Oestrogens and androgens in blastocoelic fluid and cultures of cells from equine conceptuses of 10-22 days gestation.

Six samples of blastocoele fluid recovered between 10 and 22 days gestation were tested in human clinical radioimmunoassay systems measuring total oestrogens and total androgens. The results were erratic but in 5 cases measurements for oestrogen equivalent to between 1000 and 70,000 pg/ml and for androgen between 1000 and 85,000 pg/ml were recorded. Cells from two blastocysts were cultured in medium 199 with and without horse serum. When the used media were assayed, values equivalent to at least 8000 pg oestrogen/ml were obtained on 7 of 11 occasions. In 9 of 11 samples the androgen concentration exceeded 700 pg/ml. In control media the oestrogen and androgen concentrations did not exceed 120 and 360 pg/ml respectively.     

Initiation of superovulation in mares 5 or 12 days after ovulation using equine pituitary extract with or without GnRH analogue

Cyclic mares were assigned to 1 of 3 treatments (n=15 per group): Group 1 received equine pituitary extract (EPE; 25 mg, i.m.) on Day 5 after ovulation; Group 2 received EPE on Day 12 after ovulation; while Group 3 received 3.3 mg of GnRH analogue (buserelin implant) on the day of ovulation and 25 mg, i.m. EPE on Day 12. Mares in each group were given 10 mg PGF(2)alpha on the first and second day of EPE treatment. The EPE treatment was continued daily until the first spontaneous ovulation, at which time 3,300 IU of human chorionic gonadotropin (hCG) were given to induce further ovulations. Mares in estrus with a >/=35 mm follicle were inseminated every other day with pooled semen from 2 stallions. Embryo recovery was attempted 7 days after the last ovulation. Follicular changes and embryo recovery during 15 estrous cycles prior to treatment were used as control data. During treatment, the number of follicles >/=25 mm was higher (P<0.05) for Day 5 than for Day 12 or control mares, but the number for Day-5 mares was similar (P>0.05) to that of mares treated with buserelin implants (Group 3). Initiation of EPE treatment on Day 5 resulted in a greater (P<0.05) number of ovulation (2.9) than on Day 12 (1.1) or in the control mares (1.3) but not in the buserelin-treated mares (1.8). The number of embryos recovered from mares in the Day 5 (1.2), Day 12 (1.0), buserelin (0.9) and control (0.9) groups was similar (P>0.05). The conclusions were 1) EPE initiated in early diestrus increased follicular development and ovulation and 2) treatment with GnRH analogue marginally improved response to EPE treatment.     

Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus)

A novel, minimally invasive, transabdominal embryo collection method (transabdominal method) was developed as an alternative to a standard abdominal incision for embryo collection in the common marmoset. The abdominal incision method was used for 304 flushes using 36 female animals, whereas the transabdominal method was used for 488 flushes using 48 females; successful embryo collection rates were 48.0% and 48.4% (P > 0.05), respectively. These techniques were successfully duplicated at another institute (German Primate Center, DPZ). At that institution, successful embryo collection rates were 88.9% and 77.8% for the abdominal incision and transabdominal methods, respectively (P > 0.05), whereas the average numbers of preimplantation embryos obtained per flush were (mean ± SD) 1.91 ± 0.35 and 1.71 ± 0.14 (P > 0.05). The transabdominal method reduced animal stress, did not require incisional wound healing, and enabled successive embryo recoveries to be done much sooner. More embryos in early developmental stages (zygotes/morulae) were recovered using the transabdominal method (76.1%) than the abdominal incision method (52.6%, P < 0.01). In contrast, recovery of arrested or abnormal embryos was not significantly different between these two methods (9.8% and 8.3%). To verify developmental ability of embryos recovered by the transabdominal method, transfer of 28 normal embryos to 14 surrogate mothers yielded a nidation rate of 57%. Five females sustained term pregnancies and eight neonates were born. This novel transabdominal method will facilitate progress in marmoset developmental biology and embryology.     

Fertility comparison between breeding at 24 hours or at 24 and 48 hours after collection with cooled equine semen

It has become a common practice in the equine breeding industry to send 2 insemination doses for breeding with transported cooled semen, one to be used for the initial insemination upon arrival, and the other to be held a second insemination the next day. One fertile stallion and 36 fertile mares were used to determine if breeding once with 1 dose of semen cooled for 24 h would improve fertility compared with breeding twice, 1 d apart, with half the dose of semen cooled for 24 h on the first day of breeding and half cooled for 48 h on the second day of breeding. Mares were given two intramuscular injections of 10 mg PGF2 alpha 14 d apart. Following the second injection, mares were teased with a stallion and their ovaries were scanned by transrectal ultrasonography daily. When a dominant follicle (> 35 mm diameter) was detected, 1500 units hCG were injected intravenously, and the mares were inseminated. Semen was collected in advance of anticipated breeding, mixed in nonfat dry milk solids-glucose extender to a concentration of 25 million sperm/mL, and placed in 2 commercial cooling containers for 24 or 48 h of storage prior to breeding. Mares were randomly assigned to 1 of 2 insemination treatment groups: 1) Group T1 (n = 18), in which mares were inseminated on the day of hCG injection with 500 million spermatozoa cooled for 24 h, or 2) Group T2 (n = 18), in which mares were inseminated on the day of hCG injection with 250 million spermatozoa cooled for 24 h, and again on the following day with 250 million spermatozoa cooled for 48 h. Pregnancy status was confirmed by transrectal ultrasonographic examination at 14 and 16 d after ovulation. Pregnancy rates were the same for both insemination treatment groups (12/18; 67%). There was no advantage to holding half of the insemination dose for rebreeding on the following day.     

Location and status of embryos in the genital tract of superovulated cows 4 to 6 days after insemination

Sixty lactating Holstein cows were treated, in 3 groups, with Folltropin and Estrumate to induce superovulation and then bred by artificial insemination (AI). Embryos were collected at slaughter on D 4, 5 or 6 after insemination by flushing separately the oviducts, uterine tip and the remainder of the uterine horn. The embryos and ova recovered accounted for 64.6 ± 4.3% of the ovulations, and there were no differences due to day, side or group. On D 4, 60% of the embryos were found in the oviducts; on Days 5 and 6, 80 and 91%, respectively, were found in the tip of the uterine horn. Viability was independent of the site of recovery; over 91% of the embryos grew and developed in culture for at least 3 d.     

Embryo loss in cattle between Days 7 and 16 of pregnancy

Embryo loss between embryonic Days 7 and 16 (Day 0 = day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P = 0.03) as more embryos were transferred, particularly at later stages (Day 14, P < 0.01). The average length of embryos decreased by approximately 5% for every additional embryo transferred (P < 0.0001). These effects may be linked to embryonic migration. Embryo mortality inherent to the embryo during the second week of pregnancy was 24%. Additionally, 9% of Day 14 embryos were of inferior quality, as they did not contain an epiblast. Combining embryo and recipient causes but excluding infection effects, embryonic loss of IVP embryos during the second week of pregnancy amounted to 26% (heifers) or 34% (parous dairy cows). The length of embryos doubled every day between Days 9 and 16, with a 4.4-fold range in sizes representing two thirds of the variation in length. Embryos retrieved from heifers were twice the size of those incubated in parous cows (P < 0.0001), indicating faster embryonic development/trophoblast proliferation in heifers. Whereas season did not affect embryo recoveries, length was lower (50%) in winter (winter-autumn, P < 0.05; winter-spring, P < 0.001). Lastly, transuterine migration in cattle, when transferring multiple embryos, commenced at Day 14 (4%) and had occurred in all recipients by Day 16 (38% of embryos found contralaterally).     

Regression and resurgence of the CL following PGF2alpha treatment 3 days after ovulation in mares

The present study was designed to characterize and compare the physiology and ultrasonographic morphology of the corpus luteum (CL) during regression and resurgence following a single dose of native prostaglandin F2alpha (PGF) given 3 days after ovulation, with a more conventional treatment given 10 days after ovulation. On the day of pre-treatment ovulation (Day 0), horse mares were randomly assigned to receive PGF (Lutalyse; 10 mg/mare, i.m.) on Day 3 (17 mares) or Day 10 (17 mares). Beginning on either Days 3 or 10, follicle and CL data and blood samples were collected daily until post-treatment ovulation. Functional and structural regression of the CL in response to PGF treatment were similar in both the Day 3 and 10 groups, as indicated by an abrupt decrease in circulating concentrations of progesterone, decrease in luteal gland diameter and increase in luteal tissue echogenicity. As a result, the mean +/- S.E.M. interovulatory interval was shorter (P < 0.0001) in the Day 3 group (13.2 +/- 0.9 days) than in the Day 10 group (19.2 +/- 0.7 days). Within the Day 3 group, functional resurgence of the CL was detected in 75% of the mares (12 of 16) beginning 3 days after PGF treatment, as indicated by transient major (6 mares) and minor (6 mares) increases (P < 0.05 and < 0.1, respectively) in progesterone. Correspondingly, mean length of the interovulatory interval was longer (P < 0.03) in mares with major resurgence (15.8 +/- 1.6 days) than in mares with minor (11.2 +/- 1.2 days) and no resurgences (13.5 +/- 0.3 days) in progesterone. Structural resurgence of the CL in the Day 3 group and functional and structural resurgence in the Day 10 group were not detected. In conclusion, PGF treatment 3 days after ovulation resulted in structural and functional regression of the CL and hastened the interval to the next ovulation, despite post-treatment resurgences in progesterone.     

Steroid hormones in uterine washings and in plasma of gilts between days 9 and 15 after oestrus and between days 9 and 15 after coitus

Between Days 9 and 15 after oestrus, concentrations of pregnenolone, pregnenolone sulphate, dehydroepiandrosterone (DHEA), DHEA sulphate, androstenedione, oestrone and oestrone sulphate in free uterine fluid collected from non-pregnant gilts were greater than respective values in plasma (P less than 0.05). The total contents of pregnenolone, progesterone, DHEA, testosterone, oestrone and oestradiol in washings from pregnant uteri exceeded (P less than 0.05) respective non-pregnancy levels during this same period. Concentrations of pregnenolone, pregnenolone sulphate, DHEA, DHEA sulphate, androstenedione, oestrone, oestrone sulphate and oestradiol in free uterine fluid recovered from gravid uteri were also higher (P less than 0.05) than respective plasma values. By contrast, the progesterone concentration in uterine fluid from pregnant animals was lower (P less than 0.001) than the plasma value. Concentrations of DHEA, DHEA sulphate, androstenedione and oestrone sulphate in plasma of pregnant gilts between Days 9 and 15 after mating exceeded (P less than 0.05) the respective concentrations in unmated gilts between Days 9 and 15 after oestrus. Plasma levels of pregnenolone sulphate were lower (P less than 0.05) in the pregnant animals. We therefore suggest that the endometrium of the pig can concentrate steroid hormones in uterine fluid and that increases in steroid levels in this milieu between Days 9 and 15 after coitus reflect steroidogenesis by embryonic tissues and modification of enzyme activities within uterine tissues under the influence of progestagens. The pool of steroid sulphoconjugates present in uterine fluid between Days 9 and 15 post coitum could serve as an important precursor source for progestagen, androgen and oestrogen synthesis by tissues of pig embryos before implantation.     

Microscopic examination of porcine conceptus-maternal interface between days 10 and 19 of pregnancy

Conceptus-maternal interactions in the pig were examined at days 10, 13, 16, and 19 of pregnancy. Contact between the chorion and uterine epithelium was not preserved on day 10, but extensive contact occurred in 3 of 5 pregnant gilts on day 13 and was related to localized differentiation of the uterine epithelium. Attachment events occurred earlier and more rapidly than previously reported, commencing in the region of the embryonic disc and progressing toward the extremities of the chorionic vesicle, with stages from apposition through attachment occurring simultaneously along a conceptus at day 13 and thereafter. Apical protuberances on maternal epithelial cells and interposing blunt chorionic processes were evident at sites of early maternal-conceptus interaction, but were reduced at regions exhibiting microvillous interdigitation. Placentation in the pig represents a developmental process consisting of a continuum of sequential events which occur over a broad time frame rather than at specific discrete times.     

Scanning electron microscopy of the equine oviduct and observations on ciliary currents in vitro at day 2 after ovulation

There are considerable differences between mammalian species in the distribution and activity of ciliated cells within the oviduct, and limited information is available concerning either the distribution or activity of cilia within the equine oviduct. Patterns of ciliary activity were characterized in the ampulla and isthmus of oviducts recovered at 2 d after ovulation from 10 mares, and scanning electron microscopy was used to examine regional differences in the distribution of cilia in oviducts from 3 of these mares. Based upon the motility of 15 microm latex microspheres, ciliary activity was significantly (P < 0.001) greater in the ampullar oviduct compared with that of the isthmic oviduct. The direction of ciliary beat was consistently toward the uterus in all regions of the oviduct. Scanning electron microscopy revealed ciliated and secretory cells in both regions of the oviduct at 2 d after ovulation, with no apparent differences in the proportion of ciliated versus secretory cells.     

Discrepancies between Aedes aegypti identification in the field and in the laboratory after collection with a sticky trap

Currently, sticky traps are regularly employed to assist in the surveillanceof Aedes aegypti infestation. We tested two alternativeprocedures for specimen identification performed by local health agents: directly inthe field, as recommended by certain manufacturers, or after transportation to thelaboratory. A total of 384 sticky traps (MosquiTRAP) were monitored monthly duringone year in four geographically representative Brazilian municipalities. When thesame samples were inspected in the field and in the laboratory, large differenceswere noted in the total number of mosquitoes recorded and in the number of specimensidentified as Ae. aegypti by both procedures. Although fieldidentification has the potential to speed vector surveillance, these results point touncertainties in the evaluated protocol.     

Morphological characteristics of the kidney and lung in the neonatal rats observed after 16 days spaceflight.

The aim of this study is to examine the structural development in kidney and lung macroscopically which relate with cardiovascular system in rats raised in space. Twenty three nine-day old rats and six fifteen-day old rats, which were launched at these ages and nursed by their dams in the Space Shuttle Colombia for 16 days (STS-90; Neurolab). Seventeen animals of the nine-day old rats were defined as the nine-day group, and the rest was defined as the re-adaptation group, which were reared on the ground for 30 more days after landing. The organs were weighed and the ratio of the organ weight to the body weight (body weight ratio) was calculated. Both of lung and kidney in flight rats were significantly heavier than ground controls in the body weight ratio. We found that the kidney in the nine-day and the fifteen-day group tended to extend of dorsal-ventral length in macroscopic observations. However, this difference was not observed in the re-adaptation group. These results suggest that space environment may affect in kidney development. On the other hand, the lung had no differences in macroscopic structure among flight and control groups.     

Source separation,selective collection and in reactor digestion of biowaste

Biowaste or the organic fraction of domestic waste, for instance kitchen, fruit and garden waste, is collected selectively in several European communities. The complementary fraction is called the dry or non recyclable fraction. A Dutch study reported that 92% of the participants that have a weekly collection service of both fractions (biowaste and non recyclable fraction) and 80% of the participants in the alternating collection program (one week biowaste and the next week non recyclable fraction) are pleased with separate collection of biowaste. Dominating problems that arise in case of alternating collection are a repulsive odor and an infestation with flies and maggots. By expanding the definition of biowaste to include non recyclable or soiled paper like dirty newspapers, table napkins and paper handkerchiefs, most of these problems can be overcome without changing the way compostable waste is collected and processed. The expanded definition of biowaste was used in this paper. Over a 12 month period a quality survey of the collected biowaste was conducted by the composting facility Intercompost, Hoeselt, Belgium. A special aspect was the fact that in one participating community baby diapers were included in the soiled paper fraction; this is called biowaste+. The biowaste+ had a 10% non recyclable paper fraction opposed to only 1–2% of non recyclable paper present in the conventional biowaste. Baby diapers were a rather notable part (more than 80%) of this non recyclable paper fraction of biowaste+ and as a consequence might contribute to a large extent to improve the collection and treatment of biowaste. It was demonstrated that rural districts yielded about 35% more biowaste than more urban districts; resp. ± 122 kg biowaste/capita.year versus ±90 kg biowaste/capita.year. In Hoeselt the biowaste+ yield was about 130 kg/capita.year. Biowaste+ is also separately collected in another Belgium community, namely Brecht. The purity level of the biowaste+ and the amount of non recyclable paper (including diapers) were comparable with the results of Hoeselt. In Hoeselt the biowaste+ fraction is composted aerobically. In Brecht on the other hand, the biowaste+ is processed using the Dry Anaerobic Composting process (DRANCO). The latter process is discussed in more detail. The biological start-up of the dry anaerobic composting installation at Brecht, Belgium, is reported. The reactor has a total volume of 808 m³ and a design capacity of 730 m³. After 2 months of start up, the fermentor was at full loading rate, i.e. 8 kg bVS/m³ _reactor .day and the installation was working at full capacity, i.e. 40 ton/day. After 1 year of full-scale operation, the installation can handle peak loads of 60 ton/day. The average biogas production is 107 m³/ton with an average methane content of 55%. This corresponds to an annual gross energy production of approximately 600 toe (ton oil equivalent). The average biogas production rate is 4.0 m³/m³ _reactor .day. The end product of the aerobic process in Hoeselt and the end product in Brecht, the Humotex, are of similar composition. Both are of high quality and are sold as a soil conditioner or as a potting substrate. However, the double processed compost has a garanteed hygienic quality which makes it a product suitable for export.     

DNA isolation from museum and type collection slides of microsporidia

DNA from 19 species of microsporidia was isolated and amplified from infected host tissue that were originally prepared between the years 1946 and 1996. The smears, on glass microscope slides, were either Giemsa-stained or unstained. Methanol-fixed, Giemsa-stained smears proved to be suitable for DNA isolation; DNA was amplified from only two of 14 unstained slides. The isolated DNA was successfully amplified in PCRs using small subunit and large subunit rDNA primers and sequenced. The high efficiency of DNA isolation demonstrates the usefulness of archival and type collection slides for some molecular biology and molecular taxonomy studies.     

Effects of reduced suckling on days to estrus, conception during lactation and embryo survival in sows

Trials to investigate the effects of limited suckling on sow reproduction and piglet growth were conducted using 41 first parity and 32 second parity Yorkshire sows. Separation of sows from their litters (22 hrs/day, days 21-35 postpartum) induced estrus in 60% of primiparous and 72% of second parity sows during lactation. Compared to control group animals, primiparous sows had higher weaning weights and second parity sows higher rates of embryo survival on day 30 of gestation. Piglets subjected to suckling restriction had weights, at 2 weeks after weaning, equal to those weaned after 5 weeks of unrestricted suckling. We conclude that suckling restriction can provide the dual benefits of an extended piglet nursing period and a decreased breeding to breeding interval in sows.     

Evaluation of filter paper collection of urine samples for detection and measurement of organic acidurias by capillary electrophoresis

There is little doubt that mental retardation has been prevented in most babies diagnosed by newborn screening programs for inborn errors and the cost-benefit ratios of these programs have been reported as highly positive. In a previous work we optimised a CE method for quick profiling of organic acidurias, which characterize a large number of inborn errors, so that it permits the separation, detection and even identification in less than 15 min of 22 organic acids in urine samples related to a wide range of metabolic disorders. In the present work we have studied the adequacy of filter paper collection of urine samples to simplify this step, always difficult in babies, when it is not performed by training personnel. The studied parameters were: media and conditions for re-extraction to give the best sensitivity and a more simple procedure when the samples are measured by CE, interferences coming from the diaper, recoveries obtained, possible correction of recoveries with creatinine and stability of the compounds. The whole method we report has the advantages of easy sample collection, easy shipping or delivery, and rapid analysis. Moreover, this method of collection and analysis allows the identification and quantitation of fumaric, methylmalonic, N-acetylaspartic, pyroglutamic and homogentisic acids, as well as glutaric acid for which screening is considered especially advisable.     

Assessing risks of Wolbachia DNA cross-specimen contamination following mass collection and ethanol storage

Wolbachia and other intracellular bacteria that manipulate reproduction are widespread and can have major consequences on the ecology and evolution of their hosts. Several studies have attempted to assess the host range of these bacteria based on polymerase chain reaction assays on material preserved and collected using a variety of methods. While collecting in the field, mass storage in ethanol before sorting specimens in the laboratory is by far the easiest technique, and an integral component of Malaise trapping. This implicitly relies on the assumption that mass ethanol storage does not produce cross-contamination of Wolbachia DNA among specimens. Here we test this assumption. The absence of cross contamination between known positive and negative samples stored within a vial indicate there is no reason to believe collective storage of specimens creates artefactual increases in the incidence of Wolbachia or other intracellular bacteria.     

The effect of nocturnal sampling on semen quality and the efficiency of collection in bovine species

This study evaluated night and day semen collection regimes in Holstein and Brahman bulls (four bulls of each breed) that were collected weekly, each during a morning and a night collection. Ejaculates (n=64) were obtained via artificial vagina over 4 weeks. The first collection of each week alternated between night and day. Two collection teams were employed. Bull behavior parameters included reaction time to first mount, time to ejaculation, a refractory period test, and a thrust intensity test. The numbers of interruptions were counted as a managerial parameter. Pre-freeze semen parameters included total volume, initial motility and concentration. Post-freeze semen parameters measured were: 0- and 3-h post-thaw motility; percent intact acrosomes; and percent sperm abnormalities. Data were analyzed by least squares methods. The bull within breed effect differed (P<0.05) for behavior parameters. The bull within breed effect for total motile sperm harvested was not significant. The bull within breed response was mixed for post-freeze semen viability parameters. Bull within breed was not significant for sperm abnormalities. The night versus day treatment was significant for the managerial parameter (P=0.002). Although a different collection schedule for Bos indicus cattle was not warranted, the efficiency of the collection process was affected by extraneous environmental conditions.