A new method for the treatment of sperm samples for ultrastructural study based on the use of animal tissues as biological containers

The study of the ultrastructure of spematozoa by means of transmission electron microscopy often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are: changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). To avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.     

Tris buffer improves fluorescence yield of ram spermatozoa when evaluating membrane integrity

This study was designed to evaluate the effect of various buffers on the fluorescence signal intensity of two fluorochromes (IP and CFDA) when used to assess the membrane integrity of ram sperm. Second ejaculates (18) from nine adult males were collected using an artificial vagina and diluted in either MOPS, TRIS, TES, HEPES, citrate, or phosphate-based extenders. Semen samples were stored at 15°C and the membrane integrity was assessed within the first 24 h of storage. Mean fluorescence intensity (FI) of PI- and CDFA-labeled sperm heads and fluorescence background noise (FBN) were determined quantitatively using Image J software. Fluorescence contrast (FC) was expressed as the difference between FI and FBN. Significantly, higher FI and FC were recorded when TRIS diluent was used, rather than the other diluents, both in the propidium- and fluorescein-labeled cells. The citrate and phosphate-based extenders showed intermediate results of FC between those of TRIS and zwitterionic (MOPS, TES and HEPES) groups for the PI-labeled sperm. However, in the CFDA-labeled sperm, the lower values of FC were obtained in the citrate and phosphate groups due to increased levels of FBN. For the membrane-damaged sperm, fluorescent labeling was limited to the sperm heads when TRIS-buffer was used, whereas in the other groups, the sperm tail was also frequently observed. It was concluded that TRIS buffer solution markedly increases the fluorescence yield of IP/CFDA-labeled sperm cells in the ram and that this should be considered when evaluating their membrane integrity.     

酸枣仁、茯神营养果冻的研制

以酸枣仁和茯神为主要原料,辅以白砂糖、食用胶凝剂和柠檬酸等研制出果冻.通过实验确定营养果冻的最佳配方为:卡拉胶0.4%,海藻酸钠0.2%,白砂糖12%,蛋白糖1%,酸枣仁、茯神提取液15%,柠檬酸0.2%,柠檬酸钠0.1%,乳粉8%.生产产品香味特别,风味爽口,营养全面且具有较好保健功能的酸枣仁茯神果冻.产品呈乳白色,...     

Straw-thawing method interacts with sire and extender to influence sperm motility and conception rates of dairy cows

Influence of interactions of straw-thawing method with sire and extender type (milk or egg yolk-based) on postthaw sperm motility and conception rates of dairy cows was assessed. In experiment 1, sperm from 10 Holstein sires were frozen in egg yolk citrate and heated whole-milk extenders using a split-ejaculate technique. Straws were thawed in a 37°C water bath with or without 3 min of exposure to a 37°C warming plate while wrapped in a paper towel (air-thaw). Percentage of motile sperm was assessed after 1 min and 3 h of incubation at 37°C. In experiment 2, sperm from 3 Holstein sires were frozen in egg-yolk citrate and a nonheated whole milk extender using a split-ejaculate technique. Straws were thawed and motility was assessed as in experiment 1. Conception rates (n = 475) were compared in a single herd of Holstein cows after thawing straws in 35°C water for 45 s or after air-thawing by wiping straws with a paper towel upon removal from the storage vessel and placing directly into the insemination gun. In each experiment, interactions between thaw method and sire, extender type, or both, indicated that conditions may be created that facilitate sperm tolerance of air-thaw procedures. When a significant thaw method effect was detected in either experiment, air-thaw was consistently associated with the lesser measure of sperm motility, conception, or both. In conclusion, although some sire × extender combinations seem to be tolerant of air-thaw procedures, other combinations are more sensitive, resulting in reduced postthaw sperm survival, conception rates of cows, or both, in response to air-thaw.     

荔枝核淀粉磷酸酯制备工艺

以三聚磷酸钠为磷酸化试剂,在半干法反应条件下制备荔枝核淀粉磷酸酯,并考察pH值、反应时间、反应温度和酯化剂用量对所得产品取代度的影响。通过正交试验确定制备荔枝核淀粉磷酸酯的最佳条件为:反应时间1.5h,温度150℃,三聚磷酸钠用量为淀粉干重的6%,最终可得取代度为0.016 3的淀粉磷酸酯。     

Effect of simultaneous thawing of multiple 0.5-mL straws of semen and sequence of insemination on conception rate in dairy cattle

The objectives of this study were to determine 1) the effect of simultaneous thawing of multiple 0.5-mL straws of semen and sequence of insemination (first, second, third, or fourth) on conception rates in dairy cattle, 2) whether the conception rates achieved following AI by professional AI (PAI) technicians and herdsman-inseminators (HI) differed, and 3) the effect of elapsed time from initiation of thawing straws of semen to seminal deposition on conception rates in dairy cattle. Four dairies with PAI and four with HI participated in the study. Initial data recorded included beginning thaw time, cow identification number, and time of seminal deposition. Herd records were retrieved following pregnancy diagnosis. Conception rates of dairy cows (n = 1025) were not affected by sequence of insemination (first, second, third, or fourth). Conception rates for herds using PAI were 40, 47, 41, and 50%. Conception rates for herds using HI were 24, 20, 33, and 30%. Average conception rates of dairy cows differed between PAI and HI (45 vs. 27%, respectively). The difference in mean conception rate achieved by PAI and HI was not attributable to milk production, parity, service number or stage of lactation. The elapsed time from initial thaw to completion of fourth AI was shorter for PAI than for HI, 7.6 vs. 10.9 min, respectively. Although the average conception rate differed between PAI and HI, elapsed time from initial thaw to completion of fourth AI and sequence of insemination (first, second, third, or fourth) had no effect on conception rate within inseminator group.     

Isolation of Mycoplasma spp. and serological responses in bulls prior to and following their introduction into Mycoplasma bovis-infected dairy herds

With the common use of bulls for breeding following a period of artificial insemination in seasonally bred dairy herds, it is important to consider the potential role of the bull in transmission of Mycoplasma spp. within and between herds. This study aimed to assess the prevalence of Mycoplasma spp. in a population of bulls before and after use in Mycoplasma bovis-infected herds. The frequency of subclinical infection was also measured serologically postbreeding, and the association of Mycoplasma spp. on semen quality was evaluated. Mycoplasma bovis was isolated from 4 of 118 bulls after use in 4 herds infected with M. bovis. In the bulls, M. bovis seroprevalence increased from 9% prebreeding to 46% postbreeding with a total seroconversion rate of 44% across the 4 herds, with no evidence of clinical disease. There was no association of Mycoplasma spp. in the bulls' semen and abnormal palpation characteristics (enlarged or nodular) of seminal vesicular glands or poor semen quality attributes such as semen mass activity, sperm motility, and morphology. These results demonstrate a high degree of subclinical exposure of the bulls to M. bovis in infected herds and highlight the potential for bulls to be mycoplasma carriers within and between herds. Herd biosecurity protocols and control programs should take into account the potential role of bulls in the introduction and spread of Mycoplasma spp.     

中药决明子的研究进展

主要从决明子的化学成分、药理作用及作用机制进行综述,为该药的进一步研究和开发奠定了基础。     

Evaluations for service-sire conception rate for heifer and cow inseminations with conventional and sexed semen

Service-sire conception rate (SCR), a phenotypic fertility evaluation based on conventional (nonsexed) inseminations from parities 1 through 5, was implemented for the United States in August 2008. The SCR model contains the categorical fixed effects of parity for lactations 1 to 5; state-year-month of insemination group; 6 standardized milk yield groups; service number for inseminations 1 to 7; cow age; and herd-year-season-parity-registry status class. Covariate effects for service-sire and mating inbreeding coefficients were linear regressions fit as deviations from the overall mean. Random effects included service-sire age group; AI organization-insemination year group; individual service sire; cow's genetic ability to conceive; cow's permanent environmental effect; and residual. Using insemination data from 2005 through 2009, the SCR procedure was applied separately for nulliparous heifer inseminations with conventional semen (SCRHconv), cow inseminations with conventional semen (SCRCconv), nulliparous heifer inseminations with sexed semen (SCRHsexed), and cow inseminations with sexed semen (SCRCsexed). Holstein and Jersey bulls with ≥300 and ≥200 artificial inseminations, respectively, in ≥10 herds and with ≥100 breedings during the 12 mo before evaluation were examined. The number of bulls evaluated for SCR in January 2010 was 270 Holsteins and 16 Jerseys for SCRHconv, 2,309 Holsteins and 214 Jerseys for SCRCconv, 114 Holsteins and 6 Jerseys for SCRHsexed, and 25 Holsteins and 7 Jerseys for SCRCsexed. The mean SCR for each evaluation category was set to 0; Holstein standard deviations were 2.55% for SCRHconv, 2.21% for SCRCconv, 4.29% for SCRHsexed, and 2.39% for SCRCsexed. The mean Holstein reliabilities were 82, 79, 75, and 73%, respectively. Correlations for Holstein SCR between conventional and sexed semen averaged near zero (−0.21 to 0.18). Predicted correlations between true SCR were −0.27 to 0.24. In contrast, correlations between Holstein heifers and cows were high (0.66 to 0.76), and predicted true correlations averaged near 1.0 (0.82 to 1.03). Correlations for Jerseys were often larger, although based on fewer inseminations and service sires compared with Holsteins. Some rankings for SCR could benefit from combining cow and heifer data but should be kept separate for conventional and sexed semen inseminations.     

决明子水溶性多糖的分离纯化

用热水浸提法从决明子炒品中获得决明子水溶性粗多糖CP1，经斐林试剂络合，得到两种组分C1A与C1C。同时从决明子生品，决明叶中分别获得水溶性粗多糖CP2，CP3。各组分经DEAE－纤维素柱层析进行分析，结果表明C1A为组分较单一的中性多糖，C1C为组分较复杂的混合多糖；CP1与CP2的洗脱曲线无明显区别，主要洗脱部分为中性多糖，表明炒制对决明子多糖无明显影响，CP3洗脱曲线有较明显的不同，主要洗脱部分为酸性多糖。