Ornithine Decarboxylase Activity in Brain Regulated by a Specific Macromolecul, the Antizyme

Mouse brain ornithine decarboxylase activity is about 70-fold higher at the time of birth compared with that of adult mice. Enzyme activity declines rapidly after birth and reaches the adult level by 3 weeks. Immunoreactive enzyme concentration parallels very closely the decrease of enzyme activity during the first postnatal week, remaining constant thereafter. The content of brain antizyme, the macromolecular inhibitor to ornithine decarboxylase, in turn is very low during the first 7 days and starts then to increase and at the age of 3 weeks it is about six times the level of that in newborn mice. This may explain the decrease in enzyme activity during brain maturation, and suggests the regulation of polyamine biosynthesis by an antizyme-mediated mechanism in adult brain.     

<Emphasis Type="Italic">Demodex gatoi</Emphasis> -associated contagious pruritic dermatosis in cats - a report from six households in Finland

Background  

Demodex gatoi is unique among demodectic mites. It possesses a distinct stubby appearance, and, instead of residing in the hair follicles, it dwells in the keratin layer of the epidermis, causing a pruritic and contagious skin disease in cats. Little is known of the occurrence of D. gatoi in Europe or control of D. gatoi infestation.     

Recovery Rate and Quality of Embryos from Mares Inseminated at the First Post-Partum Oestrus

The pregnancy rate is lower in mares inseminated at the first post-partum (p.p.) oestrus (40-50%) compared with pregnancy rates in subsequent oestrous cycles (55-65%). The causes of the lowered pregnancy rate are not fully understood. The aim of the present study was to examine if embryonic defects could be one of the reasons for lowered pregnancy rate. A total of 23 p.p. and 14 non-lactating control mares were flushed 7 days after detection of ovulation. Embryo recovery rate was 48% and 71% in p.p. and control mares, respectively (p=0.16). Embryos were photographed, measured, graded and stained with fluorescein diacetate to assess their viability. Thereafter embryos were bisected and stained with Hoechst 33342 to count the cell nuclei. Embryos in both groups were equally viable and the cell numbers were not significantly different. According to morphological evaluation all embryos were classified as excellent or good. Embryos aged 7.3 to 7.6 days (± 0.25 days) were smaller in the p.p. group than in the control group (p<0.05). Forty-seven (9/19) and 8% (1/13) of the uterine swabs, taken before the first insemination, yielded bacteria and neutrophils in p.p. and control mares, respectively. The amount of neutrophils and/or bacteria had no statistically significant effect on embryo recovery rate (p>0.10). Recovery of embryos was not related to histological findings in uterine biopsies taken after embryo recovery. Embryo recovery rate in p.p. mares (48%) was similar to previously reported foal heat pregnancy rates (40- 50%). Hence, early embryonic death in utero would not be the most likely reason for lowered pregnancy rate in mares inseminated at the first p.p. oestrus. Sperm transport and oviductal conditions by the time of the first p.p. oestrus would need to be studied to clarify the role of fertilisation failure as the cause of lower pregnancy rate in mares inseminated at foal heat.     

Hyaloscyphaceae in Japan (7): <Emphasis Type="Italic">Hyaloscypha albohyalina</Emphasis> var. <Emphasis Type="Italic">monodictys </Emphasis>var. nov.

 Hyaloscypha albohyalina var. monodictys, a new variety in the family Hyaloscyphaceae, Helotiales with Monodictys anamorph is described and illustrated. Received: June 26, 2002 / Accepted: July 27, 2002 Present address: Strategic Product Portfolio Department, Sankyo Co., Ltd., 3-5-1 Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8426, Japan Tel. +81-3-5255-7040 (Ext. 2528); Fax +81-3-5255-7086 e-mail: hosoya@hq.sankyo.co.jp Correspondence to:T. Hosoya     

Viability of bovine demi- and quarter-embryos after transfer

The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6 13 ) for heifers and 33.3% (4 12 ) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8 13 ) for heifers and 8.3% (1 12 ) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.     

Recovery rate and quality of embryos from mares inseminated after ovulation

The aim of this study was to evaluate the quality of embryos and their recovery rate from mares inseminated at different intervals after ovulation. Finnhorse and warmblood mares were inseminated with fresh semen 8 to 16 h, 16 to 24 h, or 24 to 32 h after ovulation. Control mares were inseminated before ovulation. Sixty-seven embryo flushings were performed between Days 7 and 9 after ovulation/insemination. Thirteen mares were not flushed, but their uteri were scanned for pregnancy on Days 14 to 16. Embryo recovery rates decreased as time from ovulation to insemination increased, although embryo quality remained normal as evaluated by morphological criteria and mitotic index. However, postovulatory insemination in this trial appeared to delay embryo development, since the embryos recovered from mares inseminated after ovulation were appreciably smaller and at an earlier stage of development than control embryos recovered from mares inseminated prior to ovulation. Part of this delay in embryo development in the postovulation group could be due to the time needed for sperm capacitation. In addition, as the time from ovulation to insemination increased, embryo development might have been further delayed by defects in the aging oocyte.     

Successful transfer of biopsied equine embryos

Embryo biopsy has been used to detect inherited disorders and to improve the phenotype by analyzing of linkages between marker loci and the desired characteristics. Unfortunately, early procedures required the removal of a large portion (one-half) of the embryo for analysis, and the transfer of bisected equine embryos has not been particularly successful. Recent discovery of the polymerase chain reaction (PCR) has made possible the detection of specific DNA sequences from only a few cells. We investigated whether the removal of a small biopsy would allow for successful PCR and normal embryonic development. In the study reported here, 14 microbladebiopsied Day 6 to 7 equine embryos were transferred nonsurgically into recipient mares. The sex of each embryo was determined from the biopsy by means of restriction fragment length polymorphism analysis of the ZFY/ZFX loci after PCR amplification. The embryos were sexed as 8 females and 6 males on the basis of PCR assay results. Two embryos were biopsied using a needle aspiration technique, but no PCR amplification products resulted from these attempts. Eight intact control embryos were transferred to recipient mares using the same method. Pregnancy rates were 3 14 and 6 8 for the microblade biopsy and control groups, respectively. All of the microblade biopsy group pregnancies were females. One was aborted for cytogenetic analysis. Two were born after normal gestation. With improved pregnancy rates, this technique could be used for preimplantation diagnostics of equine embryos. As gene mapping advances and associations between particular DNA sequences and inherited traits become established, a rapid PCR technique could be used to select embryos before transfer.     

Insemination Results with Slow-Cooled Stallion Semen Stored for approximately 40 Hours

Semen from 3 stallions was extended using 2 methods (Kenney extender and a modified Kenney extender), slowly cooled, and stored for 41 ± 6 (s.d.) h before insemination. An insemination dose (40 ml) contained 1.5-2 billion spermatozoa. In the experiment, 26 mares were inseminated in 30 cycles. The pregnancy rate per cycle obtained with sperm stored in the Kenney extender was 87% (n=15). When the semen was extended with the modified extender, centrifuged and stored, the pregnancy rate was 60% (n=15). Inseminations were done every other day until ovulation was detected. If a mare ovulated more than 24 h after the last insemination, she was inseminated also after ovulation. The single-cycle pregnancy rate was 58% when the mares were inseminated only before ovulation (n=19) but the rate was 100% when the inseminations were done both before and after ovulation (n=9) or only after ovulation (n=2). The difference in pregnancy rates was significant (p<0.05), indicating that postovula-tory inseminations probably serve to ensure the pregnancies. The extending and handling methods used in this study resulted in a combined pregnancy rate of 73%, and appear thus to be useful for storing stallion semen for approximately 2 days.     

Sperm-induced leukocytosis in the equine uterus

The objective of this study was to investigate the inflammatory reaction induced in the equine uterus by insemination with fresh and frozen semen. Eleven groups (6 to 8 mares per group) were studied during 2 breeding seasons. The mares were inseminated using raw semen, frozen semen, extended fresh and frozen semen, concentrated fresh semen, seminal plasma and seminal extenders only. One group was bred naturally. Six hours after insemination, the uteri were flushed with 50 ml of phosphate-buffered saline (PBS). Seventeen out of 104 samples (16%) exhibited slight bacterial growth. Neutrophil concentrations were significantly (P < 0.05) higher in all treated mares than in the controls. Mares infused with PBS, seminal extenders or the supernatant from centrifuged frozen-thawed semen exhibited only a mild neutrophil response. Insemination with frozen semen resulted in higher neutrophil concentrations than insemination with extended fresh semen (means of 59 vs 5 million neutrophils/ml; P < 0.05). Highest neutrophil counts were found after insemination with frozen semen or concentrated fresh semen. Bacterial contamination of uteri was insignificant 6 hours after breeding. Neutrophilia seems to be induced by spermatozoa rather than bacteria. The intensity of the neutrophil reaction seems to depend on concentration and/or volume of inseminate.     

Fast and sensitive liquid chromatography-mass spectrometry assay for seven androgenic and progestagenic steroids in human serum

A fast and sensitive LC-MS/MS method for the quantitative analysis of seven steroid hormones in 150 μl of human serum was developed and validated. The following compounds were included: 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, pregnenolone, and progesterone. Individual stable isotope-labeled analogues were used as internal standards. Sample preparation was performed by liquid-liquid extraction, followed by oxime derivatization to improve the ionization efficiency of the analytes. In contrast to the common derivatization-based methods, the reaction was incorporated into the sample preparation process and the only additional step due to the derivatization was a short heating of the autosampler vials before the sample injection. Chromatographic separation was achieved on a reversed-phase column using a methanol-water gradient. For the analyte detection, a triple quadrupole instrument with electrospray ionization was used. Total run time was 7.0 min and the lower limits of quantification were in the range of 0.03-0.34 nM (0.01-0.10 ng/ml), depending on the analyte. The method was validated using human serum samples from both sexes and applied for the serum steroid profiling of endometriosis patients.