The Study On Development Of The Testes Of Ostrich Chicks And Effects Of Boron In The Hypothalamus-pituitary-testis Axis

African ostrich,belonging to Aves class,Struthioniformes order,Struthioidae family, is the biggest bird existing in the world,whose adaptability is very strong.Compared to the other birds,African ostrich has special breeding characteristics.The sexual mature of the ostrich is much later,and the age of female ostrich reached to sexual mature is 2 to 2.5 years old,while the male is 2.5 to 3 years.The breeding characteristics of the ostrich results in the ostrich has very long breeding cycle and is infaust for cultivation of the ostrich.The ostrich has assumed significant economic importance as a farmed bird,and internal and foreign researchers have done many research on it.The morphological characteristics of some tissue and organ of the ostrich have been reported at present,but the morphological feature of sex gland axile development and the effect of microelement Boron on the hypothalamus-pituitay-testis(HPT) of male ostrich chick are still unknow.In the present study,African ostrich chicks were divided in 3 experimental groups and 1 control group at random,and the ostrich chicks of experimental groups were given Boron by drinking water.Using the methods of paraffin section,HE staining,transmission electron microscopy,immunohistochemistry(SABC),radioactive immunoassay, histochemical method and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL),the structural characteristics of ostrich chick testis,the changes of histological structure and apoptosis of HPT axis after giving Boron.The main contents and research results are as follows:1 The study on development morphology of the testis of ostrich chicksThe healthy ostrich chick testicles of 1,30,45 and 90-day-old were studied by the methods of paraffin section,HE staining,transmission electron microscopy and TUNEL. The results indicated that there were obvious variance with the testis shape of ostrich chicks and other species,but the histological structure of the testis of them was similar with that of the other birds.There were corpus highmorianum and septula testis in the testis of ostrich chicks,and the cell organ of the cytoplasm of spermiogonium was very little,but the cytoplasm of primary spermatocyte contained redundant mitochondria,free ribosome and rough endoplasmic reticulum.The spermatogenic cells were mainly primordial germ cells in testis of 1 d old ostrich chicks,and spermiogonium in testis of 30-day-old ostrich chicks.Primary spermatocytes were observed in seminiferous tubule of 45-day-old ostrich chicks,and the change of testis structure was not obvious from day 45 to day 90.Apoptosis of testicular cells took place at any developmental period from 1-day-old to 90-day-old ostrich,but more apoptosis testicular cells were observed at day 45.These results demonstrate the testis of ostrich chicks develops fast after postnatal commitment,and there is obvious variance with the morphological characteristic of testis ostrich chicks and other birds.2 The study of expression of luteinizing hormone and melatonin receptor in the testis during developmental period from 1-day-old to 90-day-old ostrichThe distribution of luteinizing hormone(LH) and melatonin receptor(MR) in the testis of 1,30,45 and 90-day-old ostrich were studied by SABC.The cytoplasm and cell membrane of LH immunoreactivity positive cells in testis of ostrich chicks showed yellow or dark brown,but there was no positive reaction.The positive substance of MR in testis was yellow or dark brown and located in cytoplasm,cell membrane and nuclei,and showed uniform or contagious distribution.The distribution of LH and MR in the testis changed with the ages of ostrich chicks.The positive signals of LH and MR were observed in 1-day-old ostrich testis,but were not detected in 30-day-old ostrich testis. There were LH immunoreactivity positive cells in the testis at day 90,but MR immunoreactivity positive substance was not observed at the developmental period.These results demonstrate that LH and MR play some role in the procedure of primordial germ cells differentiating into spermiogonium and Leydig cells differentiation.3 The change of the hormone level in serum with ages of ostrich chicksThe hormone level in serum of 1,30,45 and 90-day-old ostrich was detected by RIA. The results showed that the levels of follicle stimulating hormone(FSH),luteinizing hormone(LH),testosterone(T) and estradiol(E₂) in serum changed with ages of ostrich chicks.The level of FSH in serum increased from day 1 to day 45,and began to decreased after day 45.The level of FSH was lower in serum at day 90 than in that at day 1.The level of LH and T in serum always increased with ages of ostrich chicks.The level of E₂ in serum increased from day 1 to day 45,and began to decreased after day 45.The ratio of T and E₂ decreased from day 1 to day 45 slowly,and increased after day 45 fast.These results demonstrate the levels of reproduction hormone in serum change with ages,and change obviously at day 45.So we can presume that the testis development of ostrich chicks is very special at day 45.4 The study on histology of hypothalamus,pituitary and testis of ostrich chicksThe cytoarchitecture of arcuate nucleus(ARC),supraoptic nucleus(SON), paraventricular nucleus(PVN) and suprachiasmatic nucleus(SCN) was studied by HE staining,and the histological structure of pituitary was studied by HE staining and Orange G-Acid Fuchsin-Light Green(OFG).The results showed the acidophilic cells of antepituitary were stained yellow and those cells had the characteristic with redundant cytoplasm and limpid cells boundary,and the nuclei located aside of them.The basophilic cells of antepituitary were stained mauve and had the characteristic with limpid cells boundary,and several basophilic cells were distributed grouping.The chromophobe cells and mesenchymal tissue of pituitary were stained green and the cell boundary of chromophobe cells was unsharp.These results demonstrate the cytoarchitectue of ARC, SON,PVN and SCN of ostrich chicks are similar with that of Pekin duck in general,but the difference is the size of neurons in that.The structural characteristics of the three kind of cells in pituitary are observed and distinguished easilier by OFG staining than HE staining.5 The study on the effect of Boron on the histology of HPT axis90-day-old ostrich chicks were divided in 3 experimental groups and 1 control group at random,and the ostrich chicks of experiment groups were give Boron by drinking water.Histological changes and apoptosis characteristics of HPT axis under different Boron levels were studied by HE staining and TUNEL.The results showed that Boron could influence the histological structure of HPT axis.All organs of HPT axis of ostrich chicks in low dose Boron(100mg/L)groups adevelop much well,but middle dose Boron(200mg/L) and high dose Boron(400mg/L) both induced different degree histopathological change of HPT axis.The data by statistical analysis showed apoptosis cells were detected by TUNEL in HPT axis of ostrich chicks in control groups,and apoptosis degree changed with Boron level.Compared to the number of apoptosis cells in control groups,the number of apoptosis nerve cells increased with increasing Boron, but the number of apoptosis neuroglia cells and the cells in pituitary and testis of ostrich chicks decreased in low dose Boron groups and increase in middle dose and high dose Boron significantly(P＜0.05).These results demonstrate giving slight Boron to ostrich may promote organs of HPT axis to develop,but excess Boron will produce toxic effect on organism.So it is good to supply ostrich chicks with not so much as 100mg(B)/L (water) Boron.6 Expression of LH and MR in HPT axis of ostrich under different Boron levelsDistribution of LH and MR in HPT axis of 90-day-old ostrich under different Boron levels was studied by SABC.The results were as follows:LH immunoreactivity positive substance was observed in SON,PVN and ARC of hypothalamus,pituitary and testis of ostrich chicks.Boron effected the expression of LH,which showed the expression of LH increased in low dose Boron groups and decreased in middle dose and high dose Boron groups compared to that in control groups.MR immunoreactivity positive substance was observed in SON,PVN and ARC of hypothalamus,but not observed in pituitary and testis.Boron inhibitted the expression of MR in hypothalamus.These results indicate the distribution and expression of LH and MR in HPT axis changed with increasing Boron, and Boron could regulate the activity of HPT axis by influencing the expression LH and MR in HPT axis.7 The effect of Boron on hormone level in serum of ostrich chicksThe RIA was used to detect the hormone level in serum of ostrich chicks under different Boron levels.The data by statistical analysis showed Boron had effect on FSH, LH,T,E₂ and T/E₂ in serum,but the change regulation of them is different with increasing Boron.Compared with the hormone level in serum of ostrich chicks in control groups,the level of LH and T in serum of ostrich chicks in low dose Boron groups increased,but that in low dose and in middle dose Boron groups both decreased significantly.Moreover,the change of T level with Boron was related to the change of LH level.The levels of FSH and E₂ always increased,but the ratio of T and E₂ always decreased with increasing Boron.The results elucidate Boron influences the level of reproduction hormone in serum,and the levels of these hormones are important index to measure the gonad function,so we can presume Boron regulates the activity of HPT axis of ostrich chicks by influencing the levels of hormone of HPT axis in serum.