• Applied Microscopy
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• 제28권2호
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• pp.193-205
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• 1998

The Urechis unicinctus sperm and spermatogenic cells prepared from the testis are investigated to identify $\alpha-tubulin$ of axoneme microtubules using mouse monoclonal $anti-\alpha-tubulin$ as the first Ab and Gold(10nm) conjugated goat anti-mouse IgG as the Ab marker. The Ag-Ab reaction analyzed excellently the localization of $\alpha-tubulin$ and the gold particles incorporated with the proximal and distal centrioles, manchette microtubules, and flagellum. The gold particles can be also observed in the spermatogenic cells while the cells are still in sperm ball which is composed of a somatic cell and spermatogenic cells. The sperm ball is the functional unit of sperm production in U unicinctus testis. The spermatids are developed from the spermatogenic cells in the sperm ball and released into the testis cavity through a cortical cytoplasmic opening. The spermatid architectures are similar with the mature sperm of the testis cavity in aspects of shape of discoid acrosome, degree of nuclear condensation and ring type of mitochondrion. However, the distal centriole connecting with the flagella can be observed from the mature sperm while the both proximal and distal centrioles reveal only in the spermatids. The proximal centriole is directly connected with nuclear outer membrane during the stage of nuclear condensation and oriented perpendicularly to the distal centriole whose axis coinciding with the longitudinal axis of the spermatozoon. There are indications that the distal centriole is intimately associated with the polymerization of the flagellum. The manchette microtubules appear during spermatid development but the mature sperm have round head and no conspicuous middle piece.

• Asian-Australasian Journal of Animal Sciences
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• 제13권1호
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• pp.10-18
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• 2000

This paper describes the magnetic orientation of the intact and demembranated bull sperm treated by DTT or heparin in a 5,400 G static field. Semen samples collected from four bulls (Japanese Black) were mixed to the same sperm density. One percentage triton X-100 was used to extract the plasma membrane. The intact and demembranated sperm suspensions were treated with 20, 200, 2,000 mM DTT, 100, 1,000 or 10,000 units heparin solutions at $4{^{\circ}C}$ for 6 days. The decondensation of the sperm nuclei treated by DTT or heparin was examined by measuring the sperm head area at 1, 3, and 6 days. After measuring the area, each sperm sample was exposed to a 5,400 G static magnetic field generated by Nd-Fe-B permanent magnets for 24 hours at room temperature. Results showed that the decondensation of bull sperm nuclei was not induced by the heparin treatment, however, incomplete decondensation was induced by the DTT treatment. During the magnetic orientation, bull sperms treated by DTT or heparin had low percentages of long axis perpendicular to the magnetic lines of force. However, different aspects were obtained for long axis perpendicular orientations following treatment of DTT or heparin. Through the DTT treatment, the decline of long axis perpendicularly oriented percentages was due to the increase of long axis parallel orientation with the head of the flat plane perpendicular to the magnetic lines of force, whereas, using the heparin treatment, the decline of long axis perpendicular orientation was due to the increment of long axis parallel orientation with the head of the flat plane parallel to the magnetic lines of force. Also, percentages of the head of the flat plane perpendicular were decreased by the heparin treatment. These findings suggest that maintaining the structure of protamine in the chromatin is necessary for the sperm head to orient with its flat plane perpendicular, and maintaining the disulfide bond in the chromatin is necessary for the long axis of sperm to orient perpendicularly.

• Asian-Australasian Journal of Animal Sciences
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• 제25권2호
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• pp.189-193
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• 2012

This experiment was conducted to study variations of serum testosterone and seminal characteristics of Markhoz male goats. Blood samples were obtained via jugular vein, and semen was collected by using an artificial vagina from 14 fertile male goats (2-3 years of age), at 15-day intervals starting on 15 July and ending on 30 October 2010 (during breeding and non-breeding season). Semen volume, total sperm (volume${\times}$concentration), live sperm (%), abnormal sperm (%) and semen pH were significantly superior during the late summer and early autumn (breeding season). Variation of sperm density, motility and progressive motility was not significant during the sampling period. The results presented show that the lowest and highest levels of lactate dehydrogenase in the seminal plasma were recorded in late October (2.82 U/ml) and in late August (4.81 U/ml), respectively. Moreover, the study indicated that the serum testosterone concentration was higher during late summer and early autumn (p<0.05) than at any other of sampling period. There were negative correlations between volume and sperm density (-0.135, p<0.05), and positive correlations between volume and percentage live sperm (0.224) and percentage progressive motility (0.194, p<0.01). Sperm density was correlated with live sperm (0.200, p<0.05) and progressive motility (0.202, p<0.01). The correlation between live sperm and progressive motility was 0.554 (p<0.01). Furthermore, the results in this study indicated a significant positive correlation between live sperm and LDH (0.450) and a negative correlation between sperm density and LDH concentration (-0.272) (p<0.01). Significant, but positive correlations were found between sperm motility and LDH (0.542) and testosterone concentration (0.522), respectively (p<0.05). In conclusion, this study demonstrated that the best obtained semen was collected in late summer (during decreasing photoperiod) and early autumn (September and October). This also coincides with the natural breeding season of Markhoz goats in Iran.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.133-133
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• 2003

Recently, sperm has been used as a vector to carry exogenous genes for the production of transgenic animals. However, the success in cattle is low, due to deficiencies in oocyte activation and sperm decondensation caused by high disulphide bond (S=S) content in mature sperm. This study was carried out to develop an effective method for producing transgenic animals with round spermatids (RS). Two methods of embryo production - electric fusion (EC) or intracyto-plasmic injection (IC) and three activation treatments were compared. RS were isolated from bull testes by Percoll density gradients (20, 35, 40, 45 and 90%). Fusion between ooplast and RS was performed with a single DC electric pulse (1.0 KV/cm, 45 sec) in 0.28 M mannitol solution supplemented with 100 M CaCl2 and 100 M MgCl$_2$. (중략)

• Asian-Australasian Journal of Animal Sciences
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• 제16권2호
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• pp.165-171
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• 2003

Sixty semen ejaculates collected at weekly interval from four Murrah Buffalo bulls over a period of seven months (Nov.1999 to May 2000) were used in the present study. Three buffer medium (sodium citrate, TES and Tris) were used for soaking of sephadex. Three grades of sephadex (G-15,G-100, and G-200) were used for preparation of columns. Columns of three different height (one, two and three cm) were used for separation of semen. Twenty semen ejaculates were used in each project. In the first experiment each semen ejaculates was divided into four parts. One part was kept as control and other three parts were passed thought one cm column of sephadex G-15 prepared in three different buffers. There was significant (p<0.05) increase in percent progressive sperm motility and percent live spermatozoa and decrease in percent abnormal spermatozoa and percent spermatozoa with damaged acrosome as well as sperm numbers after filtration through all the three columns. Sperm quality obtained in the filtrate of column prepared in Tris buffer was better in comparison to other two buffers. So the Tris buffer was used in the second trial. Twenty semen ejaculates were used in this experiment. Each semen ejaculate was divided into four parts. One part was kept as control (non-filtered) and other three parts were passed through columns of different grade of sephadex (G-15, G-100 and G-200). Progressive sperm motility and live sperm percentage improved significantly while decline in percent abnormal spermatozoa and percent spermatozoa with damaged acrosome and sperm concentration was observed after filtration through all the columns as compared to control (non-filtered) semen. Since post filtration quality of semen was better in the sephadex G-100 column, therefore it was selected for the next experiment. In third experiment, Tris buffer and sephadex G-100 were used for preparing columns of different height (one, two and three cm) and twenty semen ejaculates were filtered. The quality characteristics of semen (percent progressive sperm motility, percent live spermatozoa and sperm concentration) after filtration through one cm column were significantly (p<0.05) higher than after filtration through columns of two and three cm height. However non -significant (p>0.05) difference due to height of columns was observed for percent abnormal and percent damaged acrosome but 1 cm column comparatively gave better result than of 2 and 3 cm column height.

• Asian-Australasian Journal of Animal Sciences
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• 제13권1호
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• pp.6-9
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• 2000

The purpose of this study was to evaluate the effect of lower numbers of sperm $(3{\times}10^9)$ per dose liquid semen and type of semen used in artificial insemination (AI) on sow reproductive performance in subtropical area. Semen was supplied by two commercial AI centers. A total of 671 female pigs from seven farms were inseminated with either $3{\times}10^9$ or $5{\times}10^9$ sperm per dose. Two types of semen were used: heterospermic semen from two boars of the same breed and homospermic semen from a single boar. After insemination, conception rate, farrowing rate, total litter size, and number of dead piglets were recorded. The analysis of variance indicated that there was no significant effect of interactions between pig farm, type of semen, or number of sperm on any of the traits measured. There were significant differences in conception rate, farrowing rate, and total litter size among pig farms (p<0.05). The effect of number of sperm per dose liquid semen ($3{\times}10^9$ or $5{\times}10^9$) was not significant. Sows inseminated with homospermic semen showed significantly higher conception and farrowing rates but significantly lower total litter size (p<0.05). In conclusion, the number of sperm per dose liquid semen for AI could be lowered to $3{\times}10^9 $ without affecting reproductive performance in subtropical areas like Taiwan.

• Asian-Australasian Journal of Animal Sciences
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• 제17권6호
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• pp.755-759
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• 2004

The present investigation was carried out to assess the effect of Sephadex (G-15) filtration on the post thaw bull semen quality and conception rate. Post thaw unfiltered (control) and Sephadex filtered semen from four healthy bulls (three cross bred and one pure bred Holstein Friesian) were subjected to microscopic examination viz. sperm concentration, individual motility, live sperm count and sperm morphology. Sixty-two healthy, normal cycling crossbred cows were inseminated with post thaw unfiltered (n=32) and filtered semen (n=30). Sephadex filtration of post thaw semen significantly (p<0.05) decreased total sperm concentration and sperm with abnormal head, mid piece and tail. The overall average total sperm concentration, head and tail defects in filtered semen decreased significantly (53.4, 1.2 and 6.4 million) than in the unfiltered semen (80.4, 2.4 and 15.7 million, respectively). However, after filtration significant (p<0.05) increase in overall average motile and live sperm concentration were observed (38.8 and 38.0) as compared to unfiltered semen (29.2 and 32.0 million, respectively). The overall conception rate recorded was 21.9% with post thaw unfiltered semen and 56.7% with filtered semen. It was concluded that Sephadex filtration of post thaw semen improved its quality and conception rate.

• Animal cells and systems
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• 제14권4호
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• pp.267-274
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• 2010

The decrease in sperm quality under heat stress causes a great loss in animal husbandry production. In order to reveal the mechanism underlying the sperm quality decrease caused by heat stress, we first established a mild heat-treated mouse model. Then, the sperm quality was identified. Further, the testicular proteome profile was mapped and compared with the control using 2D electrophoresis and mass spectrometry. Finally, the differential expressed proteins involved in the heat stress response were identified by real-time PCR and Western blotting. The results showed that heat stress caused a significant reduction in mouse sperm quality (P<0.05). Further, 52 protein spots on the 2D gel were found to differ between the heat-shocked tissues and the control. Of these spots, some repair proteins which might provide some explanation for the influence on sperm quality were found. We then focused on Bag-1, Hsp40, Hsp60 and Hsp70, which were found to be differently expressed after heat shock (P<0.05). Further analysis in this heat-shocked model suggests numerous potential mechanisms for heat shock-induced spermatogenic disorders.

• Asian-Australasian Journal of Animal Sciences
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• 제21권3호
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• pp.358-363
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• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.

• Asian-Australasian Journal of Animal Sciences
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• 제21권2호
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• pp.190-197
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• 2008

This study was designed to analyze boar sperm to compare motility, acrosome morphology, viability and ATP by various preservation methods between Duroc boar A with cold shock resistance sperm and Duroc boar B with cold shock sensitivity sperm. Semen volume, sperm concentration, motility and normal acrosome between Duroc boar A and B did not show any differences within 2 h after collection. There were no differences in sperm motility and normal acrosome between boar A and B at 1 day of preservation at $17^{\circ}C$ and $4^{\circ}C$, respectively. However, sperm motility and normal acrosome from 2 day of preservation at $17^{\circ}C$ and $4^{\circ}C$, respectively, were higher for boar A than boar B. The frozen-thawed sperm motility and normal acrosome were higher for boar A than boar B. The sperm viability and ATP concentration according to storage period of liquid semen at $17^{\circ}C$ and $4^{\circ}C$ were higher for boar A than boar B. Also, the sperm viability and ATP concentration of frozen-thawed semen were higher for boar A than boar B. In conclusion, we found out that the original quality of boar semen with cold shock resistance sperm played an important role.