Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm.

Cytometric sorting of frozen-thawed sperm can overcome difficulties caused by the unavailability of sorting facilities on farms where semen is collected from male livestock. In order to optimize the cytometric sex-sorting procedure, effects of Hoechst33342 staining on the viability and cytometric sorting efficiency of frozen-thawed ram sperm were evaluated. The frozen-thawed sperm were stained with Hoechst33342 at various dye concentrations (80 µM, 120 µM, 160 µM, 200 µM, 240 µM, or 320 µM) for 45 min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160 µM Hoechst33342 for various durations (0 min, 15 min, 30 min, 45 min, 60 min, 75 min, or 90 min) to evaluate effects of staining duration. Sperm motility and moving velocity were analyzed using a computer-assisted sperm analysis system (CASAS). Acrosome status, membrane integrity, and distribution of phosphatidylserine (PS) in Hoechst33342-stained sperm were analyzed using flow cytometry after staining with fluorescein isothiocyanate-labeled lectin from pisum sativum (FITC-PSA), Annexin V, or propidium iodide (PI). The fertility of Hoechst33342-stained sperm was analyzed by in vitro fertilization (IVF). A high-speed cell sorter was used to evaluate effects of Hoechst33342 staining on cytometric sex-sorting of frozen-thawed sperm. The motility, moving velocity, membrane integrity, and PS distribution of Hoechst33342-stained sperm were significantly different from that of immediately thawed sperm (P<0.05). However, there is no significant difference existing among the Hoechst33342-stained groups with respect to the above evaluated parameters. Additionally, along with the staining durations, the adverse effects of the staining procedure on sperm showed a steady increase. However, Hoechst33342 staining did not damage acrosome and in vitro fertilizing capability of frozen-thawed ram sperm. Results of cytometric sorting indicated that frozen-thawed sperm can be efficiently sorted into two sperm populations with X and Y chromosome when the Hoechst33342 concentration was 160 µM. Moreover, when the staining duration was equal to or longer than 45 min, the frozen-thawed sperm can be successfully sorted in the presence of 160µM Hoechst33342. In conclusion, Hoechst33342 staining can detrimentally influence viability of frozen-thawed ram sperm except acrosome and in vitro fertilizing capability. Accordingly, the minimum values of Hoechst33342 concentration and staining duration can be set at 160 µM and 45 min respectively. However, the maximum values of Hoechst33342 concentration and staining duration were not determined based on the current study. Further research on how to reduce injuries caused by freezing, thawing, and Hoechst33342 staining on frozen-thawed ram sperm is needed.

Establishment and cryopreservation of a skin fibroblast cell line derived from Yunnan semi-fine wool sheep in the presence of synthetic ice blocker.

In this study, the fibroblasts cell line derived from ear marginal tissue of Yunnan semi-fine wool sheep was successfully established using the primary explants technique and cryopreservation technology. Additionally, the protective effect of synthetic ice blocker (SIB) including 1, 3-cyclohexanediol (1, 3-CHD) and 1, 4-cyclohexanediol (1, 4-CHD) on frozen fibroblast cells was also assessed and compared. Propidium iodide (PI) was used to stain the dead cells following cryopreservation and thawing. The results showed that compared with Medium 199 (M199) and Dulbecco's modified Eagle's medium : Nutrient Mixture F-12 (1 : 1) Mixture (DMEM/F12), Dulbecco's modified Eagle's medium (DMEM) may be more suitable for the primary culture of fibroblast cells of Yunnan semi-fine wool sheep. The growth curve of cells is a typical "S" type. After subculture for four days, the cells entered the plateau phase and began to degenerate. Biological analysis showed that the population doubling time (PDT) for subculturing fibroblast cells was approximately 26h. The Karyotyping data indicated that the percentage of fibroblast cells with normal chromosome number 2n = 54 was over 90% following subculture for 10 passages. Moreover, the tests for bacteria, fungi, viruses and mycoplasma were negative. After serial subculture for 5 generations, the fibroblast cells were cryopreserved in the presence or absence of 1, 3-CHD or 1, 4-CHD. The data indicated that with increase of the synthetic ice blocker concentrations, the viability of frozen-thawed fibroblast cells was firstly increased and then decreased. When the concentration of 1, 3-CHD or 1, 4-CHD was 50 mM, the viable percentage of frozen-thawed fibroblast cells was 91.93% +/- 2.24% and 94.13% +/- 0.55% respectively and significantly higher than that of the cells frozen in the absence of synthetic ice blockers (88.10% +/- 1.49%, P < 0.05). In conclusion, the skin fibroblast cell line of Yunnan semi-fine wool sheep was firstly established in this study. Additionally, the presence of synthetic ice blocker can increase the viability of frozen-thawed sheep fibroblast cell line.

[Effects on electro-scaip acupuncture on cerebral dopamine transporter in patients with Parkinson' s disease].

OBJECTIVE: To investigate the effect of electro-scalp acupuncture (ESA) on dopamine transporter (DAT) in basal ganglia (BG) of patients with Parkinson's disease (PD) by single photon emission computed tomography (SPECT). METHODS: Ten patients with primary PD were divided equally into two groups randomly. Patients in the treated group received ESA at points of MS6, MS4, MS8, MS9 and MS14, for patients with unilateral lesions, ESA was applied on contralateral points, while for those with bilateral lesions, points of both sides were stimulated, the depth of puncturing was made to arrive at the galea aponeurotica. Patients in the control group took medopa orally in the dosage of 125 mg, 3 times per day for mild, 250 mg, 3 times per day for moderate and 250 mg, 4 times per day for severe cases. All the patients were examined with 99mTc-TRODAT- 1 SPECT before and after 6 weeks' treatment to analyze the activity of DAT by ratio of basal ganglia/occipital (BG/OC) using regional of interesting (ROI) technique. RESULTS: The uptake of 99mTc-TRODAT-1 increased after treatment in both groups. In the treated group, the homolateral (of the affected limbs, the same below) BG/OC ratios before and after treatment were 1.1883 +/- 0.1471 and 1.2416 +/- 0.3146 respectively, and those in the control group 1.1597 +/- 0.0568, 1.2053 +/- 0.1153, showing no significant difference between them (P > 0.05); while the contralateral BG/OC ratios before and after treatment were 0.9019 +/- 0. 1210 and 0.9496 +/- 0.2522 respectively in the treated group, and 1.0436 +/- 0.1393 and 1.2972 +/- 0.2612 respectively in the control group. The difference value of contralateral BG/OC before and after treatment in the control groups was significantly different (P<0.05). But at other cerebral regions, 99mTc-TRODAT-1 SPECT showed no difference between the two groups in changes before and after treatment, therefore, showed no difference in effect of improving DAT between the two groups (P > 0.05). CONCLUSION: ESA could reduce the loss of DAT in basal ganglia and improve its activity in patients with PD to protect the dopamine neuron, so as to delay the progression of PD and alleviate clinical symptoms of patients.

[Effects of needling in Baihui (DU20), Shuigou (DU26) and Shenmen (HT7) on glucose metabolism in the lentiform nucleus in patients with vascular dementia].

OBJECTIVE: To observe the effect of needling in Baihui (DU20), Shuigou (DU26) and Shenmen (HT7) on glucose metabolism in the lentiform nucleus in patients with vascular dementia. METHODS: Ten patients with vascular dementia were randomized equally into control group to receive the treatment of needling in the routine acupoints for hemiplegia (chosen from 6 Yang meridians of hand and foot) and treatment group with needling in 3 additional acupoints of Baihui (DU20), Shuigou (DU26) and Shenmen (HT7) for dementia. All the patients were examined by positron emission tomography (PET) to detect glucose metabolism in the bilateral lentiform nuclei before and after treatment. RESULTS: Glucose metabolism in the lentiform nucleus in normal lateral brain was obviously enhanced in the treatment group, but not in the bilateral lentiform nuclei in the control group after treatment. CONCLUSION: The effect of needling in Baihui (DU20), Shuigou (DU26) and Shenmen (HT7) for treatment of vascular dementia may involve the enhancement of glucose metabolism in the lentiform nucleus.

[Effect of scalp acupuncture on glucose metabolism in brain of patients with depression].

OBJECTIVE: To observe the effect of scalp acupuncture (SA) on the glucose metabolism in different regions of brain in patients with depression by positron emission computed tomography (PET). METHODS: Twelve depressive patients were treated by scalp acupuncture on middle line of vertex (MS5), middle line of forehead (MS1) and bilateral lateral line 1 of forehead (MS2), once a day for six days per week, and received PET detection on different region of brain before and after 6 weeks acupuncture treatment. Semiquantitative analysis was used to compare the average values of radioactive count gotten from various brain regions before and after treatment, which could reflect the condition of glucose metabolism at the brain region detected. RESULTS: SA could increase the glucose metabolism at bilateral frontal lobes, bilateral parietal lobes, right occipital lobe, right caudate nucleus, right cingulated gyrus and left cerebellum and decrease that at right temporal lobe and bilateral thalamus. CONCLUSION: SA on MS5, MS1 and MS2 in depressive patients could influence the glucose metabolism in various brain regions. It primarily illustrated that the mechanism of SA in treating depression is related with its regulation on cortex-limbic circuitry dysfunction and increase the glucose metabolism in various brain regions.

[Effect of electro-scalp acupuncture on cerebral dopamine transporter in the striatum area of the patient of Parkinson's disease by means of single photon emission computer tomography].

OBJECTIVE: To probe the mechanism of electro-scalp acupuncture in treatment of Parkinson's disease (PD) by single photon emission computer tomography (SPECT). METHODS: Five cases of PD received electro-scalp acupuncture at Dingnie Qianxiexian (MS 6), Epangxian III (MS 4), Dingpangxian I (MS 8), Dingpangxian II (MS 9) and Zhenxia Pangxian (MS 14). Contralateral points were selected for pathologic change on one side and bilateral points were selected for pathologic lesion on both sides. All the patients received 99mTc-TRODAT-1 SPECT examination before and after acupuncture treatment of 6 weeks. And activities of dopamine transporter (DAT) were analyzed by the ratio of striatum/occipital lobe (ST/OC), which was evaluated by means of technique of regional of interesting (ROI). RESULTS: The ratio of ST/OC on the same side of the affected extremity before and after treatment were 1.19 +/- 0.15 and 1.24 +/- 0.31 respectively. And on the other side were 0.90 +/- 0.12 and 0.95 +/- 0.25 respectively. They were increased after treatment (P > 0.05). CONCLUSION: Electro-scalp acupuncture can decrease the loss of DAT and improve the activities of DAT in the striatum of the patient of PD.