCD (~\u0026thinsp;32 mg/rat\u0026thinsp;=\u0026thinsp;0.2 ml of gel) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIntravaginal gel\u003c/h2\u003e \u003cp\u003eThe 2.5% (w/v) chitosan gel was prepared by manually mixing, with a mortar and pestle, chitosan and distilled water acidified with 1% lactic acid. The homogenate was left to rest for approximately 12 hours, under refrigeration. For the treatment group, the chemical compound 4-VCD was slowly incorporated, with a mortar and pestle, into the base gel, resulting in a final concentration of 160 mg/g of 4-VCD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEstrous cycle\u003c/h2\u003e \u003cp\u003eThe rats were subjected to estrous cycle assessment daily from the first day of acclimatization. As an exclusion criterion, rats that had irregular estrous cycles prior to treatment were not used in the study. To this end, daily vaginal washes were performed at the same time, prior to inoculation of the gel. Washings were carried out by placing and retrieving 10 \u0026micro;L of intravaginal saline solution and immediate cytological evaluation using optical microscopy (40x) according to the protocol and classification established by Goldman et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and Marcondes et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The stages of the estrous cycle were classified as proestrus, estrus and diestrus (1 and 2) based on the predominance of cell types [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For each group, the number of days in each phase of the estrous cycle of each rat were counted and added, for later determination of the average. Data were compared between CG and 4-VCD in M1 and M2. The number of estrous cycles of each rat/group were added and the average number of cycles/group was determined and compared between the CG and 4-VCD at both times of the study (M1 and M2). The estrous cycle was classified according to its length according to Goldman et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and consecutive episodes of \u0026ge;\u0026thinsp;4 days of diestrus and \u0026ge;\u0026thinsp;3 days of estrus were considered abnormal/irregular cycles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eOvarian follicle count\u003c/h2\u003e \u003cp\u003eAfter euthanasia, the collected ovaries were fixed in 10% formalin, processed, and embedded in paraffin. The slides were mounted in 3.5 \u0026micro;m thick sections with 40 \u0026micro;m intervals between each, avoiding multiple counts of the same follicle [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The first three slides of each ovary were evaluated. On each slide, the preantral follicles containing nuclei in the oocyte were classified as primordial (oocytes surrounded by a single layer of flattened granulosa cells) and primary (oocytes surrounded by a single layer of cuboidal cells) [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Follicular quality was assessed according to Kao et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and Springer et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], for classification into atretic or healthy follicles. All slides were analyzed with an optical microscope (40 and 100x) by three evaluators in consensus, blindly. The number of healthy and atretic primordial and primary follicles were counted and added together in each rat/group, establishing the average for each group. Therefore, GC and 4-VCD were compared in M1 and M2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEuthanasia\u003c/h2\u003e \u003cp\u003eTo collect the ovaries, euthanasia was performed at two different times: M1 on the 16th day of the experiment, 12 hours after the last inoculation of the intravaginal gel, with five rats from the GC and five from the 4-VCD group; and M2, on the 30th day of the experiment, that is, 15 days after the end of treatment, containing the rest of the animals. Euthanasia was performed with an overdose of propofol intraperitoneally and, after anesthesia, IV potassium chloride was injected. After euthanasia, the ovaries were collected for follicular counting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data obtained from the different stages of each phase, the number of estrous cycles, number of healthy and atretic primordial and primary follicles were statistically analyzed using GraphPad Prism version 8.0.1 (Graphpad Software, San Diego, USA) and expressed as median, lower and upper quartile. Comparisons were made between GC and 4-VCD according to the time of euthanasia (M1 and M2). Differences between GC and 4-VCD were considered significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. As data did not follow a normal distribution, they were analyzed using the non-parametric Mann-Whitney test. For each group, at each time point (M1 and M2), the total number of follicles was computed, the proportion (percentage) for each follicular phase was determined and proportions were calculated using Minitab Inc. (Software, version 18.1). For the proportion test, the 95% confidence interval was considered, and significant differences were considered when P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffects of 4-VCD on estrous cycle analysis\u003c/h2\u003e \u003cp\u003eIn M1, the 4-VCD group showed shorter proestrus phases and longer diestrus phases (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the CG (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of estrous cycles was lower in the 4-VCD group when compared to the CG (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of the rats in the 4-VCD group in M1, all showed an abnormal estrous cycle, with prolongation of the diestrus phase (data not shown).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026ndash; Number of days in each phase of the estrous cycle and number of estrous cycles.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProestrus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEstrous\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDiestrus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN of estrous cycle\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eM1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eGC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5/4\u0026ndash;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5/3\u0026ndash;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5/4,5\u0026ndash;5,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3/2\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4-VCD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1/0.5\u0026ndash;2.5*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3/1.5-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11/9.5\u0026ndash;11*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/0\u0026ndash;2*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eP value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,0119*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,1105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,0112*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,0290*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eM2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eGC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 /5.5\u0026ndash;8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/7.5\u0026ndash;11.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12/11-16.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/3-6.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4-VCD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3/2.5\u0026ndash;3.5*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8/5.5\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20/16\u0026ndash;22*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2/2\u0026ndash;3*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eP value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,0079*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,2463\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,0362*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,0459*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eComparison of the number of days in each phase of the estrous cycle and the number of estrous cycles in the control (CG) and treated (4-VCD) groups at two different times (M1\u0026thinsp;=\u0026thinsp;day 16; M2\u0026thinsp;=\u0026thinsp;day 30). Values are expressed as median/lower and upper quartile, n\u0026thinsp;=\u0026thinsp;5. * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical significance was determined using the Mann-Whitney test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn M2, there was a decrease in the proestrus phase and an increase in the number of days in diestrus in the 4-VCD group when compared to the CG (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, the 4-VCD group presented a significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower number of estrous cycles when compared to the CG (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). One rat in the GC presented an abnormal estrous cycle, with prolonged diestrus (during the treatment period). In the 4-VCD group, 100% of the rats had an estrous cycle classified as abnormal, with a prolongation of the diestrus phase during treatment, and four had prolonged estrus in the 15 days after treatment (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOvarian follicle count\u003c/h2\u003e \u003cp\u003eIn M1, regarding the global count of healthy and atretic primordial and primary follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), there was no statistical difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the CG and the 4-VCD group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was detected between CG and 4-VCD group regarding the proportions of healthy and atretic primordial follicles (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At this time, a higher proportion of healthy primordial follicles was noted in the CG when compared to the 4-VCD group (P\u0026thinsp;=\u0026thinsp;0.004; 21% of healthy primordial follicles in the CG and 9% of healthy primordial follicles in the 4-VCD group). At the same time, a higher proportion of atretic primordial follicles was noted in the 4-VCD group compared to the CG (P\u0026thinsp;=\u0026thinsp;0.039; 35% of atretic primordial follicles in the GC group and 46% of atretic primordial follicles in the 4-VCD group). Regarding the comparison between the proportion of healthy and atretic primary follicles, no significant difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) was detected between the groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026ndash; Global count of primordial and primary follicles (healthy and atretic) in the treated and control groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimordial\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrimary\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAtretic (primordial)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAtretic (primary)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eM1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eGC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6/2.5\u0026ndash;8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/0\u0026ndash;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8/4.5\u0026ndash;14.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7/6.5\u0026ndash;15.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4-VCD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2/0.5\u0026ndash;4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/0\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12/6.5\u0026ndash;16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8/4.5\u0026ndash;17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e \u003cb\u003evalue\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,1161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,4633\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,8340\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eM2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eGC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3/3\u0026ndash;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/0\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8/7-8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14/4.5\u0026ndash;20.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4-VCD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2/0.5-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/0\u0026ndash;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12/10.5\u0026ndash;18*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17/7-21.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e \u003cb\u003evalue\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,3398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,3447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,0112*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,6905\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eComparison of the global count of healthy and atretic primordial and primary follicles in the control (CG) and treated (4-VCD) groups at two different moments (M1\u0026thinsp;=\u0026thinsp;day 16; M2\u0026thinsp;=\u0026thinsp;day 30). Values are expressed as median/lower and upper quartile, n\u0026thinsp;=\u0026thinsp;5. * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical significance was determined using the Mann-Whitney test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProportion of healthy and atretic primordial and primary follicles in the treated and control groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4-VCD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4-VCD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrimordial (healthy)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28/132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12/125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25/133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13/158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e \u003cb\u003evalue\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0,004*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0,004*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrimordial (atretic)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46/132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57/125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39/133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e69/158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e \u003cb\u003evalue\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0,039*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0,005*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrimary (healthy)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5/125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5/133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2/158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e \u003cb\u003evalue\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0,31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0,092\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrimary (atretic)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51/132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51/125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64/133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e74/158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e \u003cb\u003evalue\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0,362\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0,587\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eComparison of the proportion of healthy and atretic primordial and primary follicles in the control (CG) and treated (4-VCD) groups at two different moments (M1\u0026thinsp;=\u0026thinsp;day 16; M2\u0026thinsp;=\u0026thinsp;day 30). Values are expressed as number of follicles phase/total follicles counted per group at each time point and as a percentage, n\u0026thinsp;=\u0026thinsp;5. * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical significance was determined using the proportion test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn M2, the number of healthy primordial follicles, atretic primaries and healthy follicles showed no statistical difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) comparing CG and 4-VCD group. It was noted that the number of atretic primordial follicles was higher in the 4-VCD group compared to the CG, with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It was observed that the CG had a higher proportion of healthy primordial follicles (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to the 4- VCD group (P\u0026thinsp;=\u0026thinsp;0.004; 19% of healthy primordial follicles CG and 8% of healthy primordial follicles in group 4- VCD) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the same time, the proportion of atretic primordial follicles was higher in the 4-VCD group compared to the CG (P\u0026thinsp;=\u0026thinsp;0.005; 29% of atretic primordial follicles in the CG and 44% of atretic primordial follicles in the 4-VCD group) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Regarding the proportion of healthy and atretic primary follicles, no statistically significant difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) was detected when comparing the groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOther findings\u003c/h2\u003e \u003cp\u003eOnly one rat in the 4-VCD group presented, on the 15th day of intravaginal gel inoculation, the presence of serosanguineous exudate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) in the vulva, without further clinical or behavioral changes. On the 16th day of the experiment, the day of euthanasia in M1, the same rat presented bloody exudate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). It is worth noting that the exudate had no odor. This rat was euthanized with the other rats from the same group in M1. After euthanasia, during macroscopic abdominal exploration, the presence of structures with a cystic appearance was noted in the body and right uterine horn (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The body and right uterine horn were edematous, with congested vessels and a hemorrhagic appearance. Among the other rats in both groups of the experiment, none showed noteworthy changes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe use of 4-VCD intraperitoneally in rodents promotes selective ovotoxicity, follicular depletion, premature ovarian failure, and changes in the estrous cycle [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The beginning of ovotoxicity was evidenced after ten days of treatment with 4-VCD intraperitoneally [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], detected by the increase in the population of atretic primordial follicles and, from the 12th day of treatment, a significant decrease is detected. in the counting of healthy primordial and primary follicles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the present study, when evaluating the ovotoxicity of 4-VCD through a less invasive route than the intraperitoneal route, it was noted that the use of intravaginal 4-VCD, using chitosan gel as a vehicle, promoted toxicity only to the primordial follicles and affected the estrous cycle of treated rats. However, there was no evidence of sterility during the study period, suggesting that the ovotoxic action of 4-VCD via the intravaginal route is less intense than intraperitoneal route. The findings are, however, promising, considering chemosterilization and support future studies.\u003c/p\u003e \u003cp\u003eFirstly, chitosan is a natural polymer and was chosen for the development of the gel, as it presents good biocompatibility, biodegradability, and easy absorption [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For inoculation, the intravaginal gel containing 4-VCD based on chitosan was easily expelled and applied, with an administration volume of 0.2mL, showing good muco-adhesive characteristics and local retention. These characteristics enhance its use since the chitosan-based gel (positive charge) performs electrostatic interactions with the vaginal mucus (negative charge), thus allowing good mucus adhesion and local permanence, guaranteeing drug release [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Besides, the gel has antibacterial properties [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, 24 hours after applying the compound, it was possible to perform vaginal washes without gel residue or interference in the vaginal cytological analysis. In view of this, the chitosan-based gel appeared to be a suitable means of delivering 4-VCD intravaginally.\u003c/p\u003e \u003cp\u003eRegarding observations related to the estrous cycle, it is known that rats treated with compounds that are harmful to the reproductive system, may present periods of estrus or diestrus lengthened in each cycle (\u0026ge;\u0026thinsp;4 days of diestrus and \u0026ge;\u0026thinsp;3 days of estrus), causing long-lasting estrous cycles [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the present study, the evaluation of the estrous cycle showed that the rats in the 4-VCD group at both moments (M1 and M2) presented a cycle classified as abnormal, with a greater number of days in diestrus. As a result, a prolongation of the estrous cycle and a lower number of cycles were noted in relation to the GC. These findings corroborate the findings of studies that used 4-VCD in doses of 40 and 80 mg/kg intraperitoneally in rats that presented irregularities in the estrous cycle at the beginning of treatment, characterized by the prolongation of days in the diestrus phase and thus, also a lower number of estrous cycles in the given period [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These changes begin to be observed in the first 30 days of treatment, starting from the third estrous cycle [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the long term, Mayer et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] observed that the cyclicity is interrupted 360 days after the start of treatment with 4-VCD, after the exhaustion of antral follicles. In the present study, due to the short evaluation period, it was not possible to determine whether the same evolution would occur with the use of 4-VCD in intravaginal gel, even so, interference with cyclicity was noted, justifying future studies that include long-term evaluation.\u003c/p\u003e \u003cp\u003eFurthermore, it was noted that one of the rats in the 4-VCD group (1/10) showed signs of inflammation in the uterus and vagina in M1, noted macroscopically during data collection. It is known that insults to the vaginal mucosa can trigger a local inflammatory response, and leukocytes can be found in vaginal washes up to 48 hours after the event, thus mimicking the diestrus phase and confusing the assessment of vaginal cytology for up to 14 days [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Clear signs of vaginal inflammation were not evident in the other animals in the treated group (9/10), and the possible interference of the gel containing 4-VCD in the local inflammatory response was not completely ruled out and could have been inferred in the analyzes of the estrous cycle in M1. The 4-VCD has corrosive characteristics that cause insults to inoculating tissues; cases of inflammation of mucous membranes, skin, muscles, and other organs have already been observed [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, to confirm this hypothesis, studies that include histopathological evaluation of the involved tissues are necessary for further conclusions. However, it is important to highlight that 80% of the rats in the 4-VCD group maintained irregular cycles in M2 after the gel inoculations were suspended, characterized by periods of prolonged estrus and a smaller number of estrous cycles.\u003c/p\u003e \u003cp\u003eInterestingly, the irregularities in the estrous cycle detected at the beginning of 4-VCD treatment appear to be related to the onset of 4-VCD ovotoxicity to small preantral follicles. This is because the prolongation of estrous cycle phases occurs in parallel with the decrease in the population of healthy follicles and the increase in the number of atretic follicles [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is known that follicular damage caused by 4-VCD in rats begins after 10 days of intraperitoneal administration at a dose of 80 mg/kg, confirmed by the degradation of oocyte DNA, without there being yet a significant loss in the number of primordial and primary follicles [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]; and with 12 to 15 days of intraperitoneal treatment with 4-VCD at the aforementioned dose, a significant decrease in the global count of primordial and primary follicles is detected [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, in the present study it was observed that one day after the end of intravaginal treatment with 4-VCD (M1) it was not possible to detect a significant difference in the number of healthy or atretic follicles between the groups. But, at this moment, the proportion of healthy primordial follicles was higher in the CG, as well as the proportion of atretic primordial follicles was higher in the 4-VCD group. Thus, these findings corroborate those of other articles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], in which 4-VCD was able to promote a decrease in the population of healthy follicles, increasing follicular atresia. Although the results indicate an ovotoxic action of 4-VCD in M1, the data suggest that through the intravaginal route the ovotoxicity of the compound does not occur with the same intensity or speed as observed through the intraperitoneal route [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferently from what was observed in M1, in M2 a significant increase in the number of atretic primordial follicles was noted in the 4-VCD group. However, no difference was observed in the number of healthy primordial and primary follicles, nor in the number of atretic primary follicles. These findings are in accordance with literature data on the onset of ovotoxicity induced by 4-VCD. According to Kao et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the initial phase of follicular damage induced by the compound is noted by an increase in the population of atretic follicles, followed by a decrease in the number of healthy follicles. Therefore, it is believed that, for there to be a significant decrease in the number of healthy follicles, a greater dosage and/or time of exposure to the compound would be necessary. Nevertheless, Hinds et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] when using 4-VCD orally at different doses (150\u0026ndash;450 mg/kg) for five days in mice, they did not notice ovotoxicity, attributing the inefficiency of the compound to the short exposure time and doses considered low for this route. In parallel, a study conducted by Abolaji et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], when using 4-VCD in rats orally, at doses of 100, 250 and 500 mg/kg, ovotoxicity of 4-VCD was demonstrated, but exposure to the compound lasted 28 days. Reiterating the findings in M1, in M2 it was found that in the 4-VCD group, a significantly higher proportion of atretic primordial follicles and a lower proportion of healthy primordial follicles was observed in relation to the CG, demonstrating the ovotoxic action of the compound via the intravaginal route with just 15 days of exposure.\u003c/p\u003e \u003cp\u003eIt is known that 4-VCD has an ovotoxic nature through different routes and doses [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and when associated with other chemical compounds [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. However, the route of inoculation can influence its mechanism of action [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. According to the findings of the present study, the intravaginal route for administration of 4-VCD showed signs of ovotoxicity, but there was no complete damage to the primordial follicles in the groups evaluated. It is known that for chemosterilization to occur and fertility to be compromised, the entire population of primordial follicles must be affected [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. As growing and antral follicles are recruited from primordial and primary follicles, their population will only be affected when the small preantral follicles begin to decrease significantly [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to Hinds et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], up to 30 repeated doses of 4-VCD orally are necessary for it to affect the fertility of rats. In the study conducted by Reis et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the administration of 4-VCD at a dose of 160 mg/kg for 15 consecutive days subcutaneously in rats significantly decreased the population of healthy antral follicles. However, ovotoxicity induced by 4-VCD to primordial follicles, at a dose of 80 mg/kg intraperitoneally, occurs at a constant rate, in response to daily/repeated dosing, between 10 and 30 days, and is not a single event [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Because of this, it is believed that longer exposure (more than 15 days) to 4-VCD intravaginally is necessary for ovarian atresia and, thus, sterility to occur. Future studies should be carried out to determine whether a longer exposure time and/or a higher dose of 4-VCD via the intravaginal route will be able to promote infertility in rats. However, the present study shows important evidence that this pathway has the potential to be better studied.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIt was noted that the administration of intravaginal gel composed of 4-VCD at a dose of 160 mg/kg for 15 consecutive days promoted toxicity to the primordial follicles, as well as affecting the estrous cycle of treated rats. The studied gel applied intravaginally showed promise and caused atresia of primordial follicles, encouraging future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEAPS and EMG carried out the experimental protocol, evaluation and data collection of the estrous cycle and assisted in the collection of biological samples from rats, together with FWSC and MLAM. ACFR and SH prepared the intravaginal gels (control and treatment). EAPS, EMG and MET performed the processing and histological analysis of ovarian tissues (classification and follicular count). All authors contributed to writing the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES) \u0026ndash; Finance Code 001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Committee on the Ethics of Animal Use of the Federal University of Pampa (No. 020/2018). All methods were carried out in accordance with relevant guidelines and regulations. The study was carried out in compliance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrior publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData have not been published previously.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES) \u0026ndash; Finance Code 001.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdulkarim A, Khan MA, Aklilu E. Stray animal population control: methods, public health concern, ethics, and animal welfare issues. 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Compromised fertility in free feeding of wild-caught Norway rats (Rattus norvegicus) with a liquid bait containing 4-vinylcyclohexene diepoxide and triptolide. J Zoo Wildl Med. 2017;48:80\u0026ndash;90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1638/2015-0250.1\u003c/span\u003e\u003cspan address=\"10.1638/2015-0250.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHinds LA, Tran TT, Blome AK. Effect of Different Periods of Treatment with 4-vinylcyclohexene Diepoxide on Fertility of Female Rats. J Zoo Wildl Med. 2013;44(4s). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1638/1042-7260-44.4S.141\u003c/span\u003e\u003cspan address=\"10.1638/1042-7260-44.4S.141\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioone.org/doi/full/10.1638/1042-7260-44.4S.141\u003c/span\u003e\u003cspan address=\"http://www.bioone.org/doi/full/10.1638/1042-7260-44.4S.141\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chemosterilization, female chemical castration, follicular atresia, ovotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-4649606/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4649606/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eFor the population control of stray dogs and cats, alternative methods to surgical castration are being studied and developed, such as chemosterilization. In this context, substances that lead to irreversible degeneration of the tissue administered directly to the gonads are being investigated, with most studies focusing on males due to the ease of access to the testicles. In females, ovarian degeneration is reported with the use of a chemical compound called 4-vinylcyclohexene diepoxide, a selective ovotoxic agent, used in experimental studies in rats to mimic menopause. However, it requires fifteen consecutive days of intraperitoneal application. Envisioning its use for the population control of dogs and cats, a less invasive and effective route is necessary. Thus, the aim of this study was to evaluate the effect of 4-vinylcyclohexene diepoxide on the ovaries and its chemosterilizing potential by intravaginal gel inoculation in rats. Twenty Wistar female rats were used, randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;10/group): control and treatment. The control group received 0.2 mL of chitosan-based gel, and the treatment group received the same gel containing 4-vinylcyclohexene diepoxide (160 mg/kg) for fifteen days. The rats were evaluated daily by vaginal cytology until euthanasia. The animals were euthanized at two time points (n\u0026thinsp;=\u0026thinsp;5/group): Sixteen (M1) and thirty (M2) days after the start of treatment. The ovaries were collected, prepared, and evaluated by optical microscopy for counting of primordial and primary follicles.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the group treated with 4-vinylcyclohexene diepoxide, a prolongation of the diestrus phase (M1 and M2), as well as a prolongation of the estrus phase (M2), was noted after inoculations of the intravaginal gel. Even so, in the treated group, there was an increase in the number of estrous cycles compared to the control group (M1 and M2). In the follicular count, a higher median number of atretic primordial follicles was noted in the group treated in M2. Follicular evaluation in M1 and M2 revealed a higher proportion of healthy primordial follicles in the control group, as well as an increase in the proportion of atretic primordial follicles in the treated group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIt was possible to observe evidence of ovotoxicity of chitosan gel containing 4-vinylcyclohexene diepoxide, applied intravaginally, due to the increase in the population and proportion of atretic primordial follicles and interference in the estrous cycle.\u003c/p\u003e","manuscriptTitle":"Influence of intravaginal 4-vinylcyclohexene diepoxide on ovarian follicles and estrous cycle in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 16:50:21","doi":"10.21203/rs.3.rs-4649606/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1daef4af-5c24-4d99-ae4b-66fc8eab96c8","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-01T10:55:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 16:50:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4649606","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4649606","identity":"rs-4649606","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}