Semen Collection and Polymerase Chain Reaction‐Based Sex Determination of Black‐Headed and Straw‐Necked Ibis

This study aimed to develop a polymerase chain reaction (PCR)‐based sexing and effective semen collection methods for black‐headed and straw‐necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black‐headed and 4 straw‐necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W‐chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro‐ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw‐necked ibis using the massage method. Limited motility, viability and concentration of straw‐necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR‐based sexing proved to be an accurate molecular sexing method for black‐headed and straw‐necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw‐necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.     

Dual potential of microalgae as a sustainable biofuel feedstock and animal feed

The rise in global population has led to explorations of alternative sources of energy and food. Because corn and soybean are staple food crops for humans, their common use as the main source of dietary energy and protein for food-producing animals directly competes with their allocation for human consumption. Alternatively, de-fatted marine microalgal biomass generated from the potential biofuel production may be a viable replacement of corn and soybean meal due to their high levels of protein, relatively well-balanced amino acid profiles, and rich contents of minerals and vitamins, along with unique bioactive compounds. Although the full-fatted (intact) microalgae represent the main source of omega-3 (n-3) polyunsaturated fatty acids including docohexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the de-fatted microalgal biomass may still contain good amounts of these components for enriching DHA/EPA in eggs, meats, and milk. This review is written to highlight the necessity and potential of using the de-fatted microalgal biomass as a new generation of animal feed in helping address the global energy, food, and environmental issues. Nutritional feasibility and limitation of the biomass as the new feed ingredient for simple-stomached species are elaborated. Potential applications of the biomass for generating value-added animal products are also explored.     

Novel prediction method of beer foam stability using protein Z, barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and yeast thioredoxin

Foam stability is an important quality trait of beer. Our previous results of two-dimensional gel electrophoresis (2DE) analyses of beer proteins implied a relationship between barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and beer foam stability as judged by the NIBEM-T analyzer. To develop a novel prediction method of beer foam stability under different conditions of barley cultivar and malt modification, multiple linear regression analysis was applied. The spot intensities of major beer proteins on 2DE gel were quantified and used as explanatory variables. The foam stabilities of 25 beer samples each brewed from malt with different malt modification in one of the three cultivars (cultivars A, B, and C) were explained by the spot intensities of BDAI-1 at the 5% significance level ( r = 0.421). Furthermore, two other major protein spots (b0 and b5) were observed on the 2DE gels of Japanese commercial beer samples with different foam stability. Then, multiple regression for foam stability was calculated using these three spot intensities as explanatory variables. As a result, 72.1% of the beer foam stability in 25 beer samples was explained by a novel multiple regression equation calculated using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. To verify the validity of the multiple regression equation and the explanatory variables, the beer foam stability in practical beer samples was analyzed. As a result, 81.5% of the beer foam stability in 10 Japanese commercial beer samples was also explained by using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. Mass spectrometry analyses followed by database searches revealed that protein spots b0 and b5 were identified as protein Z originated from barley and thioredoxin originated from yeast, respectively. These results confirm that BDAI-1 and protein Z are foam-positive factors and identify yeast thioredoxin as a possible novel foam-negative factor.     

热杀菌条件对NFC冬枣汁成分及抗氧化活性的影响研究

为了研究不同热杀菌条件对NFC冬枣汁理化性质、活性成分及抗氧化活性的影响,本文以冬枣为研究对象,设置了低温长时杀菌(75℃,5、10、15、20、25 min)和高温短时杀菌(90℃,0.25、0.50、0.75、1.00、1.50 min)两个处理,考察了NFC冬枣汁的可溶性固形物含量、pH值、总酸含量、VC含量、DPPH清除率、亚铁离子还原率等指标。研究结果表明,两种杀菌方式对NFC冬枣汁基本理化性质影响不显著(P>0.05)。相比于低温长时杀菌,高温短时杀菌的△E值更低,更能保持冬枣汁原有的色泽。两种杀菌处理后多酚、VC含量均显著降低(P<0.05),低温长时杀菌25 min后VC含量未检出,抗氧化活性也显著低于高温短时杀菌(P<0.05)。因此,通过对NFC冬枣汁品质变化的分析,发现高温短时杀菌能更好地保持原有的色泽和营养成分。     

Affected homologous chromosome pairing and phosphorylation of testis specific histone, H2AX, in male meiosis under FKBP6 deficiency

A gene for FK506 binding protein 6 (Fkbp6) expresses during a specific stage of male and female meiosis. Disruption of the gene influences male reproduction, i.e. arrests spermatogenesis, but not female reproduction. Using the mouse model (targeted disruption), the role of the gene in homologous chromosome pairing has been demonstrated in a previous study. For further understanding the function of Fkbp6 in chromosome synapsis, we evaluated chromosome pairings during male meiosis in the as/as rat, a spontaneous null mutation, and compared them with those of the mouse model. Electron microscopy of the pachytene nuclei unveiled several types of abnormal chromosome pairing in the rat model, as shown in the mouse previously. The frequencies of aberrant pairings in the knockout mice and mutant rats were 42 of 67 nuclei (62.7%) and 20 out of 74 nuclei (27.0%), respectively. In order to clarify the mechanism of male specific infertility in Fkbp6 deficiency, the localization of gammaH2AX, a marker protein of XY chromosome inactivation during male meiosis, was examined. Immunostaining of gammaH2AX unveiled normal localization of the molecule to XY chromosomes (XY body) in both models, showing the independency of FKBP6 in sex chromosome inactivation. Besides the XY body, focal localization of gammaH2AX was observed in accordance with the unsynapsed chromosomes in both types of null animal. These results indicate the fundamental role of Fkbp6 in homologous chromosome synapsis during male meiosis. In conclusion, male specific infertility under Fkbp6 deficiency remains unsolved.     

Endocrine status and development of porcine testicular tissues in host mice

Clarification of the endocrine status of host mice provides us with basic knowledge with which we can manipulate the growth and function of xenografted testicular tissues. We investigated the hormonal profiles of castrated mice grafted with porcine immature testicular tissues from 30 to 210 or more days after grafting (day 0=castration and grafting). The serum follicle stimulating hormone (FSH) concentrations of the host mice declined (P<0.05) from day 60 compared with those of the castrated, ungrafted mice. The serum inhibin and testosterone levels were higher (P<0.05) than those in the castrated, ungrafted mice from days 30 and 90 days, respectively. The inhibin levels further increased (P<0.05) from day 90, during which time the levels were higher (P<0.05) than those in the intact male mice. In the grafts, formation of lumens occurred in the seminiferous cords on day 90 and spermatozoa appeared in the lumens from day 120. However, spermatogenesis in the grafts did not reach the qualitatively normal levels observed in adult boars. The intensity of the immune reaction to inhibin alpha subunits in the Sertoli cells of the grafts decreased with differentiation of the seminiferous tubules. The present findings indicate that a feedback loop was established between the mouse hypothalamo-pituitary axis and the grafted porcine tissues from 60 days post-grafting. The results also indicate that the serum inhibin levels in the host mice remained high even after the appearance of lumens in the seminiferous tubules of the grafted tissues; this is strikingly different to the situation in normal male animals, in which the serum inhibin levels decline at around the time of tubular differentiation. The lack of efferent ducts in the tubules of the grafted tissues probably caused the accumulation of inhibin to be released into the lumens, resulting in high concentrations of circulating inhibin. These high levels of inhibin may directly affect spermatogenic activity and suppress FSH secretion.     

Secretion of inhibin in male Japanese quail (Coturnix japonica) from one week of age to sexual maturity

The objective of this study was to investigate the changes in secretion of inhibin and cellular localization of the inhibin alpha and inhibin/activin (beta(A) and beta(B)) subunits in male Japanese quail from 1 to 7 weeks after hatching. The post-hatch profile of plasma luteinizing hormone (LH), immunoreactive (ir) inhibin and testosterone were measured by radioimmunoassay. Testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against inhibin alpha, inhibin/activin beta(A) and inhibin/activin beta(B) from one week of age to sexual maturity. Testicular weight increased gradually until 4 weeks and abruptly increased from 5 weeks of age onwards. The plasma concentrations of LH and ir-inhibin increased significantly at 5 weeks of age, and the plasma concentration of testosterone increased significantly at 6 weeks of age. Pituitary contents of LH showed a steady increase until 6 weeks of age and then abruptly increased at 7 weeks of age. Coincident to the increase in plasma testosterone, the testicular contents of testosterone significantly increased from 5 weeks through sexual maturity. Immunohistochemically, localization of the inhibin/activin alpha, beta(A) and beta(B) subunits was found in the Sertoli and Leydig cells at all ages of development from one week of age to sexual maturity. These results suggest that Sertoli and Leydig cells are the major source of inhibin secretion during development in male Japanese quail.     

Relationships among age, body weight, scrotal circumference, semen quality and peripheral testosterone and estradiol concentrations in pubertal and postpubertal Holstein bulls

In the present study, the correlations among age, body weight, scrotal circumference (SC), semen quality and peripheral testosterone and estradiol-17beta (E(2)) concentrations were investigated in pubertal (n=5) and postpubertal (n=7) groups of Holstein bulls over a 6 week period. There were significant positive correlations (P<0.01) among age, body weight and SC in both groups, and similar significant correlations between sperm motility and SC in pubertal bulls (P<0.01) and between sperm concentration and SC in postpubertal bulls (P<0.05). The sperm motility after collection (P<0.05) and after freezing and thawing (P<0.01) of the postpubertal bulls correlated positively with the E(2) concentration. Estrogen may be important for the function of postpubertal bull testes, in which it may regulate spermatozoa motility in vivo.     

Secretion of inhibin in female Japanese quails (Coturnix japonica) from hatch to sexual maturity

To clarify the cellular source and secretory pattern of inhibin in the Japanese quail during follicular development, the plasma concentrations of immunoreactive (ir) inhibin were measured from 1 to 7 weeks after hatching. Localization of the inhibin/activin alpha, beta A and beta B subunits was investigated by immunohistochemistry. To monitor development of the pituitary and ovarian functions, the plasma luteinizing hormone (LH) and progesterone concentrations were also measured. Ovarian weight increased gradually until 6 weeks of age and then abruptly increased at 7 weeks of age just at the onset of egg production. Plasma concentrations of LH increased significantly at 6 weeks of age. The plasma concentrations of ir-inhibin and progesterone and the pituitary contents of LH also increased significantly at 7 weeks of age. Immunohistochemically, the inhibin/activin alpha, beta A and beta B subunits were localized in the granulosa cells of all follicles during different stages of development from 1 to 7 weeks after hatching. The inhibin alpha, beta A and beta B subunits were also found in the interstitial cells but not theca cells of all follicles. These results demonstrated that the plasma concentrations of ir-inhibin of the female Japanese quails rose with ovarian development. The immunohistochemical results suggested that granulosa and interstitial cells are the major source of ovarian inhibins in female Japanese quails.     

Expression of nerve growth factor and its receptors trkA and p75 and inhibin alpha-subunit in the ovarian interstitial cells of lactating golden hamsters

Characteristic daily increases in the plasma levels of luteinizing hormone (LH) are present every afternoon during lactation in golden hamsters. The objective of this study was to investigate the effect of the diurnal rhythm of increases in LH on expression of nerve growth factor (NGF), its receptors trkA and p75 and inhibin alpha-subunit in the ovarian interstitial cells of lactating golden hamsters. Both lactating and non-lactating groups of postpartum golden hamsters were used in this study. The expression of NGF, its receptors trkA and p75 and inhibin alpha-subunit were determined by immunohistochemistry. Positive staining of NGF, trkA and p75 was found in the interstitial cells of the lactating group, and no immunoreactivity for NGF, trkA or p75 was observed in the ovarian interstitial cells of the non-lactating group. In addition, immunostaining of inhibin alpha-subunit was also observed in the interstitial cells of the lactating group but not in those of the non-lactating group. Immunostaining of the inhibin/activin beta(A)- and beta(B)-subunits was observed in the granulosa cells of antral follicles, but not in the interstitial cells of the lactating and non-lactating animals. These results suggest that the diurnal rhythm increases in LH can induce expression of NGF, trkA, p75 and inhibin alpha-subunit in the ovarian interstitial cells of lactating golden hamsters and that NGF, its receptors trkA and p75 and inhibin alpha-subunit may have the capacity for autocrine or paracrine modulation of interstitial cell differentiation in golden hamsters.