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Kvalita spermatu ovlivňuje oplození u sladkovodních ryb
CHENG, Yu
Short-term storage of sperm is a widely used technique during artificial fertilization in hatchery practice due to its convenient, inexpensive and practical value. Chapter 2, three egg incubation techniques were compared to supply one simple and efficiency method to incubate fertilized sterlet eggs. The percentage of cleavage, neurulation and hatching rates of embryos and larvae were examined to compare the effects of incubation systems consisting of placing Petri dishes 1) in the hatchery tank, 2) on a table in small incubation box in an air-conditioned room or 3) in a large thermostatic incubator box. No significant differences were found among these egg incubation systems. The experience gained allowed us to practice this simple method of egg incubation in Petri dishes on other fish species such as carp or zebrafish. Chapter 3, attention was paid to processes of egg activation and fertilization, which are highly important issues in fish reproductive biology. In the sterlet, multiple treatment groups with different gradient of spermatozoa number and volume of activation solution were used to fertilize eggs and further determine the minimum number of spermatozoa with optimal volume of activation water. The artificial fertilization experiments revealed that a total of 50,000 spermatozoa in 8 ml or 32 ml of activation water was sufficient for successful fertilization. It was obtained that a very low concentration of 100-1,300 spermatozoa per ml in activation water can achieve efficient fertilization with good quality sperm. Chapter 4, sperm managements including prevent sperm spontaneous movement, artificial seminal plasma composition, use of natural seminal plasma from good sperm were explored. The fertilization optimizations with different methods, spermatozoa: egg ratio and minimum sperm concentration were also examined in zebrafish. Results indicated that extender E400 was efficient during zebrafish sperm storage; a test tube with a well-defined amount of 6,000,000 zebrafish spermatozoa in E400 extender per 100 eggs and 100 ?L of activation solution has proven to be more successful than using a Petri dish. Sperm of European catfish can be collected and stored in immobilization solution for as long as 1 week for successful fertilization. Sterlet sperm with bad quality can be revitalized by incubation with seminal plasma from good sperm. The results help to establishing a sperm in vitro storage method and fertilization procedures in freshwater fishes. Chapter 5, activation solution is one of the key factors providing sperm and egg performance during fertilization. Different activation solutions were employed to test the practical application efficiency in a hatchery jar with sperm that was aged but diluted in artificial seminal plasma. The large-scale artificial fertilization trials indicated that fertilization and hatching rate were high (78-87%) after one week of sperm storage in a carp artificial seminal plasma in activation solutions as hatchery water and Perchec solution. Results offered the practical basic for the further application of aging sperm in a hatchery. Chapter 6, in this study, aged sperm in common carp was used to evaluate the sperm motility and kinematics, sperm viability, osmolality and pH of seminal plasma sperm short-term in vitro storage. DNA methylation analysis based on the whole-genome bisulfite sequencing was also performed to reveal the molecular changes of aging sperm. The results showed that aging spermatozoa in common carp has a considerable negative impact on their performance and as well as artificial fertilization success. the level of methylation at the CpG sites increased significantly with 24 hours post stripping spermatozoa compared to the fresh group, but then reduced significantly at 96 hours post stripping. The results contribute to investigate the sperm phenotype and functional changes in common carp and providing clues for understanding the molecular changes during sperm storage
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Analýza morfologických změn spermií kanců a jejich vliv na plodnost prasnic
ŠTVERÁK, Martin
The aim of the work was to evaluate the quality of boar ejaculate in terms of sperm morphology and the influence on fertility of sows. The data came from 58 sperm collections from 8 boars of one line. The boars were housed in the semen collection centre under the same conditions and were in age from 11 to 21 months. In the ejaculate analysis, the evaluation of the pathologically changed sperms was performed by determining the frequency of the finding of individual morphological changes. To evaluate the effect of semen on the litter size, data from 123 successful inseminations and subsequent births were processed. The results showed that boars in most cases produced ejaculate with an average volume with a lower sperm concentration. The incidence of morphologically abnormal sperms was normal for almost all boars. The most common sperm abnormalities were immature sperms and defects of sperm flagella. It was confirmed that more piglets were born after using insemination doses made from sperm ejaculate with the higher sperm count. Furthermore, when using ejaculate with a morphologically abnormal sperm count below 15%, more piglets were born than when the sperm count was 1525%. In the case of immature sperms, a negative correlation with the number of born piglets has been proven.
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Odběr spermatu pomocí katetru a jeho využití při výtěru štiky obecné (Esox lucius L.)
PLAŇANSKÝ, Tomáš
The aim of this diploma thesis is to compare quality of northern pike sperm collected by different methods. First method is collection of stripped sperm by abdominal massage of the belly. Second method is collection of stripped sperm with special catheter to eliminate sperm contamination by urine. The last method is collection of testicular sperm. Differently collected sperm was evaluated and compared its quality. The main observed parameters were sperm volume, spermatozoa concentration, spermatozoa motility and velocity and osmolality of seminal fluid. Sperm samples were used for eggs fertilization. In fertilized eggs, the fertility of eggs and larvae hatching rate were observed.
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Znečištění životního prostředí a endokrinně podmíněná neplodnost samců ryb
GOLSHAN, Mahdi
There are a large number of natural and synthetic environmental contaminants (ECs) known or suspected to mimic or interfere with male reproductive endocrine functions. Our current knowledge is largely addressed to ECs that induce oestrogen-induced feminization. However, there are several ECs that cause alternations in androgen production similar to oestrogenic ECs, but they do not induce vitellogenin-induced feminization. The mechanisms of action of these chemicals are still unclear due to the fact that androgen receptor (AR) functions in androgen-mediated male reproductive physiology are largely unknown. In the present study, we examined the effects of vinclozolin (VZ) (100, 400 and 800 ?g/L) and di-(2-ethylhexyl)-phthalate (DEHP) (1, 10 and 100 ?g/L) on male reproductive performance in goldfish following 30 days of exposure. Since both VZ and DEHP act as agonists and/or antagonists of hormonal receptors, estrogen receptor subtypes (er) and androgen receptor (ar) mRNA levels were studied. For studying their oestrogenic activity, one group of goldfish were exposed to 17?-estradiol (E2). In DEHP treated goldfish, sperm production, motility and velocity were decreased at 1, 100 and 10 ?g/L, respectively. Our previous study has shown that sperm production, motility and velocity were decreased in goldfish exposed to 800 ?g/L VZ. These suggest that DEHP and VZ are capable of interfering with spermatogenesis and sperm maturation. In E2 treated goldfish, none of the males produced sperm, indicating E2-suppressed spermatogenesis. Similar to E2 treated goldfish, 11-KT levels were decreased in goldfish exposed to ?10 and ?1 ?g/L DEHP at day 15 and 30, respectively. In VZ treated goldfish, 11-KT levels were decreased in goldfish exposed to 800 ?g/L VZ following 7 d, but increased in goldfish exposed to 100 ?g/L VZ following 30 d of exposure. E2 levels remained unchanged and increased in DEHP and E2 treated goldfish, respectively. In VZ treated goldfish, LH levels were increased at 100 ?g/L. In contrast, LH levels were decreased in DEHP and E2 treated goldfish following 15 d of exposure. There were also positive relationships between LH and 11-KT levels suggesting that inhibition or stimulation of androgen production were mediated by DEHP- or VZ-induced alternations in pituitary function. In VZ treated goldfish, gnrh3 mRNA levels were decreased following 7 d of exposure and increased at low dose following 30 d of exposure. kiss1 or kiss2 mRNA levels were also increased in VZ treated goldfish, while gpr54 mRNA remained unchanged. In DEHP treated goldfish, gnrh3, kiss1 and its receptor (gpr54) mRNA levels did not change during the period of exposure. In E2 treated goldfish, gnrh3 mRNA levels were decreased at day 7, but kiss1 and gpr54 mRNA levels were increased at day 30 of exposure. These results suggest that, in contrast to DEHP, VZ effects on pituitary and testicular functions are mediated by disruption of hypothalamus function and upstream neuroendocrine regulators. The brain ar and testicular lhr mRNA levels were changed in VZ and E2 treated goldfish depending on dose and period of treatment, however they remained unchanged in DEHP treated goldfish. These differences suggest that DEHP may act through an independent hormonal receptor pathway, while VZ acts through a receptor pathway. vtg, er and cyp19a1b remained unchanged in DEHP and VZ treated goldfish but increased in E2 treated goldfish. These indicate that neither VZ nor DEHP acts as oestrogenic compound to impair male fertility. In conclusion, DEHP and VZ reduced sperm quality in goldfish due to stimulation and inhibition of 11-KT production which were mediated by alternations in pituitary function to produce LH or by disruption of the transfer of Cholesterol to steroidogenesis. Upstream neuroendocrine regulators (gnrh3 and kiss-1/gpr54) were disrupted in VZ treated goldfish. Taken together, VZ and DEHP differentially act on brain and testis to impair fertility endpoints.
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