In vitro culture and interferon-tau secretion by ovine blastocysts.

Embryos were collected surgically from superovulated ewes on days 7, 8, 9 and 10 (oestrus=day 0) to evaluate the long-term culture and interferon-tau (IFN-tau) secretion of ovine blastocysts. Embryos were cultured in 2 ml Dulbecco's modification of Eagle's medium (DMEM) supplemented with 15 mg/ml BSA in 5% CO(2) in air or DMEM without BSA in 5% CO(2), 7% O(2), and 88% N(2) at 39 degrees C, examined daily for morphological features and diameter and each day placed into fresh culture medium to enable daily measurement of IFN-tau secretion. Nine day-7 and two day-9 embryos were cultured in DMEM with BSA and nine continued to develop. The day-7 embryos reached a mean maximum diameter of 370.0+/-50.25 microm after 4 days in culture. Nineteen day-7, 12 day-8 and five day-10 embryos were cultured in DMEM without BSA but only six of the day-7 and one day-8 embryos survived for at least 7 days with the former reaching a mean maximum diameter on day 7 of 357+/-43.75 microm whereas all five day-10 embryos survived for at least 7 days reaching a mean maximum diameter on day 6 of 1038+/-155.8 microm. An anti-viral assay and a ELISA for IFN-tau were developed. There was a considerable variation in the time of onset and amount of IFN-tau secreted that did not seem to be related to embryo morphology. Of 28 day-7 embryos cultured, 60.7% were secreting IFN-tau after 1 day of culture whereas 87.5% of day-8 embryos were secreting IFN-tau after 1 day in culture. The mean concentration of IFN-tau secreted by day-8 embryos after 1 day in culture (10.99+/-2.55 ng/ml) was not significantly different to day-7 embryos after 2 days in culture (8.8+/-1.75 ng/ml).

Induction of multiple chemokine and colony-stimulating factor genes in experimental Burkholderia pseudomallei infection.

Melioidosis is a disease of the tropics caused by the facultative intracellular bacterium Burkholderia pseudomallei. In human infection, increased levels of IFN-gamma in addition to the chemokines interferon-gamma-inducible protein 10 (IP-10) and monocyte interferon-gamma-inducible protein (Mig) have been demonstrated. However, the role of these and other chemokines in the pathogenesis of melioidosis remains unknown. Using BALB/c and C57BL/6 mice as models of the acute and chronic forms of human melioidosis, the induction of mRNA was assessed for various chemokines and CSF (G-CSF, M-CSF, GM-CSF, IP-10, Mig, RANTES, MCP-1, KC and MIP-2) in spleen and liver following B. pseudomallei infection. Patterns of chemokine and CSF induction were similar in liver and spleen; however, responses were typically greater in spleen, which reflected higher tissue bacterial loads. In BALB/c mice, high-level expression of mRNA for all chemokines and CSF investigated was demonstrated at day 3 postinfection, correlating with peak bacterial load and extensive infiltration of leucocytes. In contrast, increased mRNA expression and bacterial numbers in C57BL/6 mice were greatest between 4 and 14 days following infection. This paralleled increases in the size and number of abscesses in liver and spleen of C57BL/6 mice at days 3 and 14 postinfection. Earlier induction of cytokine-induced neutrophil chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) mRNA was demonstrated in spleen, while MIP-2, MCP-1, IP-10 and Mig were demonstrated in liver of BALB/c mice when compared to spleen and liver of C57BL/6. The magnitude of cellular responses observed in the tissue correlated with increased levels of the chemokines and CSF investigated, as well as bacterial load. Compared with C57BL/6 mice, greater infiltration of neutrophils was observed in liver and spleen of BALB/c mice at day 3. In contrast, early lesions in C57BL/6 mice predominantly comprised macrophages. These results suggest that the inability of BALB/c mice to contain the infection at sites of inflammation may underlie the susceptible phenotype of this mouse strain towards B. pseudomallei infection.

Non-invasive in vivo magnetic resonance imaging assessment of acute aflatoxin B1 hepatotoxicity in rats.

Acute aflatoxin B1 (AFB1)-induced hepatotoxicity was assessed in vivo in male Sprague-Dawley rats (150-300 g) using magnetic resonance imaging (MRI). MRI results were compared to serum enzyme levels, histology and electron microscopy. Twenty-four hours following intraperitoneal delivery of AFB1 (3 mg/kg body weight in a saline/dimethyl sulfoxide (DMSO; 0.03 ml/kg body weight) solution), regions of damage, characterised by increased proton signal intensities in T2-weighted images, were observed in the vicinity of the hepatic portal vein (HPV) and in the right medial regions of the liver. Image analysis of regions of apparent damage around the HPV and right medial regions, following 24 h of AFB1 exposure, indicated statistically significant (P<0.05) increases in proton image signal intensities, when compared to saline/DMSO-treated rats. No significant difference in proton image signal intensities were observed 1-2 h following AFB1 exposure. Twenty-four hours following AFB1 exposure, histopathological assessment was characterised by portal/central vein/artery congestion, sinusoid congestion, nuclear pyknosis and karyolysis, and hepatocyte vacuolation; electron microscopy (EM) examination indicated nuclear debris, swollen cytoplasmic compartments, vacuolation, and the disappearance of the smooth endoplasmic reticulum, and elevated levels of serum aspartate aminotransferase and alanine aminotransferase were found to be significantly different (P<0.01) than controls.

Effects of 17beta-oestradiol or oestrous stage-specific cow serum on the ability of bovine oviductal epithelial cell monolayers to prolong the viability of bull spermatozoa.

The effect of 17beta-oestradiol and oestrous stage-specific cow serum on bovine oviductal epithelial cell monolayers to extend the viability of co-cultured bull spermatozoa was examined. Monolayers of cells from ampullary and isthmic segments were pre-treated with medium containing either oestrous cow serum, luteal-phase cow serum, 1 microg/ml 17beta-oestradiol + foetal bovine serum or foetal bovine serum alone (control) before the addition of motile frozen/thawed spermatozoa. Motility was visually assessed throughout a 48 h co-incubation period, while fertilising ability of spermatozoa was evaluated by adding in vitro matured bovine oocytes. Pre-treatment with 17beta-oestradiol or oestrous cow serum resulted in a higher percentage of motile spermatozoa after 18 h in isthmic and after 36 h in ampullary cultures compared with the control, but pre-treatment did not affect fertilisation rates. Only at 42 h in ampullary cultures was motility higher in luteal serum pre-treated cultures compared to the control. Motility was also assessed in medium conditioned by pre-treated monolayers. Pre-treatment with 17beta-oestradiol enhanced the ability of conditioned medium to prolong motility and medium conditioned with oestrous cow serum was superior to medium conditioned by luteal-phase serum at maintaining motility. In conclusion, the ability of oviductal epithelium to prolong the motility of spermatozoa is enhanced by 17beta-oestradiol.

Effects of bovine oviductal proteins on bull spermatozoal function.

The effects of bovine oviductal proteins on bull sperm viability, acrosome reaction and motility were studied. Motile frozen/thawed spermatozoa from Percoll gradients were incubated with 1.0 mg/mL oviductal proteins (>8 kDa) extracted by ammonium sulphate precipitation from oviductal extract (OE) or serum-free oviductal epithelial cell-conditioned media (CM), treated in the presence (CM+) or absence (CM-) of 1 microg/mL 17beta-estradiol. Inclusion of oviductal proteins had a significant beneficial effect on sperm viability (76.3 to 80.6%+/-5.3) compared with the control (without oviductal proteins; 57.8%+/-5.3) immediately after the commencement of incubation. After 5 h, viability was significantly higher for CM- and OE treatments than for the control, although no differences were observed at 24 h. Acrosomal status only differed among treatments after 24 h, when higher percentages of acrosome- reacted spermatozoa were found in the control (46.0%+/-2.5) than in the oviductal protein treatments (33.1 to 38.2%+/-2.5). No differences in percentages of motile spermatozoa occurred within the first hour of incubation, although inclusion of CM proteins decreased sperm velocities, beat cross frequency, linearity, and straightness but increased values for mean angular displacement. These findings suggest that proteins secreted by oviductal epithelium promote viability, delay the acrosome reaction and suppress the motion of spermatozoa.

Reproductive performance of sows in the tropics.

A retrospective study was undertaken on 6 piggeries in north Australia and South Vietnam to define the reproductive performance of sows in the tropics. Reproductive performance was analysed in respect to age of first mating, time of the year, breed and management practices. There was a significant effect of the time of the year at mating on farrowing rate. Highest farrowing rates were achieved when sows were mated during the cool time of the year in the Australian piggeries and during the dry-hot period as opposed to the wet-hot period in Vietnamese piggeries. The number of matings per oestrus and interval between matings influenced both farrowing rate and number of piglets born although an increased litter size at birth was accompanied by an increase in stillbirths and mummified foetuses.

Requirement of inner cell mass for efficient chorionic gonadotrophin secretion by blastocysts of common marmosets (Callithrix jacchus).

The role of the inner cell mass in the induction of chorionic gonadotrophin synthesis and secretion by the trophoblast of the peri-implantation primate blastocyst was studied in common marmoset monkeys. An in vitro system for the culture of blastocysts commencing with blastocysts collected 8 days after conception was developed. Chorionic gonadotrophin measured in the spent culture fluid was first detected in most blastocysts after 3 or 4 days (day 11 or 12) of culture at a time equivalent to implantation in vitro. Initial secretion of chorionic gonadotrophin coincided with development of parietal endoderm and histological appearance of syncytiotrophoblast in the polar trophoblast. Little chorionic gonadotrophin was secreted by blastocysts with a poorly developed, or absent, inner cell mass. Mural trophoblast removed from blastocysts after 2 days of culture (day 10) grew in vitro as a unilaminar vesicle but failed to secrete significant amounts of chorionic gonadotrophin. However, mural trophoblast from older blastocysts (days 13 and 14) after chorionic gonadotrophin secretion had commenced continued to secrete chorionic gonadotrophin, with trophoblast from day 14 blastocysts secreting significantly more than that from day 13. It was concluded from these studies that while mural trophoblast from marmoset blastocysts will proliferate in vitro in the absence of an inner cell mass, efficient induction of chorionic gonadotrophin secretion requires the presence of the inner cell mass or its derivatives. Once chorionic gonadotrophin secretion has commenced, secretion will continue in the absence of the inner cell mass.

Changing responsiveness of luteal cells of the marmoset monkey (Callithrix jacchus) to luteotrophic and luteolytic agents during normal and conception cycles.

Dispersed marmoset luteal cells were incubated for 2 h and progesterone production measured after exposure to hCG, cloprostenol, dibutyryl cAMP, PGF-2 alpha, PGF-2, adrenaline or melatonin. The cells were studied on Days 6, 14 and 20 after ovulation in conception and non-conception cycles. Luteal cells from Day 14 non-pregnant marmosets were compared with human luteal cells taken in the mid-luteal phase. All the treatments stimulated progesterone production including cloprostenol, which is luteolytic when administered to the marmoset in vivo, but the degree of response varied with the stage of the cycle or pregnancy and between marmoset and human luteal cells. In the marmoset, overall analysis of the effect of the treatments showed that, on Day 6 after ovulation, there was no significant effect of any of the treatments in cells from pregnant or non-pregnant animals. In contrast, luteal cells from non-pregnant animals on Day 14 showed a significant response to the treatments (F (8,41) = 2.79, P less than 0.0145) whereas cells from pregnant Day-14 animals were responsive; in cells from pregnant animals, the control production of progesterone was high and already equivalent to the levels stimulated by the treatments. By Day 20, cells from pregnant animals produced lower control concentrations of progesterone than did those on Day 14 and there was a significant overall effect of the treatments (F (8,33) = 3.78, P less than 0.003). These results show that the marmoset CL gains responsiveness to treatment between Days 6 and 14 after ovulation in the non-pregnant cycle. In pregnancy, on Day 14, 2 days after attachment of the embryo, the high control concentrations of progesterone and absence of response to treatment suggest that an embryo message may have affected the CL, providing an endogenous stimulus.

Births following the transfer of cultured embryos obtained by in vitro and in vivo fertilization in the marmoset monkey (Callithrix jacchus).

The aim of this study was to determine whether marmoset monkeys are a suitable primate model for in vitro fertilization (IVF), embryo culture, and transplantation studies. A prostaglandin analogue given in early pregnancy and human chorionic gonadotropin given near the end of the ensuing follicular phase were used for controlling the reproductive cycle, timing oocyte collection, and synchronizing the cycles of oocyte donors and embryo recipients. Five embryos obtained by IVF were transferred at early stages to the uterus of three recipients, and two gave birth to live infants. Some of the embryos were cultured to advanced blastocyst stages. In vivo fertilized oocytes were also cultured and transferred to two recipients, and one gave birth. We concluded that the marmoset is one of the best primates for such investigations.

Quality management: program design. An interdisciplinary approach.

Concepts of quality management related to program design and implementation are relatively new to the health care field. The process used to satisfy the requirements of program evaluation, business planning, and clinical care quality assurance within the framework of quality management is discussed. Using an interdisciplinary approach, the authors trace the development of a specific rehabilitation program. Such programs can allow the health care provider to be more fruitfully involved in a health care service design beneficial to the patients served.

Normal in-vivo development of marmoset monkey embryos after trophectoderm biopsy.

A procedure was developed to remove trophectoderm cells from day-8 blastocysts of the marmoset monkey. Using micromanipulative techniques, a tear was made in the zona pellucida opposite the inner cell mass which facilitated the controlled herniation of trophectoderm cells as the blastocysts developed in vitro. After 24 h (day-9 blastocysts) and 48 h (day-10 blastocysts) of culture approximately 20% and approximately 50% respectively of the blastocyst had herniated. The herniated trophectoderm was cut off by freehand and the biopsied blastocysts transferred to recipients. Normal offspring were born but pregnancies could be established from day-10 blastocysts only if the recipients were treated with human chorionic gonadotrophin during early pregnancy. One pregnancy was established after the transfer of three frozen biopsied day-10 blastocysts. Biopsies of 30-50 cells from day-10 blastocysts could be readily grown in vitro as trophoblast vesicles to in excess of 1000 cells but biopsies of less than 20 cells from day-9 blastocysts formed a monolayer of binucleated and multinucleated cells with limited cell replication. Assuming human trophectoderm cells have a similar capacity to the marmoset to grow in vitro, the application of this technique to human blastocysts would provide sufficient cells on which the preimplantation diagnosis of a genetic disorder could be made.

Embryonic signals during the peri-implantation period in primates.

During the peri-implantation period of pregnancy in primates, chorionic gonadotrophin (CG) is the first clear signal of the embryo's presence and viability. In the marmoset (Callithrix jacchus) implantation begins on Day 11-12 after ovulation and CG is secreted by the embryo from this time. The inner cell mass is necessary for the normal secretion of CG by the trophoblast. Implantation can be disrupted both in vivo and in vitro by antisera to the hCG-beta subunit. The secretion of a platelet activating factor by the preimplantation embryo has yet to be confirmed, as has the physiological function of this and other preimplantation signals. Local sampling by perfusion of the corpus luteum allows a direct measurement of the interactions between luteotrophins and luteolysins, as well as a method of screening potential new agents for regulating the function of the corpus luteum.

Controlled ovulation in the marmoset monkey (Callithrix jacchus) with human chorionic gonadotropin following prostaglandin-induced luteal regression.

Controlled induction of ovulation in the marmoset monkey was attempted with a single injection of human chorionic gonadotropin (hCG; 50 IU) given on day 7 after prostaglandin-induced luteal regression. Animals given hCG (n = 12) ovulated within a 2-day period (days 9 and 10 after prostaglandin) compared with a 4-day period (days 9 to 12) in the control group (n = 12). The mean interval to ovulation was similar in both groups. There was no difference in the timing of the preovulatory estradiol (E2) peak between groups, although E2 levels on the day of hCG injection were lower than in controls on the day of the onset of the luteinizing hormone surge. All animals given hCG ovulated and 11 of 12 became pregnant. Ten of 11 embryos recovered surgically from six of these animals were normal blastocysts; 5 of the remaining 6 animals carried pregnancies to term. The results are of practical importance for experiments involving follicular and oocyte maturation and the collection and transfer of embryos.

Characteristics of trophoblastic tissue derived from in vitro culture of preimplantation embryos of the common marmoset monkey.

The features of trophoblastic tissue derived from the in vitro culture of marmoset monkey embryos have been described. Long-term trophoblast cultures (in excess of three years in one case) were established from the primary trophoblast monolayer of four of 38 embryos; division of one of these embryos produced two long-term cultures. The trophoblast cells retained their ability to synthesize and secrete chorionic gonadotrophin (CG) during maintenance in vitro and were capable of prolonging the luteal phase when transferred to the uterus of marmosets. A characteristic feature of the cultures was the formation of multiple fluid-filled vesicles enclosed by a single layer of cytotrophoblast cells and attached to the culture dish by a small monolayer of syncytiotrophoblast cells. The tissue was propagated by cutting vesicles into small pieces and placing into a fresh culture dish; attempts to subculture using single-cell suspensions were unsuccessful. These cultures provide a convenient source of marmoset CG for purification as well as an in vitro system for studying other secretory products of primate trophoblast.

Partial purification and characterization of chorionic gonadotrophin in plasma and in culture medium of trophoblast cells from the marmoset monkey (Callithrix jacchus).

A biologically active gonadotrophin has been purified from the media of long-term cultures of trophoblast cells of the common marmoset monkey by a combination of precipitation and chromatography. Marmoset chorionic gonadotrophin (CG) is a glycoprotein which binds Concanavalin A and wheat germ agglutinin. The protein purified from culture media exists as several isoelectric species with pI in the range pH 3.5-4.5. On gel filtration it eluted with an apparent molecular weight of 68-72,000 but on PAGE migrated as if it was 58-65,000. A glycoprotein with similar characteristics has been recovered from plasma samples of pregnant marmosets. Biological activity of partly purified CG from media, as determined by a mouse testicular cell bioassay, was 1-3 i.u./mg protein.

Successful transfer of the embryos of Przewalski's horses (Equus przewalskii) and Grant's zebra (E. burchelli) to domestic mares (E. caballus).

Blastocysts were collected non-surgically from 2 Przewalski's horse and 2 Grant's zebra mares and transferred extra-specifically to domestic horse and donkey recipients. Nine Przewalski's horse embryos were transferred surgically, and 2 non-surgically, to domestic Welsh-type pony mares. After surgical transfer, 7 (77.8%) pregnancies were established and 4 foals were born. Twelve Grant's zebra embryos were transferred surgically to 5 pony and 7 domestic donkey recipients respectively and 1 non-surgically to a donkey; 3 (60%) zebra-in-horse pregnancies were established and 2 went to term. Only 2 (28.6%) zebra-in-donkey pregnancies were established but neither went to term, although one zebra foal was aborted alive at Day 292 but failed to survive. No pregnancies resulted from the non-surgical transfers. Measurement of chorionic gonadotrophin concentrations and parental-specific lymphocytotoxic antibodies in the serum of the recipient animals indicated a pronounced maternal immunological response to the extra-specific embryo, but this could not be correlated with success or failure of pregnancy. The results indicate that extra-specific embryo transfer may be a useful aid to breeding exotic equids in captivity.