The extract of the Broussonetia papyrifera can be used as a raw material source for the sterility agent of Microtus fortis

doi:10.21203/rs.3.rs-5305445/v1

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The extract of the Broussonetia papyrifera can be used as a raw material source for the sterility agent of Microtus fortis

https://doi.org/10.21203/rs.3.rs-5305445/v1

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Background: The plant sterilants are studied to control rodent populations with their minimal environmental risk and other ethical considerations. However, the practical utilization was unsatisfactory and stagnant due to high costs and process difficulties. Broussonetia papyrifera is a potential plant material, that showed inhibition to reproduction of Microtus fortis, which usually caused serious damages to crops around the Dongting Lake region, China. The M. fortis were treated with different doses of B. papyrifera leaves methanol extract.

Results: The results showed growth of sex organs were inhibited, male testosterone level and sperm quality reduced. Though there were some positive effects on female, the reproductive parameters of couple voles were also inferior, most treated couple spent more reproductive time, fetal counts and weight were worse. It is also found that M. fortis would obviously negative respond to the extract after the single treatment or long-term repeated treatment rather than the short-term repeated treatment. B. papyrifera leaves showed the more applicative potential of sterilant for male rodents. Conclusions: These findings enrich the study of plant sterilants,and provide insights into the utilization of B. papyrifera and management of rodents. Owing to effectiveness and accessibility of the leaves, the derived sterilant may be more economic in the practice that controlling rodent pest.

methanolic extract

Microtus fortis

sterilants

organ coefficient

sperm teratogenicity

Rodents are the most species-rich mammals, which comprise approximately 42% of all living mammals and over 2200 defined species nowadays (Romanenko et al. 2012), include various kinds of mice, rats, voles and others. Some of they are significant pests that associated with the destruction of crops and the spread of disease worldwide (Meerburg et al. 2009, Díaz et al. 2010, Buckle 2013). Amongst vertebrates, rodents are primarily responsible for damage to forests and agriculture (Mwanjabe et al. 2002, Mulungu et al. 2007, Guo et al. 2013). The development of methods for controlling rodent pests has fostered a rich literature on rat population biology (Keiner 2005, Pages et al. 2013). In recent years, chemical methods have been the mainstay of the rodent pests control such as the anticoagulant rodenticides, which are quick action and high toxicity to rodents (Pelz et al. 2005, Zhou et al. 2013, Chen et al. 2020). However, rodents are mammals with sophisticated behavior, it is essential to allow for behavioral responses that have evolved by natural selection, thereby increasing individual fitness (Baker et al. 2007, Pianalto and Yool 2017), which enable them to resist these chemical rodenticides over time. Though the lethal chemical rodenticides may deal with the immediate pest problem rapidly. Lethal control may, however, be followed by rapid immigration of rodents such that the damage reduction is short lived (Smith and Meyer 2015). This consequence has led, not only to potentially harmful on rodent predators and environment (Zhang 2000, Geduhn et al. 2015), but also to the evolution of resistance with rodents to these chemical rodenticides (Ishizuka et al. 2008).

Recent attempts to control rodent populations have included fertility management, which is the hot topics in pest prevention. Most of these studies have advocated sterilization to control fertility (Shi et al. 2002, Jacob et al. 2008, Massawe et al. 2018). The conventional sterilization methods may be immunotherapy, surgical sterilization, and gene drives (Kirkpatrick 2007, Drury et al. 2017, Massawe et al., 2018). For example, immunocontraception uses an individual’s immune system to inhibit fertility (Hardy et al. 2006), whereas chemosterilization uses a chemical or hormonal compound (Alemany et al. 2008). Though they may be potentially be very effective, resistance of rodents to these sterilization methods evolves for the same reasons that resistance evolves to rodenticides based on the strong potency (Drury et al., 2017, Shuster et al. 2018), meanwhile, they are really expensive, and may impose ecological risks (Norbury 2000, Churchill 2014). The plant-derived sterilants mainly be produced from plants and their extract, which are more environmental and economic, and have a more persistent control effects compared with the conventional chemical agents (McGee et al. 2020). At present, abundant plant species have been known for the effects on fertility regulation of animals (Kumar et al. 2012). Among them, the triptolide and curcumol are the most familiar that used in rodent control. Triptolide is extracted from Tripterygium wilfordii, study revealed its antifertility effect of triptolide on male rats intensified with rising dose (Hikim et al. 2000). Curcumol is an important component of the essential oil of Rhizoma curcuma, and has antifertility effects on both male and female rodents (Wen et al. 2022).

The Microtus fortis is a small herbivore species widely distributed around the Eurasia, it often causes damage to agriculture in many countries include China, Russia, Mongolia, Korea (Zhu et al. 2019). Meanwhile, the vole is the host of some pathogenes like hantavirus which cause hemorrhagic fever with renal syndrome in human (Zou et al. 2008). This rapid-reproduction specie is the dominant rodents around the Dongting Lake region, where is located in the middle of the Yangtze valley, China (Zhang et al. 2007, Zhang et al. 2014). During the dry season, the vole primarily lives on the beaches of Dongting Lake. However, the available space reduce, and density of population increase during wet season, the voles migrate to the surrounding village and cropland (Zhang et al. 2016), then cause the damages to crops and healthy events. To avoid these adverse result, rodent-proof walls have been built around Dongting Lake wetland that prevent the voles migrate to outside of the wetland (Zhang et al. 2012). When population density of the voles breaks out during wet seasons, they are usually killed using artificial behaviors or chemical rodenticides (Ye et al. 2006). These methods are rapid and effective while lead to the subsequent environmental problems. For example, the high toxic rodenticides like anticoagulants are effective to controlling rodent pest, but also cause the death of their predators and other species due to the broad-spectrum toxicants (Campbell et al. 2015, Ma et al. 2019). Therefore, developing some affiliative rodenticides such as plant-inhibitors is needful which are more friendly to ecosystem.

Broussonetia papyrifera is the deciduous tree belonging to the Moraceae family with fast growth and high adaptability (Xi et al. 2013), and also widely distribute around the Dongting Lake region. In wet season, we also have observed that numbers of M. fortis feed B. papyrifera leaves during their migration from beaches to croplands. Owing to the good palatability of B. papyrifera leave, it has been used to feed livestock massively for a long time (Zhang et al. 2009, Tao et al. 2020). However, compounds contained in B. papyrifera leaves showed the inhibitive effect on estrogen biosynthesis in mammalian ovarian granulosa (Yang et al. 2014). According to our previous feeding experiment, the growth and fertility of M. fortis were inhibited when they were fed with B. papyrifera leaves (Wang et al. 2023). Combining with our subsequent study, revealed that the B. papyrifera leaves have strong feeding attraction to M. fortis, and the defense of B. papyrifera leaves were sensitive to bite of the voles (Wang et al. 2024). This reminder us the substances of B. papyrifera leaves are abundant and changeful base on the environment, and suggested the leaves were the potential new resource to develop plant-inhibitors that control reproduction of rodents. Thus, the present study aimed to explored the inhibitory effects of methanol extract from B. papyrifera leaves on the reproductive parameters of laboratorial M. fortis. Meanwhile, considering that M. fortis has been evolved as the suitable model animal, this study verified the antifertility activity of B. papyrifera leaves, and may provide a scientific basis for comprehensive rodent control and management.

Plant material

Fresh leaves of B. papyrifera were collected from the trees that natural grow with health soil. The leaves were first thoroughly washed using ultrapure water and allowed to dry under shade with optimal ventilation, then accelerated the efficiency of evaporation at 60 ℃ using a constant temperature oven. Dried leaves were ground into coarse powder, then macerated in a tenfold (w/v) volume of 100% methanol for 3 days at room temperature, the solvent was filtered using a screen (0.075 mm). To reduce the content of organic solvent, the clarified solvent was hundredfold evaporated under reduced pressure to afford the B. papyrifera leaves methanol extract. The extract was 10-fold, 100-fold, 500-fold, 1000-fold and 5000-fold dissolved with ultrapure water, and made five doses of extract, respectively.

Animals and experimental design

A total of 180 mature M. fortis with half males and half females were used for present study, which were the offspring of wild-caught M. fortis from the Dongting Lake area, that maintained in the laboratory, and outbred stock. The voles were assigned into six groups, including one control group and five treatment groups, each group contained 15 males and 15 females. All M. fortis were maintained under standard conditions with 12-hour light/dark cycles at 20–22 ℃, and allowed free access to water and standard pellet laboratory diet (Hunan SJA Laboratory Animal CO., LTD) (Zhu et al. 2011). The voles were weighed before the experiment to record the original weight, then given the different treatment. Voles of the control group (CK) were extra given 1 ml ultrapure water per day by gavage. Voles of the five treatment groups were extra given 1 ml 5000-fold (group Ⅰ), 1000-fold (group Ⅱ), 500-fold (group Ⅲ), 100-fold (group Ⅳ), and 10-fold (group Ⅴ) dissolved extract per day, respectively (Fig. 1).

Anatomy and morphological index of organs

For both male and female, three cages with four individuals in each group were chosen for anatomy after treating for 1 days, 10 days and 20 days, respectively. That is four males and four females of each group were sacrificed in once experimental periods. Voles were weighed and then excessively anaesthetized by diethyl ether (Han et al. 2015) after the last gavage for 24 hours, the hearts, livers, lungs, kidneys, male testes, female uteruses and ovaries were excised, washed and weighed. Moreover, the lengths of female uteruses were measured, the volume of male testes were also estimated according the following equation:

V = 4π/3(long diameter/2×transverse diameter/2×anteroposterior diameter/2)

Hormone assessment

Blood samples were collected immediately after the voles were narcotized and weighed by removing the eyeballs (Deng et al. 2018). The blood samples were kept at room temperature for 1 hour, then they were centrifuges twice at 5000 rpm for 10 min to obtain the serums which were transferred into new tubes and stored at -20 ℃ for subsequent measure. Male serum testosterone and luteinizing hormone (LH), female estradiol, follicle-stimulating hormone (FSH) and LH were quantified using enzyme-linked immunosorbent assay (ELISA) kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) following the manufacturer’s instructions.

Sperms analysis

Left caudal part of epididymis was carefully separated from each testis, after PBS washing, caudal epididymis was cut in 2 ml normal saline with 10% BSA and incubated at 37 ℃ for 15 min. The sperm suspension was analyzed by a computer-aided sperm analysis system (CASA, Song Jing Tian Lun Biotechnology Co., Ltd., Nanning, China). At least 5 sights of each vole were captured and the index including sperm count, vitality were examined and recorded. For sperm morphology assessment, a smear of sperm samples was fixed and stained using Diff-Quik staining method (Rurangwa et al. 2004). The slides were observed under the light microscope under 40× magnification, at least 20 sights of each vole were inspected randomly for the record of abnormal and total count of sperms.

Mating and breeding assay

M. fortis in cages with three individuals were used for breeding in each group, after treating for 20 days, males and females were matched randomly into three cages of couple voles, and stop treatment. Then, the reproduction situation in cages were inspected every day, when fetal voles appeared, the time of reproduction, count and weight of fetuses were recorded.

Data statistics

The organ coefficients were calculated using the weights of the organs divided by the weights of bodies. The mean value of indexed were calculated using SPSS software and expressed as means ± standard error (Mean ± SE). The significant differences among the mean weights of cages were analyzed using One-way Analysis of Variance (ANOVA), last weight variations of cages through the experiment were analyzed using Paired Samples T test method. Focus on reproductive indexes, we laid emphasis on the comparisons between CK group and each treatment group, respectively, and the Independent Samples T test was used to evaluate the differences. P-value was lower than 0.05 (P<0.05) was considered statistically significant.

Variation of body weight respond to extract

To confirm the influence of B. papyrifera leaves extract on weight of these voles, comparisons were carried out among different groups. The results showed the weight of them changed fractionally during treatment (Fig. 2). The weight range of CK voles were stable, their discrepancies were lower than 2%. According to the comparisons among groups and CK, the weights of treated groups were at similar level refer to CK. The slight variations possibly be related to food intake and digestion. Therefore, the B. papyrifera leaves extract showed a marginal effect on body weight of M. fortis.

Influence of extract on the organ coefficient

Overall, there were not obvious influence of B. papyrifera leaves extract on development of hearts, livers, lungs, kidneys of M. fortis. These organ coefficients of male voles were almost at the similar levels (Fig. S1A), a few significant differences were found between some treatment groups and CK group (P<0.05), while most organ coefficients showed no significant difference in three periods. Also fewer differences found in female comparisons (Fig. S1B), there was no significant difference of lung coefficients and liver coefficients between treatment groups and CK (P>0.05). Only two significant differences of heart coefficients and one of kidney coefficients were found from the comparisons between treatment and CK (P<0.05).

Figure 3 summarizes the comparison among sex organs of M. fortis between each treatment groups and CK. As a whole, B. papyrifera leaves extract showed the more obvious inhibiting effects on male testes and female uteruses than on the female ovaries. The testis coefficients of CK were higher than all treatment groups after 1 day and 20 days (Fig. 3A), among which the testis coefficients of group Ⅳ was significant lower than CK after 1 day (P<0.05). After 10 days, testis coefficients group Ⅰ, Ⅳ, Ⅴ rose and were higher than CK, the significant differences (P<0.05) were found between CK and group Ⅱ, and between CK and group Ⅳ.

Compared with CK, uterus coefficients of some groups were insignificant higher (P>0.05), when that of group Ⅳ was significant lower (P<0.05). However, it can be seen that CK showed the major superiority both after 1 day and 20 days (Fig. 3B). Nonetheless, female ovary coefficients rose after extract treatment (Fig. 3C). The ovary coefficients of CK were inferior compared with treatment groups especially after treating for 1 day, when the ovary coefficients of group Ⅰ, Ⅲ, Ⅳ, Ⅴ were respective higher than CK (P<0.05). But the inferiority of CK weakened over time.

Being similar with organ coefficients, the physical development of testes and uteruses were inferior after treatment. Volumes of CK were greater after 1 day and 20 days, while it was weaker than most treatment groups after 10 days (Fig. 3D). Testicular volumes of group Ⅲ was significant lower than CK after 1 day (P<0.05), and that of group Ⅳ was significant higher than CK after 10 days (P<0.05), while other comparison showed no significant differences (P>0.05). From the comparison of uterine lengths, CK group demonstrated superiority over time (Fig. 3E). Focus on uterine lengths, CK showed significant inferiority after 1 day (Fig. 3B), its length was shortest and significant weaker than group Ⅲ (P<0.05), group Ⅳ and Ⅳ (P<0.01). The tendency changed after 10 days, only uterine lengths of group Ⅰ significant higher than CK (P<0.01) while other were lower than CK, wherein, group Ⅳ was significant lower than CK (P<0.05), group Ⅲ and Ⅴ were extremely significant lower than CK (P<0.01). There was no statistically significant difference found after 20 days, however, the uterine length of CK was higher than all treatment groups.

Effect of extract on the sex hormones

The extract of B. papyrifera leaves showed positive effects on the measured sex hormones except male testosterone (Fig. 4). Testosterone levels of group Ⅰ maintained a tiny superiority with no significant (P>0.05) compared with that of CK through the experiment, while other four groups were generally weaker than CK. (Fig. 4A). Though group Ⅲ and Ⅴ rose and were higher than CK to certain degree after 10 days, group Ⅴ was significant greater than CK (P<0.05), then they reduced on the day 20. It was obvious testosterone levels of CK had the obvious superiority, especially compared with group Ⅱ and Ⅳ.

The LH levels were measured both in male and female, which demonstrated that stimulative effects were less than inhibitive effects over time (Fig. 4B, E). Their LH levels emerged fluctuant phenomenon with changing dose. On the day 1 and day 20, male LH level of CK was still higher than groups Ⅱ and Ⅳ, and showed significant superiorities. However, LH level of CK reduced after 20 days, when treatment groups showed the preponderant superiorities compared with CK. Unlike male, female LH levels of CK were lowest from the day 1, most groups had the significant superiority than that of CK during the experiment.

Compared with CK in female, the sex hormone levels of treatment groups demonstrated the remarkable superiority, as a whole, the extract enhanced the levels of E2 and FSH. The E2 of CK were lower than treatment groups since treated for 1 day (Fig. 4C), and its inferiority enlarged after 20 days, though it seemingly got better from 1 day to 10 days. The beneficial effect of extract on FSH levels was not significant as above two female hormones (Fig. 4D), variations of the two higher content of extract groups (group Ⅳ, Ⅴ) were extraordinary. FSH levels of group Ⅳ and Ⅴ were similar, which were rose exceeding though the periods, they were even significant lower than that of CK after 1 day (P<0.05), remarkably, their FSH levels exceeded CK after treating for 10 days, and has the significant superiority after 20 days (P<0.05).

Variation of male sperm parameters

There was no significant variation (P>0.05) of sperm counts of treatment groups compared with CK (Fig. 5A). The fluctuation of CK was wide with large standard error, which caused that CK showed no significant superiority though its mean counts were more than most treatment groups after 1 day and 20 days. Nonetheless, we could consider the extract had no positive effect on sperm count of the male voles.

The results demonstrated that the extract could reduce the activity of sperms over time (Fig. 5B). After treating for 1 day, activity of most groups were higher than CK except group Ⅳ, but did not has the significant different (P>0.05). The activities of group Ⅱ, Ⅲ, Ⅳ reduced obviously while group Ⅰ and Ⅴ rose slightly after 10 days, and CK was significant higher than group Ⅱ and Ⅲ (P<0.05). Afterward, sperm activities of all groups were lower than CK after 20 days, when CK showed the greater superiority, and kept significance with group Ⅱ and Ⅲ (P<0.05).

Based on the information of abnormal sperms, it was found the extract has the effect of teratogenicity (Table 1, Fig. 6), the types of abnormal sperm were showed in Fig. 6. Though there were a few significant differences found between CK and treatment groups, most of the abnormal proportion in treatment groups were higher than that of CK. After 1 day, the abnormal proportion of CK was 16.59% ± 0.87%, only group Ⅳ was a bit lower than CK, while other groups all were higher, in which group Ⅰ was 23.55% ± 0.48% and significant higher than CK (P<0.01). However, the abnormal proportion of group Ⅰ and Ⅳ fluctuated largely after treating for 10 days, and group Ⅰ was the only group that lower than CK. At the period, the proportion of CK was higher than four group, and showed the significant differences with group Ⅲ (P<0.05) and Ⅳ (P<0.01), respectively. After 20 days, the abnormal proportion of CK was lower than 10% while other were higher, meanwhile, groups Ⅳ still showed the significant inferiority compared with CK (P<0.01).

Table 1

The abnormal proportion of sperm and comparison between treatment groups and CK.
Group	Abnormal proportion (mean ± SE %, P value = group/CK)
Group	1 Day	10 Day	20 Day
CK	16.59 ± 0.87	9.77 ± 1.05	8.29 ± 1.94
Ⅰ	23.55 ± 0.48 **	9.15 ± 0.92	10.45 ± 3.94
Ⅱ	17.69 ± 1.95	15.45 ± 5.51	18.17 ± 3.16
Ⅲ	25.15 ± 5.33	19.01 ± 2.10 *	17.54 ± 3.47
Ⅳ	16.15 ± 2.03	22.79 ± 1.32 **	25.71 ± 2.94 **
Ⅴ	18.42 ± 1.13	11.42 ± 2.76	12.91 ± 0.98
Data presented as Note: * represent P<0.05, ** represent P<0.01

The reproductive characteristic of couple voles

The reproduction parameters of CK were greater than most groups (Table 2). Though the reproductive time of CK was a bit more than group Ⅳ and Ⅴ, but its count of fetuses also was more than the two groups, while weights were close. The difference between CK and group Ⅰ, Ⅱ, Ⅲ were more obvious. Reproduction of group Ⅲ was the only one that superior to CK with the less reproductive time and bigger weight of fetuses. Based on the close reproductive time and the same count fetuses, group Ⅰ showed the obvious inferiority of fetal weight, which was only 2.900 ± 0.091 g, and much less than that of CK (3.900 ± 0.102 g). Though fetal weight of group Ⅱ was 3.922 ± 0.083 and little bigger than CK, its fetal count was less. Meanwhile, the major inferiority of group Ⅱ was that couple voles spent much more reproductive time compared with CK and other groups.

Table 2

Characteristics of reproduction in different groups
Group	Reproductive time(day)	Count of fetuses	Weight of fetuses(g)
CK	24	4	3.900 ± 0.102
Ⅰ	22	4	2.900 ± 0.091
Ⅱ	28.333	3	3.922 ± 0.083
Ⅲ	21.667	4	4.117 ± 0.139
Ⅳ	22.667	3.333	3.990 ± 0.129
Ⅴ	22.5	3	3.967 ± 0.080

The present study attempts to reveal the antifertility effect of methanol extract from B. papyrifera leaves on M. fortis orally. Before that, the study also evaluated the potential effects on other physiological parameters, and found the body weight of the voles treated with extract remained approximately unchanged, growths of heart, liver, lung and kidney also did not get the obvious influence. Which showed that these doses of methanol extract from B. papyrifera leaves exert hardly any harmful effects on these parameters, the metabolic processes of these organs were normal. Overall, the comparison of reproductive parameters in groups suggest that methanol extract of B. papyrifera leaves have the potential function that inhibit the growth of reproductive organs of M. fortis, and the efficient teratogenicity to male sperm. Though there some positive response from female indexes. Studied also revealed the extract of plants inhibit the fertility of male, include reducing sperm motility and testosterone level, increasing sperm deformity (Jalili et al. 2014, Tu et al. 2020). Which were similar with the results on male voles in this study. As a whole, the fertility of female did not suffer the obvious inhibition, based on their sexual organs and hormones. Steroid hormones have the close correlation, and important for develop of sexual organs. In our results, growths of female ovaries were better after treated, this may because of the higher hormone levels. However, there is no certain evidence that proving the positive correlation between sexual hormones and reproduction, the mating behavior can occur with very low levels of sexual hormones (Crews 1984). According to the inferior reproduction of treated couple voles, there must be some undetected effects in our experiment, such as oestrous cycles, mating behavior, pregnancy rate, these may also attribute to the males.

In the 21st century, fertility control has become a humane, sustainable technology with few negative effects for controlling rodent damage and thus has attracted substantial attention. Scholar proposed that control rodent damage by making male sexually sterilants (Shuster et al., 2018). Base on polygamy of reproductive strategy, it may be the efficient measure that control the population abundance of most kinds of rodents with fewer expenditure. At this viewpoint, B. papyrifera leaves could reach at least the condition that inhibiting fertility of male rodents. Though developing an antifertility agent from a plant source is cost-effective and has also low toxicity, once being involved in the practical application, it still was faced with the high costs and production difficulties. In China, sterilants from plant sources that have been applied in the fertility control of pest rodents include Tripterygium wilfordii, gossypol, Ruta graveolens, Camellia oleifera, radix trichosanthis (Trichosanthes kirilowi), colchicine, semen ricini (Ricinus communis), aaruginous turmeric rhizome (Curcumae aeruginosae) and neem (Melia azederach). Though all of them had been made into baits for animal experiment in the laboratory or sprayed in the wild for animal foraging, which had certain effects, the practical applications have been unsatisfactory (Fu et al. 2016). Thus, the application of fertility control remained stagnant. Compared with these plants, the advantages of B. papyrifera leaves might be obvious. First, it is widely planted and naturally grows in China (Xi et al., 2013, Han et al. 2016), which explains the available resource is much abundant, even people could draw the leaves materials locally where usually suffer from rodent pests. It could immensely save the cost of plant materials and transportation. Second, the crude extract of B. papyrifera leaves can already has the efficient inhibitive effect on M. fortis according to present research, it means this plant resource is more economical in the process of production. Though in terms of controlling rodents, fertility control has greater potential than conventional rodenticides (Singleton et al. 2002, Shilova and Tchabovsky 2009, Coeurdassier et al. 2014). It is undeniable that advantages and disadvantages are relative as scholar propose, the effects of sterilants are not immediate because they do not directly cause rodent mortality (Wen et al., 2022). People would rather to kill all rodents in small environment, while it may be desirable to controlling their population abundance in ecosystem (Dominguez et al. 2021). In this case, neither sterilants nor rodenticides are the excellent measure that against rodent pests, however, the combined use of they may be applicable and efficient sometimes. For example, when rodent populations outbreak, rodenticides could be firstly used to eliminate most rodent individuals, then rendered the remaining rodents sterile by sterilants.

The excavation of contributing substances is also significant for evolution of plant sterilants. As the compounds, the substances in methanol extract of B. papyrifera leaves have determined by a metabolomics analysis. A total of 5170 metabolites were detected in methanol extract of B. papyrifera leaves, and classified into 17 categories (Figure S2). Studies demonstrated that plants rich in secondary metabolites such as flavonoid, terpenes and alkaloids, showed the antifertility activity on animals, include impeding ovulation, impeding spermatogenesis, reducing sperm activity and count, disturbing breeding cycle (Peng et al. 2003, Sindhia and Bairwa 2010). These kinds of secondary metabolites have been identified in the methanol extract. Study revealed that plant extracts which contain toxic compounds like glycosides, anthraquinones, tannins, organicacids and toxic minerals, have the toxic effects on livers and testes (Toson et al. 2014). Most of these metabolites are also found in our determination, thus, it is indeed challengeable that understand the effective substance due to the huge quantity of active substance in the methanol extract of B. papyrifera leaves. In addition, the dose and treatment pattern caused the variation of effects (Passos et al. 2000), in this study, when dose rose gradually and reached particular degree, the effect tendency changed oppositely. There were difference effects on voles between single treated and repeated treated, the intake of group Ⅳ after treating for 10 days was the same with group Ⅴ after 1 day. However, the effect showed some difference, the voles responded more effectively with the single treated in the early stage of experiment, otherwise they should be treated for long term, at least more than 20 days according to our results. Therefore, application of sterilants should be skillful with doses and measures.

This study reports the potential of B. papyrifera leaves as an economic and effective sterilant for M. fortis. Crude methanol extract showed the potent inhibition on especially male fertility include impeding growth of their sex organ, and reducing sperm quality. It supports that B. papyrifera leaves would be more applicable to involve male contraceptive agent. The success of B. papyrifera leaves also validates a new idea for developing rodent sterilants, it provides stimulus for the stagnant application with effectiveness and low cost. The further organic experiment and field verification are required to reveal the effective mechanisms and practicability of B. papyrifera leaves sterilants. Moreover, we also think the serendipitous plant discovery with antifertility activity should be encouraged and supported to explore their feasibility as the pest chemosterilants, to enrich the communications and practices of pest management.

Ethics approval and consent to participate

The animal study protocol was approved by the Ethics Committee of Institute of Subtropical Agriculture, The Chinese Academy of Sciences.

Funding

The research was supported by National Natural Science Foundation of China (U20A20118), Natural Science Foundation of Hunan Province (2024JJ5064), Chinese Universities Scientific Fund (2452022018), National Key R&D Program of China (2022YFD1400400), Doctoral fund of Guiyang Healthcare Vocational University (GKDK2024-12).

Author Contribution

S. W., Z. X. and conceived and designed the experiments. The optimization of the method and work were performed by Y. Z. and M. Z. S. W., W. L and Z. H. carried out the experiment. The data analysis conducted by S. W., T. L. and Z. H., with checked by Y. Z., T. H. and Z. X. The manuscript was primarily written by S. W., and all authors provided and approved the final manuscript.

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No competing interests reported.

FigureS1.jpg
Fig. S1. The usual organ coefficients of male M. fortis (A: male, B: female, 1: heart, 2 lung, 3: liver, 4: kidney, * represent P＜0.05).
FigureS2.jpg
Fig. S2. The annotation of metabolites in methanolic extract of Broussonetia papyriferaleave (A: amount and classification, B: secondary classification).

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The extract of the Broussonetia papyrifera can be used as a raw material source for the sterility agent of Microtus fortis

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Materials and methods

Plant material

Animals and experimental design

Anatomy and morphological index of organs

Hormone assessment

Sperms analysis

Mating and breeding assay

Data statistics

Results

Variation of body weight respond to extract

Influence of extract on the organ coefficient

Effect of extract on the sex hormones

Variation of male sperm parameters

The reproductive characteristic of couple voles

Discussion

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