Antifertility effects of Azadirachta indica methanol seed extract on canine spermatozoa in vitro

Objective:To investigate the effects of Azadirachta(A.)indica methanol seed extract on canine male fertility in vitro.Methods:The effect of the A.indica methanol seed extract(2.5,5.0,10.0 and 20.0 mg/mL,respectively)on canine spermatozoa was evaluated in vitro at 1.0,2.5,5.0 and 10.0 min post-treatment.The two controls were comprised of sperm sample in phosphate-buffered saline,and sperm sample in phosphate-buffered saline and 1%dimethyl sulfoxide,respectively.The experiment was repeated(n=3)in duplicates with semen collected from 3 adult Basenjis.The effect of A.indica methanol seed extract treatments on canine spermatozoal total and progressive motility(phase-contrast microscopy),sperm vitality(eosin-nigrosin vital staining method)and total abnormalities(phase contrast microscopy,eosin-nigrosin staining and Papanicolaou staining methods)were determined.Results:A.indica methanol seed extract treatment caused a concentration and time-dependent decrease in sperm percentage total motility,progressive motility and vitality.The absence of sperm revival post-treatment provided evidence of a permanent or irreversible incapacitation of sperm by A.indica methanol seed extract.Conclusions:The inhibition of sperm motility and significant spermicidal effect observed post-treatment suggest that A.indica methanol seed extract may produce a potent antifertility or contraceptive effect in dogs if applied as an intra-vaginal cream pre-coitus.

Preliminary study on antifertility activity of Enicostemma axillare leaves and Urena lobata root used in Indian traditional folk medicine

Objective:To evaluate the possible antifertility activity of Enicostemma axillare(E.axillare) leaves and Urena lobata(U.lobata) root in adult male Wistar albino rats.Methods:Six groups of rats were treated with ethanolic(70%v/v) extracts of E.axillare(375 and 750 mg/kg body weight) and U.lobata root(300 and 600 mg/kg body weight) once daily for 55 days.Control groups received the distilled water and vehicle.All the treated rats had corresponding recovery groups.At the end of each treatment periods,animals were killed and organ weights,sperm characteristics,testicular and epididymal biochemicals as well as testicular enzymes were assessed.Results:The E. axillare and U.lobata at tested doses did not decrease body weight,whereas the weight of testes, epididymides and seminal vesicles were significantly(P【0.01) reduced.Significantly(P【0.01) more reductions in the sperm motility,viability and counts,epididymal and testicular protein contents were noted in the rats treated with higher dose of both the plants.Both the plants at the higher dose caused a marked increase(P【0.01) in sperm morphological abnormalities,testicular cholesterol and ascorbic acid contents were remarkably increased(P【0.01),while,the activities of testicular glucose-6-phosphate dehydrogenase(G-6-PDH) andΔ~5-3β-hydroxy steroid dehydrogenase(Δ~5-3β-HSD) were significantly reduced(P【0.01).However,reversal of these changes occurred after 55 days of treatment withdrawal.Conclusions:This study suggests that the ? axillare leaves and U.lobata root reversibly inhibited spermatogenesis and steroidogenesis indicating reversible antifertility activity which could partially support the traditional of these plants as male contraceptives.

Antifertility effects of 60-day oral gavage of ethanol extract of Spondias mombin leaves in guinea pigs: A biochemical, reproductive and histological study

Objective:To investigate the impact of ethanol extract of Spondias(S.)mombin leaves on the biochemical parameters of testicular function,hormonal profile,sperm profile and reproductive outcomes of guinea pigs.Methods:Sexually matured male[(1000.40±8.12)g]and female[(810.00±7.09)g]guinea pigs were used.In testicular function study,a total of 24 male guinea pigs were randomised into 4 groups of 6 guinea pigs each.Group A(control)was orally administered 1 mL of physiological saline,once daily for 60 days while groups B,C and D were treated like the control group except that they were orally administered 100,250 and 500 mg/kg body weight of ethanol extract of S.mombin leaves.For the fertility study,the same animal groupings and treatments in the testicular function study were adopted.The male guinea pigs were paired with the females(1:1)and afterwards examined for pregnancy outcomes.Results:The ethanol extract of S.mombin leaves contained saponins,alkaloids,flavonoids,tannins,steroids,phenolics,phlobatannins,cardiac glycosides,cardenolides and dienolides with saponins(4.80 mg/mL)occurring the most whilst cardenolides and dienolides(0.08 mg/mL)were the least.The ethanol extract of S.mombin leaves significantly and dose dependently reduced the activities of alkaline phosphatase,glutamate dehydrogenase,3-hydroxy-3-methylglutaryl coenzyme A reductase,malic enzyme,17-β-hydroxysteroid dehydrogenase,lactate dehydrogenase,catalase and superoxide dismutase as well as levels of testosterone,glycogen,total protein and ascorbic acid in the testes when compared with the control group(P<0.05).All the doses of ethanol extract of S.mombin leaves also reduced the levels of sorbitol dehydrogenase,3-β-hydroxysteroid dehydrogenase,glucose-6-phosphate dehydrogenase and sialic acid whereas the levels of testicular acid phosphatase,gamma glutamyl transferase and cholesterol increased dose dependently(P<0.05).The serum luteinising hormone,testosterone and estradiol were reduced after the administration of ethanol extract of S.mombin leaves whereas levels of serum follicle stimulating hormone increased significantly.The 100 and 200 mg/kg body weight of ethanol extract of S.mombin leaves increased the testosterone/estradiol ratios whilst the 500 mg/kg body weight of ethanol extract of S.mombin leaves decreased it.The sperm motility,sperm count,normal sperm morphology,sperm density,sperm viability and semen viscosity were significantly reduced in the ethanol extract of S.mombin leaves-treated guinea pigs(P<0.05)whereas the head-,tail-and neck-defects increased significantly when compared with the control group(P<0.05).In contrast,the semen volume and pH were not significantly altered by the ethanol extract of S.mombin leaves(P>0.05).The ethanol extract of S.mombin leaves at both 100 and 250 mg/kg body weight significantly reduced the total number,circumference,weight and length of the pups whereas the 500 mg/kg body weight of ethanol extract of S.mombin leaves-treated rats did not produce any pup.The 250 and 500 mg/kg body weight of ethanol extract of S.mombin leaves induced degenerative and necrotic changes in the seminiferous tubules with vacuoles in the germinal epithelium and a few to complete absence of spermatozoa.In all of these,the 500 mg/kg body weight of ethanol extract of S.mombin leaves produced the most pronounced alteration on the parameters.Conclusions:S.mombin leaves have induced infertility in the male guinea pigs via endocrine dysregulation,anti-spermatogenic activity,testicular dysfunction and oxidative stress and made possible by the presence of saponins,alkaloids,flavonoids,tannins,steroids,phenolics,and cardiac glycosides.

Antifertility properties ofCentella asiatica ethanolic extract as a contraceptive agent: Preliminary study of sperm proteomic

Objective:To investigate the antifertility properties ofCentella asiatica L. ethanolic extract and identify the sperm proteomic changes in treated male rats.Methods: A total of 32 male Sprague-Dawley rats were divided into two groups: control group (n=16) received distilled water and treatment group (n=16) which received 300 mg/kg ofCentella asiatica L. extract for 42 d. After the treatment period, the number of implantation sites was recorded and the sperm proteomic changes were analysed by 2D gel electrophoresis. In addition, the expression of protein spots was quantified by MALDI-TOF analysis.Results:Centella asiatica L. extract resulted in low number of implantation sites in the treatment group (100.00±2.82) compared to the control group (183.00±2.14). The percentage of infertile male rats in the treatment group was higher (43.75%) compared to the control group (18.75%). Proteomic analysis showed the expression of protein spots identified in the treatment group decreased with 234 spots compared to the control group with 282 spots.Conclusions:The results revealed that there was antifertility activity in the male rats with the administration ofCentella asiatica L. ethanol extract and the identified proteins could provide understanding on the adverse effect ofCentella asiaticain male reproductive system.

Phytochemical analysis and antifertility potential of Cynodon dactylon in female Wistar rats:A herbal approach towards contraception

Objective:The objective of the present study was to evaluate the antifertility activity of ether(ErCD),chloroform(CeCD)and ethyl alcohol(EyCD)extracts of the whole plant of Cynodon dactylon in female Wistar albino rats.Methods:Acute oral toxicity and an antifertility study were performed in female Wistar rats with two dose levels(200 and 400 mg/kg,orally)of EyCD.The estrogenic and progestogenic effects of EyCD were further observed by administering it to immature Wistar rats by investigations of vaginal cornification,hormonal level,uterus weight,biochemical parameters,histopathology of the uterus and deciduoma formation,respectively.Isolation of EyCD was carried out by Flash Chromatography and isolated fraction was estimated by HPLC.Results:No toxicity with any of the extract was found up to the dose of 2000 mg/kg body weight.EyCD treated rats exhibited maximum reduction in pregnancy(83.33%).Estimation of EyCD on vaginal cornification,estrogen-induced uterotrophic assay and deciduoma model demonstrated vaginal cornification,significant(P<0.01)increase in uterine weight and uterine proliferation in histopathology and reduced deciduoma formation respectively.Hormonal and biochemical parameters confirmed the above findings indicating estrogenic potential and antiprogestogenic potential of EyCD that might be attributed to the presence of phytoestrogen(apigenin)in EyCD.Conclusion:The results suggested that extracts of C.dactylon possess significant antifertility activity,which is consistent with the literature reported in folk medicine of this plant in fertility regulation.

Antifertility effects of quinestrol in male lesser bandicoot rat, Bandicota bengalensis, and potential in managing rodent population under field conditions

Integrating fertility control techniques using steroid hormones after lethal control can help reduce post control rebuildup of rodent populations.The current study is thefirst to assess the antifertility effects of quinestrol in male lesser bandicoot rat,Bandicota bengalensis which is the predominant rodent pest species in Southeast Asia.Rats in different groups were fed bait containing 0.00%,0.01%,0.02%,and 0.03%quinestrol for 10 days in laboratory and evaluated immediately,and 15,30,and 60 days after treatment discontinuation for effect on reproduction and other antifertility parameters.Effect of 0.03%quinestrol treatment for 15 days was also observed in managing rodent populations in groundnut cropfields.Treatment resulted in average consumption of 19.53±1.80,67.63±5.50,and 246.67±1.78 mg/kg bwt active ingredient by three treated groups of rats,respectively.No reproduction was observed in female rats mated with male rats treated with 0.03%quinestrol,even 30 days after cessation of treatment.Post-mortem examination showed a significant(P<0.0001)effect of treatment on organ weights(testis,cauda epididymis,seminal vesicles,and prostate gland)and different sperm parameters(sperm motility,sperm viability,sperm count,and sperm abnormality)in the cauda epididymalfluid with partial reversibility after 60 days.A significant(P<0.0001)effect of quinestrol on the histomorphology of testis and cauda epididymis was observed,suggesting its effect on spermatogenesis.Affected cell association and cell count in seminiferous tubules did not fully recover within 60 days of stopping treatment.Evaluation of the effects of quinestrol treatment in groundnutfields showed greater reductions in rodent activity infields treated with 2%zinc phosphide followed by 0.03%quinestrol treatment as compared tofields treated with 2%zinc phosphide alone.Research concludes that quinestrol has the potential to reduce fecundity and post control rebuildup of B.bengalensis populations,but long-term studies of the effectiveness of quinestrol under large-scalefield conditions are needed to use it as part of an integrated pest control program for rodents.

The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein(tNASP)could result in reproductive failure,we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP(mtNASP).Using mouse as a model,recombinant mtNASP(rmtNASP)and a synthetic peptide,human tNASP393-408(htNASP393-408),were investigated for their antifertility effect.Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice.Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice.Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP.There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide.The effect on fertility in the mice immunized with the synthesized peptide was reversible.Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.

Effect of Solanum surattense seed on the oxidative potential of cauda epididymal spermatozoa

Objective:To evaluate the effect of aqueous seed extract of Solarium suratlense(S.suraltense)on the oxidative potential of cauda cpididymal spermatozoa.Methods:S.surattense seed extract was orally administered at the dosage of 10 mg/kg b.w.for 15 days,after which aspartate transferase(AST),alanine transferase(ALT),glutamate dehydrogenase(GDH),citric acid and isocitrate dehydrogenase(ICDH)were assayed.Results:The activity levels of the enzymes AST and ALT,which are considered to be the androgenicity in the sperm suspension,were depleted in the extract fed rats.The activity level of the enzyme ICDH,was reduced significantly in the treated group(P<0.00l).Conclusions:It can be concluded that the oral administration of the aqueous seed extract of S.surattense can deplete the oxidative stress of cauda epididymal spermatozoa in albino rats.

GENERATION AND PARTIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SOLUBILIZED MEMBRANE FRACTION OF HUMAN SPERMATOZOA

Human sperm membrane antigens extracted by deoxycholate (DOC) were used to immunizeBALB/c mice.Hybrid cell lines secreting sperm-specific monoclonal antibodies were generatedby cell fusion in a semi-solid medium and screened by indirect immunofluorescent assay usinglive and methanol-fixed sperm.Out of 850 hybrid clones from cell fusion,28 were shownto secrete sperm-specific antibodies which reacted with the acrosome,equatorial segment,whole surface plasma membrane or tail of spermatozoa.Finally,seven hybrid cell lineswere established and shown to secrete monoclonal antibodies which had no cross-reactivitywith arty human tissues other than testis and sperm.The majority were also shown toinhibit fertilization of mouse oocytes in vitro and human sperm penetration of zona-freehamster ova.Western blot analysis revealed that some of these antibodies reacted withsperm membrane antigens of distinct molecular size.

Genome-wide identification of gene expression in the epididymis of infertile rat induced by alpha-chlorohydrin using oligonucleotide microarray

Objective To establish a rat model of sterility associated with epididymis and epididymal gene expression profiles relation to fertility by alpha-chlorohydrin.Methods Rats were treated with 10 mg·kg-1·d-1.alpha-chlorohydrin for 10 consecutive days.Sperm maturation and other fertility parameters were analyzed.The sperm motility and morphology were evaluated by computer-assisted sperm analysis(CASA);sperm survival rate was assessed by SYBR-14 and propidium iodide(PI)fluorescent staining;the weights of testes,epididymides,prostates and seminal vesicles were determined by electronic balance;histological examination of above tissues were evaluated by HE staining;and serumal dihydrotestosterone(DHT)and testosterone(T)of rats were detected by enzyme-labeled immunoassay.Each male rat was paired with 2 female rats in proestrus.Female rats were examined the next morning for the presence of sperm in vaginal smears and underwent a cesarean section on day 12 of gestation.Finally the reproductive indices were calculated as follows:copulation index(number of sperm positive females /number of pairings),pregnancy index(number of pregnancies /number of sperm positive females),and fertility index(number of pregnancies /number of pairings).After that we used Affymetrix Rat 230 2.0 oligo-microarray to identify epididymal special genes associated with fertility.Finally,we validated some of these genes by Real-Time quantitative polymerase chain reaction.Results The motility of spermatozoa from the cauda epididymidis of treated rats showed a significant decrease in percentage of motile,progressively motile sperm,and sperm survival rate.At the same time,the morphology of cauda epididymal spermatozoa was also adversely affected by the treatment.In addition,the serumal androgen levels of treated animals weren't changed compared with the control group.Accordingly,matings with treated males resulted in no successful pregnancy.Then,we classified general functions of the down or up regulated epididymal genes by chlorhydrin with the GeneSpring gene ontology(GO)analysis,which are involved in macromolecular metabolism and transport,primary metabolism process,cell metabolism,biological process regulation,immunology regulation,ion combination,hydratase and oxidoreductase activity.Among all the different expressed genes,we analyzed and screened the down-regulated genes associated with glucose,lipid,protein and other energy metabolism,which are considered as the major ACH action targets.Simultaneously,the up-regulated genes by chlorhydrin were detected and their characters of negative regulated sperm maturation and fertility analyzed,which are apoptosis and immune-related genes and not reported before.Conclusions We established male infertile rat model with ACH(10 mg·kg-1·d-1,po,10 days)through evaluating changes of sperm motility and morphology,mating index,fertility index and pregnancy index.Simultaneously,the ACH didn't affect the major androgen(T and DHT)metabolism and sexual ability,which is considered as the best way for male contraception.Then we determined the down-regulated epididymal genes relation to substance metabolism,which can affect the epididymal sperm maturation and presumed the major antifertility targets by ACH.Further more,we found and analyzed the epididymal up-regulated genes associated with apoptosis and immune functions,which maybe the new possible sites of action by ACH and other male antifertility agents.

Conventional treatment options and herbal remedies for male infertility:An overview

Male infertility is responsible for an estimated 50%of all cases of infertility.Treatments for male infertility include surgery,in-vitro fertilization,hormone therapy,and herbal remedies.Assisted reproductive technologies and methods have made it possible to identify and treat previously untreatable causes of male infertility.Currently available treatments for male infertility are prohibitively expensive,difficult to obtain,necessitate a lengthy course of treatment,and have a host of side effects.Herbal therapy offers male infertility treatment that is less expensive and has fewer side effects than other treatments.The current review focuses on the various treatment options for male infertility.