Cryopreservation of Immature Bovine Oocytes to Reconstruct Artificial Gametes by Germinal Vesicle Transplantation

Joining immature gamete cryopreservation and germinal vesicle transplantation (GVT) technique could greatly improve assisted reproductive technologies in animal breeding and human medicine. The present work was aimed to assess the most suitable cryopreservation protocol between slow freezing and vitrification for immature denuded bovine oocytes, able to preserve both nuclear and cytoplasmic competence after thawing. In addition, the outcome of germinal vesicle transfer procedure and gamete reconstruction was tested on the most effective cryopreservation system. Oocytes, isolated from slaughterhouse ovaries, were stored after cumulus cells removal either by slow freezing or by vitrification in open pulled straws. After thawing, oocytes were matured for 24 h in co-culture with an equal number of just isolated intact cumulus enclosed oocytes, and fixed in order to evaluate the stage of meiotic progression and cytoskeleton organization. Our results showed that after warming, vitrified oocytes reached metaphase II (MII) in a percentage significantly higher than oocytes cryopreserved by slow freezing (76.2% and 36.5% respectively, p < 0.05). Moreover, vitrification process preserved the organization of cytoskeleton elements in a higher proportion of oocytes than slow freezing procedure. Therefore vitrification has been identified as the elective method for denuded immature oocytes banking and it has been applied in the second part of the study. Our results showed that 38.3% of oocytes reconstructed from vitrified gametes reached the MII of meiotic division, with efficiency not different from oocytes reconstructed with fresh gametes. We conclude that vitrification represents a suitable method of GV stage denuded oocyte banking since both nuclear and cytoplasmic components derived from cryopreserved immature oocytes can be utilized for GVT.     

Validation of the FACSCount AF System for Determination of Sperm Concentration in Boar Semen

A flow cytometric method has been developed for rapid determination of sperm concentration in semen from various mammalian species. * * Patent Pending, Int. Publication Number WO/00/54026.
All cells containing DNA are stained with SYBR‐14 or propidium iodide (PI) and sperm concentration is determined in relation to an internal standard of fluorescent microspheres (beads). Satisfactory staining can be achieved within 2–3 min and the following flow cytometric analysis on the FACSCount AF System rapidly provides the user with a precise and accurate assessment of the sperm concentration. In this study, the FACSCount AF System and Sperm Counting Reagent (BD Biosciences) was compared with microscopic counting using a Bürker–Türk haemocytometer. In addition, sperm concentration was determined using the Corning 254 spectrophotometer which is used routinely by Danish artificial insemination stations for boars. The results show that the agreement between flow cytometry and microscopic counting is very high. The slope for the regression line was 1.12 (SE = 0.03) with an estimated intercept with the Y‐axis of 22 × 10⁶sperm/ml (SE = 10 × 10⁶ sperm/ml) and an estimated error of the model of 10 × 10⁶ sperm/ml. For the spectrophotometer, the slope of the regression line was 1.09 (SE = 0.07) with an estimated intercept of 137 × 10⁶ sperm/ml (SE = 25 × 10⁶ sperm/ml). The average error made by the spectrophotometer was 55 × 10⁶ sperm/ml. In addition, the results obtained using flow cytometry was highly repeatable (CV = 2.7%) in comparison with the spectrophotometric method (CV = 6.3%). These results indicate that the FACSCount AF System is a valuable tool for precise and accurate assessment of sperm concentration in boar semen and that use of this system may lead to production of more uniform insemination doses containing a specific number of sperm per dose.     

Identification and changes in the seasonal concentrations of heat shock proteins in roe deer (Capreolus capreolus) epididymides

Heat shock proteins (HSPs) act as molecular chaperones with important regulatory functions. HSPs are considered to be essential factors in animal reproduction. In view of seasonal variations in the secretory activity of the reproductive tract of mature roe deer (Capreolus capreolus), the aims of this study were to identify HSPs in the epididymides and compare the expression of the identified proteins in three periods of the reproductive season. Two‐dimensional polyacrylamide gel electrophoresis revealed the highest number of polypeptides in homogenates of epididymal tissues and in caput, corpus and cauda epididymal fluids throughout the reproductive season. Epididymal tissue homogenates and epididymal fluids were analysed by tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with enzymatic activity, including polypeptides with antioxidant properties, structural and cell signalling functions. Moreover, among the identified polypeptides, five of them were similar to heat shock proteins: endoplasmin (Grp94); heat shock protein 90 kDa (HSP90); 78‐kDa glucose‐regulated protein (Grp78); chain A, the crystal structure of the human HSP70 ATPase domain and heat shock protein beta‐1 isoform X. The concentrations of the analysed polypeptides, expressed in optical density units (ODU), differed significantly (p ≤ .05) across the examined periods of the reproductive season. The highest ODU values for almost all analysed proteins were observed during the rutting period. The presence of HSPs in the epididymal tissues and fluids of roe deer in different periods of the reproductive season could indicate that those proteins play an important role in sperm maturation in the epididymis.     

The Usefulness of Captive Kept Capercaillie (Tetrao urogallus L.) as the Semen Donors for Artificial Insemination and Gene Pool Preservation In vitro

Captive breeding of birds threatened by extinction in zoological gardens or other closed aviary centres is one of the methods allowing their protection and gene pool preservation ex situ in vivo. Such birds are usually kept in captivity lifelong and serve as parents of several new generations that can be further released into natural environment, or males are used as semen donors for artificial insemination and gene banking. Therefore, the fecundity of such flocks (number of laid egg and spermatozoa quantity and quality) is very important. The aim of this study was to evaluate the usefulness of captive kept capercaillie (Tetrao urogallus L.) as semen donors in three subsequent reproductive seasons, based on the assessment of manually collected semen quality. Male response to dorso‐abdominal massage, ejaculate volume, sperm concentration, motility and morphology were evaluated individually at three succeeding years. Depending on individual male properties and year of collection, the number of positive reactions to semen collection attempts (i.e. ending with ejaculation) varied from 44.4% to 100.0%; single ejaculate volume ranged from 10 to 300 μl, spermatozoa concentration from 10 × 10⁶ per ml to 3520 × 10⁶ per ml and percentage of live morphologically normal spermatozoa from 19.3 to 80.3%. The highest average value (66.7) of semen quality factor (SQF) was noted for a 2‐year‐old male (varying from 1.9 to 258.1), while the lowest for ten‐ (4.8; varying from 0.1 to 17.0) and 7‐year‐old (6.6; varying between 0.6 and 13.6). Assuming that for AI purposes, the ejaculate quality has to be at minimum 10 SQF, obtained results indicate that majority of capercaillie kept in captivity, both young (2–3 years old) and older (up to 10 years old), can be valuable semen producers in succeeding seasons.     

Rate Constants for Charge Transfer Across Semiconductor-Liquid Interfaces

Interfacial charge-transfer rate constants have been measured for n-type Si electrodes in contact with a series of viologen-based redox couples in methanol through analyses of the behavior of these junctions with respect to their current density versus potential and differential capacitance versus potential properties. The data allow evaluation of the maximum rate constant (and therefore the electronic coupling) for majority carriers in the solid as well as of the dependence of the rate constant on the driving force for transfer of delocalized electrons from the n-Si semiconducting electrode into the localized molecular redox species in the solution phase. The data are in good agreement with existing models of this interfacial electron transfer process and provide insight into the fundamental kinetic events underlying the use of semiconducting photoelectrodes in applications such as solar energy conversion.     

防疫耳标主要质量指标的检测

本着遵照标准,结合实际的原则,阐述了动物用防疫耳标强度指标和字迹附着力指标等主要质量指标的检测方法,并通过大量试验证实其具有简单、科学、有效等特点,在实际检测过程中具有一定的推广应用价值。     

Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles are Dose‐Dependent

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF‐1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α‐MEM⁺, supplemented with different concentrations of human recombinant IGF‐1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO₂ in 24‐well plates with total replacement of the medium every 2 days. Non‐cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF‐1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF‐1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF‐1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF‐1 tested. Ultrastructurally, the non‐cultured control and IGF‐1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF‐1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.