| N-Ethyl-N-nitrosourea (ENU) is generally accepted as a very strong mutagenic agent. ENU can induce large amount of random mutation phenotypes within a relatively short period, mutant strain animals with special phenotypes may be obtained after further phenotypic screening and genetic tests, and genetic basic tests can be thus carried out on special phenotypes of mutated animals. It has shown excellent prospects in the studies on mice and can be used as a reference method to confirm the functions of genes. ENU mutagenesis can also be used to establish new models for the studies on genetic diseases in human. The management database for ENU mutagenic mice of G1generation has been established now, and genetic resources of mutagenic mice are effectively preserved by using embryo cryopreservation technique, sperm freezing, in vitro fertilization and other techniques.The experimental animal carriers for ENU mutagenesis are normally mice. The results from the mutagenesis tests on mice indicate that ENU not only can induce single base mutation in DNA, but also is a kind of cytotoxic agent, which can lead to death of large amount of spermatogenous cells in male mice. After ENU is withdrawn, the genital functions can be gradually recovered. The administration dosage in the ENU mutagenesis tests is very important. High dosages may lead to increase in the mutation rate, but the treatments may lead to intolerance, infertility or death in male mice; in contrast, low dosages may decrease the mutation rate, and the results of the tests may be not satisfactory. Since the strains of mice and the administration methods utilized by the researchers are different, the research results are not comparable, and the "proper dosage" of ENU is still not determined. Normally, the dosage of ENU can be evaluated from the three aspects as below:(1) the percentage of infertile male mice;(2) the length of average infertility period;(3) the number of dead male mice within a relatively short period after the genital capacity is recovered. The experimental data for ENU mutagenesis for big experimental animals are still insufficient now. The present study was carried out to examine the effects of ENU on spermatism and spermatogenesis in Bama miniature pigs in order to investigate a proper dosage for ENU mutagenesis of Bama miniature pig. The present study firstly examined the quality of fresh sperm from90adult Bama miniature pigs and explored the method for sperm cryopreservation. Ejaculation amount, sperm vitality, density, abnormal rate and resistance of adult Bama miniature pigs were95.31ml,75.33%,0.78×108,5.13%and581, among which the ejaculation amount, sperm vitality and density were all lower than Duroc, Yorkshire and other commercial available pigs, indicating that the cryopreservation technique for its sperm still had certain difficulty. Orthogonal experimental design was used for the sperm cryopreservation studies on Bama miniature pigs, and the program for sperm cryopreservation of Bama miniature pigs was optimized on the basis of investigating the functions of LDL concentration, glycerol concentration and trehalose concentration by combining with the effects of equilibrium time at15℃and thawing method on the motility rate after freezing and thawing. The results showed that the program1(A3B4C2D3E1) was the optimal program, and the motility rate after thawing was52.26%. In order to analyze the protective effects of different combinations of LDL, glycerol and trehalose in the dilution and the refrigerating solution, four kinds of combinations were designed, and sperm motility, acrosomal integnity, plasma membrane integrity, DNA injury rate and other parameters were used as the judging parameters. The results indicated that the protective effects for the combination with9%LDL,200mM trehalose and2%glycerol (the combination4) were significantly better than those of other combinations (P<0.05), which further indicated that the program1was the optimal program for sperm cryopreservation of Bama miniature pigs.During the administration stage after ENU mutagenesis for Bama miniature pigs, the food intake for the pigs in different dosage groups on the day of administration extremely significantly decreased in comparison to that in the control group (P<0.01), afterwards, it gradually recovered (5-6days after administration)(the difference in comparison to that in the control group was not statistically significant, P>0.05); the same tendency may appear after repeated administration on the second and third administration (at the second and third week). The food intake of Bama miniature pigs may be significantly affected during the administration with ENU, the decrease in food intake was dose-dependent, and the recovery time for food intake increased with the increase in the dosage. The growth rates of male pigs in different dosage groups after drug administration showed decreases to different extents. The mortality of Bama miniature pigs gradually increased with the increase in the administration dosage of ENU, and the fertility gradually decreased. The results indicated that Bama miniature pigs can tolerate a dosage no higher than85mg/kg.The ejaculation amount of Bama miniature pigs within12months after ENU mutagenesis showed a decreasing and subsequently increasing tendency, and the ejaculation amounts of Bama miniature pigs almost recovered in the dosage groups of no higher than65mg/kg at12months after drug administration; the ejaculation amounts in the75-105mg/kg groups could not recover the level before drug administration, but the recovery of ejaculation amount in the75mg/kg group was better than those in the85-105mg/kg groups (P<0.05), and the differences in the ejaculation amounts among the85-105mg/kg groups were not statistically significant (P<0.05). The sperm motility of Bama miniature pigs firstly decreased and then increased, and the sperm motility in the45mg/kg and65mg/kg group at12weeks after administration can recover the level before administration, while the sperm motility in the75-105mg/kg groups can not recover the level before administration, the recovery in sperm motility in the75mg/kg and85mg/kg groups was better than that in the90-105mg/kg groups (P<0.05), and the differences in the sperm motility among the90-105mg/kg groups were not statistically significant (P<0.05). The sperm density of Bama miniature pigs also firstly decreased and then increased, the sperm density in the45mg/kg and65mg/kg group can recover the level before administration, the sperm density in the75-105mg/kg groups can not recover the level before administration, among which the recovery in sperm density in the75mg/kg group was better than that in the85mg/kg group (P<0.05), the recovery in sperm density in the latter group was better than that in the90-105mg/kg groups (P<0.05). The sperm abnormality rate of Bama miniature pigs firstly increased and then decreased, the sperm abnormality rates in the45mg/kg and65mg/kg groups can recover the level before administration, while the sperm abnormality rates in the75-105mg/kg groups can not recover the level before administration, among which the sperm abnormality rates in the75mg/kg and85mg/kg groups were better than those in the90-105mg/kg groups (P<0.05). The ejaculation amount18months after administration was significantly higher than that of12months (P<0.05), indicating that the functions of accessory gonads of the male pigs recovered and no statistically significant difference was detected in other sperm parameters. The recovery in the capabilities of spermatism and spermatogenesis12months after ENU administration had become stable.The breeding records for the male pigs after ENU mutagenesis indicated that no statistically significant difference was detected in the pregnancy rate of female pigs for breeding in the45mg/kg and65mg/kg groups (P>0.05), and the differences of other dosage groups in comparison to that in the control group were all statistically significant (P<0.05), and it became more evident that the pregnancy rates of the female pigs for breeding decreased with the increase in the dosage; no statistically significant difference was detected in the litter sizes of the male pigs in different dosage groups (P>0.05); the difference in the survival rates of weaned baby pigs did not show any correlation with the administration dosage; the differences in the incidence rates of suspectable phenotypes in the pigs of F1generation reproduced by the male pigs in different dosage groups (FO generation) were all statistically significant in comparison to that in the control group (P<0.05), among which the incidence rates of suspectable phenotypes in the baby pigs in the45mg/kg and 65mg/kg groups were significantly lower than that in the105mg/kg group (P<0.05), and the differences in the incidence rates of suspectable phenotypes in the baby pigs between the75-95mg/kg groups and the105mg/kg group were not statistically significant (P>0.05). Combining the tolerance of the male pigs to ENU and the incidence rate of suspectable phenotypes in the baby pigs,75-85mg/kg were proper dosages for ENU mutagenesis of Bama miniature pigs.Histological examinations were carried out on the genital organs and the acccessory gonads in the Bama miniature pigs18months after ENU administration. The results showed that the epididymis index and the seminal vesicle index in the85-105mg/kg groups were significantly lower than those of the control group except the45and65mg/kg groups, indicating that the genital organs and the acccessory gonads had been damaged; with the increase in the administration dosage of ENU, the normal contorted seminiferous tubules in the testis of the male pigs decreased, the percentage of contorted seminiferous tubule showing partial and complete degenerative atrophy decreased; the percentages of different grades of spermatogenic cells and supporting cells in the contorted seminiferous tubules also decreased with the increase in ENU dosage. ENU administration had no significant effects on the prostate of Bama miniature pigs. It can be observed under the transmission electron microscope that vacuoles can be detected in some spermatogenous cells and sperm nucleus depolymerization can be detected, and the nuclear membrane of some spermatids disappeared. The results from the follow-up detection on hormonal level showed that the differences in serum T and E2levels in the male pigs in different dosage groups before ENU injection were not statistically significant (P>0.05); statistically significant differences can be detected in serum T and E2levels in the male pigs between the dosage groups and the control group except the45mg/kg group four months after administration (P>0.05), and the T level was significantly lower than that in the control group (P<0.05), while the E2level was significantly higher than that in the control group (P<0.05), indicating that T and E2secretion was significantly affected with the degeneration of testicular cells of Bama miniature pigs. The decrease in T level and the increase in E2level are not only the symbol for the changes in sperm quality of male animals, but also the reason inducing the changes in sperm quality.Testicles secrete male hormones and produce sperms, and these two functions should be accomplished under the regulations of related genes. Among which, StAR and P450scc are mainly related to testosterone synthesis, while GDNF, SCF and CREM are mainly related to spermatogenesis. Apoptosis of spermatogenic cells is also concurrent with spermatogenesis, and the rate of cell apoptosis may change under the effects of exogenous factors. Bax and Bcl-2are representatives for the genes promoting and inhibiting apoptosis respectively. The experimental results indicated that except that the expression level of StAR in the testicular tissues of the male pigs in the105mg/kg group was significantly lower than that in the control group (P<0.05), other dosage groups were not significantly affected; no statistically significant difference was detected in the expression level of P450scc in the45and65mg/kg groups and the control group (P>0.05), the expression levels in the75and85mg/kg groups were significantly lower than that in the control group (P<0.05), and the expression levels of P450scc in the testicles of the male pigs in the90-105mg/kg groups were significantly lower than those in other dosage groups (P<0.05, Figure6-2). The expression levels of GDNF in the testicles of the45and65mg/kg groups were not affected (P>0.05), the expression levels of GDNF and CREM were almost the same, in other words, the expression levels of GDNF in the testicles of the male pigs in the75-105mg/kg groups significantly decreased (P<0.05), while the decreasing amplitude in the expression level increased with the increase in the dosage, indicating that the self-renewal capability of stem spermatogonium in the contorted seminiferous tubules in the testicles of the male pigs gradually decreased and the mature sperm cells decreased. When the dosage reached90mg/kg or even higher, self-renewal of stem spermatogonium and sperm maturation of the male pigs were seriously inhibited. The differences in the expression level of BAX in the testicles of the male pigs in the45and65mg/kg groups in comparison to the control group were not statistically significant (P>0.05). the expression levels of BAX in the75-105mg/kg groups were significantly higher than that in the control group (P<0.05). and the differences increased with the increase in ENU dosage (P<0.05), while the differences in the expression levels of Bcl-2in different dosage groups in comparison to the control group were not statistically significant, indicating that the apoptosis of spermatogenic cells in the testicles of the male pigs increased with the increase in the dosage except the45and65mg/kg groups, and the apoptosis of spermatogenic cells was maintained at a relatively high level when the administration dosage of ENU increased to a dosage higher than90mg/kg, spermatogenic cells decreased and the sperm quality significantly decreased.In general, the conclusions as followed can be drawn from the present study:(1) Normal ejaculation amount, sperm vitality, density, abnormality rate and resistance of adult Bama miniature pigs were95.31ml.75.33%,0.78×108,5.13%and581respectively;(2) The optimal method for semen freezing of Bama miniature pigs was the program1, in other words, supplement with9%LDL,200mM trehalose and2%glycerol. equilibrium at15℃for3h and thawing at37℃for45s;(3) The food intake of Bama miniature pig decreased after administration with ENU, the growth rate decreased, the death rate and the infertility increased with the increase in the administration dosage, and they can not tolerate a dosage no lower than90mg/kg;(4) With the increase in the administration dosage of ENU, the sperm quality of Bama miniature pigs gradually decreased, the damages in testicles, epididymis and seminal vesicle of male pigs were gradually aggravated, the amplitudes in the decrease in serum T level and the increase in serum E2level of the male pigs both increased, the expression level of GDNF in the contorted seminiferous tubules gradually decreased, the apoptotic spermatogenic cells gradually increased;(5) the decrease in serum testosterone level in the male pigs after administration with ENU was mainly related to the decrease in the expression level of P450scc, the decreases in the expression levels of GDNF and CREM led to the decrease in the self-renewal capability of stem spermatogonium and the blockage of sperm maturation in the male pigs, the increase in the expression level of BAX led to the increase in the apoptotic spermatogenic cells, the decrease in the number of spermatogenic cells and the damages in spermatogenic capability;(6) According to the comprehensive analysis on the tolerance of Bama miniature pigs to ENU and the changes in spermatogenic functions, and the optimal dosage for ENU mutagenesis can be preliminarily determined as75-85mg/kg. |
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