鶏凍結精子の走査電子顕微鏡による研究

最近走査電子顕微鏡による精子の形態学的研究が行なわれるようになり, 哺乳類の精子は凍結保存により先体に著しい悪影響を被ることが知られている。一方家禽精子においても哺乳類の精子と同様に融解精子先体に著しい変化の認められることが報告されているが, 凍結融解操作によってもたらされる一連の精子の損傷について観察した報告は少ない。 本研究は光学顕微鏡と走査電子顕微鏡を用い, 錠剤化凍結法とストロー凍結法により, それぞれ二種類の希釈液を用いて凍結処理し, 融解後の鶏精子の活力および崎形精子の微細構造について観察した。その結果, 走査電子顕微鏡を用いれば, 従来の光学顕微鏡下で通常認められる首曲り畸形精子の他に精子先体の離脱や, 精子頭部末部の膨化が認められた。しかし凍結法, 希釈液によるそれら畸形精子の出現割合の差異は明らかでなかった。Recently the scanning electron microscope (SEM) is currently being used to study the morphology of normal and abnormal spermatozoa. In general, it is known that the acrosome of mammalian spermatozoa is affected by deep freezing preservation. On the other hand, HARRIS et al. also reported that the acrosome of fowl spermatozoa is influenced by deep freezing as well as in that of mammalian spermatozoa. MARQUEZ et al. reported that some swelling or distortion of the mitochondria were caused when the turkey semen has been exposed to a glycerol containing medium. But there are few reports pertaining to the series of the morphological changes in fowl spermatozoa after freezing and thawing. In the present study, the scanning electron microscope was used to observe the morphological changes observed in fowl spermatozoa before, during and after freezing and thawing of sperm cell

Factors Influencing the Number of Eggs Recovered from Rabbits Superovulated with FSH or PMSG: Analysis of Five Years of Data from 509 Rabbits

To determine the best conditions for superovulation in rabbits, we analyzed the influence of age, season and hormone treatment on the numbers of eggs collected over five years from 509 rabbits aged 4–10 months using follicular stimulating hormone (FSH) or pregnant mare serum gonadotropin (PMSG) hormone stimulation. The number of eggs recovered was significantly higher in younger rabbits in both treated groups (P < 0.01–0.05). The number of eggs collected from rabbits treated with FSH were significantly higher than from rabbits treated with PMSG at all ages (P < 0.01). Seasonal differences were not observed in either hormone treatment group as they were maintained under constant temperature, humidity and light cycle through the year. Thus, younger rabbits are more sensitive to hormonal superovulation treatment with both FSH and PMSG, and FSH offers a better regimen for egg collection

Effect of the primary cooling rate on the motility and fertility of frozen-thawed rabbit spermatozoa

[EN] In the present study, we examined the effect of primary cooling rates on the motility and fertility of frozen-thawed rabbit spermatozoa. Rabbit semen diluted with an egg-yolk acetamide extender was cooled from room temperature to 5°C at four different rates (-0.1, -0.2, -0.4, -0.8°C/min) as a primary cooling step, then semen was frozen in liquid nitrogen vapour. After thawing, sperm cooled at -0.1°C/min showed the highest motility (40.7 ± 7.3%); there were no significant differences between the motilities of the -0.1, -0.2, and -0.4°C/min groups. The motility of frozen-thawed sperm cooled at -0.8°C/min (29.2 ± 6.8%) was significantly lower than that of sperm cooled at -0.1 and -0.2°C/min. The viability (-0.1°C/min, 38.1 ± 4.0%; -0.8°C/min, 24.3 ± 7.3%) of frozen-thawed sperm was closely related to its motility (-0.1°C/min, 36.7 ± 7.2%; -0.8°C/min, 22.3 ± 4.7%). Quality of post-thaw motile sperm cooled at different rates was estimated by comparing the fertilisation ability of the -0.1 and -0.8°C/min groups following artificial insemination. There were no significant differences in pregnancy rates and mean litter sizes. These data suggest that cooling rabbit semen at rates ranging from -0.1 to -0.8°C/min affects the viability but not the fertilisation capacity of motile spermatozoa after thawing.The authors thank Ms. T. Shimazaki, Ms. R. Tsuneyoshi, and Ms. R. Eriguchi for their technical assistance. This study was partly supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 22500386).Maeda, T.; Liu, E.; Nishijima, K.; Tanaka, M.; Yamaguchi, S.; Morimoto, M.; Watanabe, T.... (2012). Effect of the primary cooling rate on the motility and fertility of frozen-thawed rabbit spermatozoa. World Rabbit Science. 20(2):65-70. doi:10.4995/wrs.2012.1080SWORD657020

Metagenomic Diagnosis of Bacterial Infections

To test the ability of high-throughput DNA sequencing to detect bacterial pathogens, we used it on DNA from a patient’s feces during and after diarrheal illness. Sequences showing best matches for Campylobacter jejuni were detected only in the illness sample. Various bacteria may be detectable with this metagenomic approach

Direct Metagenomic Detection of Viral Pathogens in Nasal and Fecal Specimens Using an Unbiased High-Throughput Sequencing Approach

With the severe acute respiratory syndrome epidemic of 2003 and renewed attention on avian influenza viral pandemics, new surveillance systems are needed for the earlier detection of emerging infectious diseases. We applied a “next-generation” parallel sequencing platform for viral detection in nasopharyngeal and fecal samples collected during seasonal influenza virus (Flu) infections and norovirus outbreaks from 2005 to 2007 in Osaka, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.1–0.25 ml of nasopharyngeal aspirates (N = 3) and fecal specimens (N = 5), and more than 10 µg of cDNA was synthesized. Unbiased high-throughput sequencing of these 8 samples yielded 15,298–32,335 (average 24,738) reads in a single 7.5 h run. In nasopharyngeal samples, although whole genome analysis was not available because the majority (>90%) of reads were host genome–derived, 20–460 Flu-reads were detected, which was sufficient for subtype identification. In fecal samples, bacteria and host cells were removed by centrifugation, resulting in gain of 484–15,260 reads of norovirus sequence (78–98% of the whole genome was covered), except for one specimen that was under-detectable by RT-PCR. These results suggest that our unbiased high-throughput sequencing approach is useful for directly detecting pathogenic viruses without advance genetic information. Although its cost and technological availability make it unlikely that this system will very soon be the diagnostic standard worldwide, this system could be useful for the earlier discovery of novel emerging viruses and bioterrorism, which are difficult to detect with conventional procedures

Motility of Japanese Quail (Coturnix japonica) Sperm Diluted with Chicken Seminal Fluid

This study examined whether chicken seminal fluid was a useful diluent for quail sperm. Both sperm motility and progressive motility, when diluted with chicken seminal fluid (90.4% and 54.6%), were significantly higher than those of Lake solution supplemented with 0.005% caffeine and 10% quail cloaca fluid (80.1 and 41.6%), respectively. This result suggested that chicken seminal fluid colud be used as an effective diluent for the analysis of quail sperm motility

Diagnosis by Microbial Culture, Breath Tests and Urinary Excretion Tests, and Treatments of Small Intestinal Bacterial Overgrowth

Small intestinal bacterial overgrowth (SIBO) is characterized as the increase in the number and/or alteration in the type of bacteria in the upper gastrointestinal tract and accompanies various bowel symptoms such as abdominal pain, bloating, gases, diarrhea, and so on. Clinically, SIBO is diagnosed by microbial culture in duodenum/jejunum fluid aspirates and/or the breath tests (BT) of hydrogen/methane gases after ingestion of carbohydrates such as glucose. The cultural analysis of aspirates is regarded as the golden standard for the diagnosis of SIBO; however, this is invasive and is not without risk to the patients. BT is an inexpensive and safe diagnostic test but lacks diagnostic sensitivity and specificity depending on the disease states of patients. Additionally, the urinary excretion tests are used for the SIBO diagnosis using chemically synthesized bile acid conjugates such as cholic acid (CA) conjugated with para-aminobenzoic acid (PABA-CA), ursodeoxycholic acid (UDCA) conjugated with PABA (PABA-UDCA) or conjugated with 5-aminosalicylic acid (5-ASA-UDCA). These conjugates are split by bacterial bile acid (cholylglycine) hydrolase. In the tests, the time courses of the urinary excretion rates of PABA or 5-ASA, including their metabolites, are determined as the measure of hydrolytic activity of intestinal bacteria. Although the number of clinical trials with this urinary excretion tests is small, results demonstrated the usefulness of bile acid conjugates as SIBO diagnostic substrates. PABA-UDCA disulfate, a single-pass type unabsorbable compound without the hydrolysis of conjugates, was likely to offer a simple and rapid method for the evaluation of SIBO without the use of radioisotopes or expensive special apparatus. Treatments of SIBO with antibiotics, probiotics, therapeutic diets, herbal medicines, and/or fecal microbiota transplantation are also reviewed

Effects of Ethynylestradiol Injection into Maternal Japanese Quail (Coturnix japonica) on Male Reproductive Function of the F1 Generation

This study examined whether administration of ethynylestradiol to laying quail affects male reproductive function in the F1 generation. Regularly laying Japanese quail were daily-injected with 50 μl of corn oil or 1 μg of ethynylestradiol dissolved in 50 μl of corn oil for 5 days. Fertilized eggs were then collected 2, 4 and 6 d after the first injection (groups C1, C3 and C5 for oil treatment ; E1, E3 and E5 for ethynylestradiol treatment). The chicks that hatched from these eggs were raised and their reproductive functions (age at sexual maturity, copulation frequency, testicular weight, semen or sperm quality and fertility) were examined. Reproductive function did not differ significantly between ethynylestradiol-treated groups and corresponding controls (C1 to E1, C3 to E3, C5 to E5) and a among treatment days within control or treatment (C1—C5 or E1—E5). These results suggest that injection of ethynylestradiol in laying birds under the conditions used in this study does not significantly disrupt male reproductive function in the F1 generation