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Biomedical subjects

K J Betteridge

Publications and source records attributed to K J Betteridge.

At least 55 records · Page 3Linked to original sources
PubMed1991

The sex ratios of bovine embryos produced in vivo and in vitro.

The male:female ratio of developing bovine embryos produced and allowed to develop in vitro and in vivo was determined retrospectively from the cytogenetic analysis of 804 embyos. The overall male:female ratio of the 307 (38%) embryos that could be sexed was 162 (52.8%):145 (47.2%) and did not differ (P>0.05) from the expected 1:1 ratio. Among premorula stage embryos produced in vivo (n = 66) and in vitro (n = 30), the ratios were 1.2:1 and 0.76:1, respectively. Among morulae and blastocysts produced in vivo (n = 74), produced and cultured in vitro (n = 106, and produced in vitro and cultured in vivo (n = 31), the ratios were 1.11:1, 1.3:1 and 0.94:1, respectively, none of which differed significantly from 1:1. There was no difference (P>0.05) in the number of cells or mitotic index between male and female morulae and blastocyst, respectively.

Journal Article↗
PubMed1991

Fertilizing capacity of bovine sperm may be maintained by binding of oviductal epithelial cells.

The ability of the bovine oviduct to maintain the motility and fertilizing capacity of bovine sperm was investigated by incubating frozen-thawed sperm with endosalpingeal epithelial cells cultured on either tissue culture plastic (nonpolarizing) or Matrigel-coated Millicell (polarizing) substrata. Sperm were also incubated in medium alone or with cultured bovine tracheal epithelial cells. Motility was determined at 6-h intervals over a 48-h period. The fertilizing capacity of sperm was evaluated after 0, 24, and 30 h of incubation by adding oocytes to the culture and determining the incidences of fertilization and polyspermy. Motility was maintained for 48 h in sperm that bound to endosalpingeal epithelial cells, but to a greater extent with polarized cells (38.4% motile) than with nonpolarized cells (0.8%). Fertilizing capacity was maintained for 30 h in sperm incubated with endosalpingeal epithelial cells on Matrigel/Millicell, but not in sperm incubated in medium alone or with tracheal cells. Only sperm incubated with oviductal cells developed hyperactivated motility. Scanning electron micrographs revealed that sperm were bound by the rostral portion of the intact acrosome to the apical surface of polarized endosalpingeal cells. These results suggest that the oviduct may not only store sperm but may also maintain sperm viability and fertilizing capacity during the preovulatory period.

Animals↗
PubMed1991

Absence of oxytocin-neurophysin messenger RNA in the day-18 bovine conceptus.

Total cellular RNA was isolated from conceptus tissue obtained from 22 superovulated cows 18 days after artificial insemination. Total RNA was also isolated from luteal tissue from 3 cyclic cows 7 and 8 days after oestrus. Luteal and conceptus RNA were simultaneously subjected to formaldehyde-agarose gel electrophoresis and transferred to nitrocellulose by bidirectional diffusion blotting. Northern blots were probed using cDNAs specific for bovine oxytocin and bovine beta-actin gene sequences. Hybridization of the oxytocin cDNA to RNA was consistently observed on autoradiographs as a 0.6 kilobase (kb) band in lanes containing corpus luteum RNA, but was not detected in lanes containing conceptus RNA. The presence of conceptus RNA on the blots was confirmed by hybridization of the actin cDNA to conceptus RNA, which resulted in a 2.0 kb band on autoradiographs. These results suggest that oxytocin is not synthesized by the bovine conceptus on Day 18 of gestation.

Actins↗
PubMed1991

Failure to detect platelet-activating factor using the splenectomized mouse bioassay.

The ability of purified preparations of platelet-activating factor (PAF), from three different suppliers, to induce thrombocytopaenia in mice after splenectomy and to activate mouse platelets in vitro was examined. Although the PAF preparations were potent activators of horse and cow platelets in vitro, injections of up to 1 microgram PAF failed to elicit thrombocytopaenia responses in either CD1 or Swiss Webster random-bred mice. However, when thrombin was injected into Swiss Webster mice, a dose-dependent decrease in the concentration of platelets was observed. Furthermore, isolated platelets from these strains and from 3 inbred lines (C3H/He, BALB/c, C57BL/6) of mice, were not aggregated by PAF in vitro but were sensitive to adenosine diphosphate and thrombin. No change in circulating platelet concentrations was observed over the initial 7 days of gestation in intact Swiss Webster and C57BL/6 or splenectomized C57BL/6 mice, suggesting either an absence of PAF production during early pregnancy in these strains or insensitivity of their platelets to PAF. These results suggest that many mouse strains are unsuitable for the bioassay of PAF.

Adenosine Diphosphate↗
PubMed1991

Glucose and glutamine metabolism in pre-attachment cattle embryos in relation to sex and stage of development.

Individual Day-7 embryos (morulae to expanded blastocysts) were incubated with radiolabelled substrates and karyotyped to determine the sex. In Exp. 1, embryos were incubated for 3 h with D-[1-14C]glucose, as a measure of the activity of the pentose-phosphate pathway (PPP) and D-[5-3H]glucose, as a measure of total glucose metabolism. The labelled products 14CO2 and 3H2O were collected throughout the measurement period. Total glucose metabolism in male embryos was twice that in female embryos and increased between the morula and expanded-blastocyst stages. Relative to total glucose metabolism, PPP activity was four times greater in female than in male embryos. In Exp. 2, embryos were cultured with D-[1-14C]glucose, and L-[3,4-3H(N)]glutamine (a measure of Krebs cycle activity) in the presence of brilliant cresyl blue, a stimulator of the PPP. Glutamine metabolism increased from the morula to expanded-blastocyst stages. Relative to the metabolism of glutamine, the activity of the PPP was one-third greater in female than in male embryos.

Animals↗
PubMed1991

Non-invasive assessment of the incidences of pregnancy and pregnancy loss in the feral horses of Sable Island.

Field observations of 400 totally unmanaged feral horses on Sable Island, Nova Scotia, were complemented by oestrogen determinations in faecal samples from 154 identified females over a 4-year period (454 mare-years). Of mares that were sampled throughout the year and subsequently produced foals, 92.1% exhibited elevated faecal oestrogens between 15 October and 30 March. The results confirm that faecal oestrogens are a useful indicator of pregnancy after approximately 120 days gestation. Distribution of foaling resembled that seen in other feral populations, with 95% of births occurring from April through July. The foaling rate for mares aged 3 years or older was 62.0%, with 50.7% of mares foaling in 3 or 4 years. Foaling rates were low (4.1%) in mares bred as yearlings and rose with age to 70.8% in those bred as 4-year-olds. Fetal loss after Day 120 was deduced from faecal oestrogens to be 26.0% overall, with marked variation from year to year (9.6-37.3%) and with age (70.0% in those bred as yearlings, decreasing to 5.6% in those bred as 4-year-olds). Of 58 mares aged 2 years or older that were sampled every year, about half (49.6%) the barren years were attributable to fetal loss after 120 days gestation. All mares conceived in at least 2 of the 4 years, suggesting that pregnancy loss, even after Day 120, is as important as failure to conceive in causing barren years.

Abortion, Veterinary↗
PubMed1990

Production of chimaeric bovine embryos and calves by aggregation of inner cell masses with morulae.

Bovine inner cell masses (ICMs) were isolated by immunosurgery at day 8 or 10, or by dissection at day 14, and combined with day-5.5 morulae. Aggregation was obtained between 89%, 62%, and 0% of the day-5:day-8, day-5:day-10, day-5:day-14 composites, respectively. Chromosome analysis of composites, respectively. Chromosome analysis of composites potentially carrying the 1/29 translocation as a chromosome marker and temporarily transferred to the bovine uterus for 8 days showed that chimaeric day-14 embryos can be obtained from day-5:day-8 aggregation. The definitive transfer of eight day-5:day-8 and 11 day-5:day-10 composites resulted in the birth of six and four calves, respectively; five of the six, but none of the four, were chimaeric. The five chimaeras showed mostly the ICM phenotype. The morphological differences between ICMs at different stages of development were examined by electron microscopy and related to the success of the aggregation technique. It is concluded that bovine embryonic cells can regulate for at least 3 days difference in development but not 5 days even though aggregation is still possible.

Animals↗
PubMed1988

Effects of the zona pellucida, agar embedding, and culture on the survival of micromanipulated bovine embryos after freezing and thawing.

Three experiments were undertaken to compare how the integrity of the zona pellucida, embedding in agar, and incubation in the sheep oviduct, respectively, affected the survival of bovine embryos after micromanipulation, freezing, and thawing. In Experiment 1, the proportions of whole day 7 embryos that continued their development in vitro after being frozen without a zona pellucida (N = 18), or with the zona pellucida opened over one-half (N = 14), or one-fourth (N = 14) of its circumference, or left intact (N = 17) did not differ significantly (44, 43, 36, and 59%, respectively). Experiment 2 compared the survival of bisected day 7 embryos frozen with or without embedding in agar cylinders. In vitro, one of eight half-embryos (17%) survived in each group. In vivo, the survival rate was 0% (N = 18) after freezing half-embryos in an agar cylinder and (11%) (N = 18) for halves frozen without agar. In Experiment 3, embryos were bisected on day 5 or 6, embedded in agar, then cultured for 2 days or 1 day, respectively, in the sheep oviduct before freezing them on day 7. Of the 54 half-embryos transferred to the sheep oviducts, 27 (50%) were recovered, frozen, thawed, and transferred to recipients, resulting in the birth of 17 calves (63%), of which six pairs were identical twins. This survival rate was significantly higher than for control embryos collected at day 7, bisected, and frozen without agar embedding (16%; N = 12).(ABSTRACT TRUNCATED AT 250 WORDS)

Agar↗
PubMed1988

Transcervical collection of equine conceptuses between 10 and 16 days after ovulation.

To recover intact Day-10.5 to Day-16.5 equine conceptuses (Day 0 = ovulation), a rigid catheter was used for 131 collections from donor mares diagnosed pregnant by ultrasonography. A total of 139 conceptuses were recovered, comprising 124 singletons, six pairs of twins and one set of triplets. Of these, 120 (86%) were intact after the collection, 14 (10%) had collapsed, and in five cases (4%), collapsed trophoblastic membranes were surrounded by an intact capsule. The recovery rate of intact conceptuses ranged from 99% on Days 10.5 to 12.5 to 40% on Day 16.5. More uterine flushes per recovery were needed to collect conceptuses on Day 14.5 than on Days 10.5 and 11.5 (x +/- SEM : 3.1 +/- 0.5 vs 1.4 +/- 0.1 and 1.3 +/- 0.2 flushes, respectively, P<0.05), and the total volume of flushing medium used was greater on Day 14.5 than on Days 10.5, 11.5 and 12.5 (1040 +/- 193 vs 406 +/- 49, 396 +/- 48 and 499 +/- 59 ml, respectively, P<0.01). Seventy of the 100 mares inseminated at the first estrus following embryo collection became pregnant, indicating that the technique used had no major effect on subsequent fertility.

Journal Article↗
PubMed1988

Assessment of immunoregulation by cultured, pre-attachment bovine embryos.

The possibility that the viability of bovine embryos might be predicted by measuring their release of immunoregulatory substances during culture has been investigated. Bovine embryos between days 2 and 19 of gestation were cultured for 24-48 h, the embryo-conditioned medium was harvested and studied for suppression of PHA-stimulated bovine leukocyte cultures. Medium incubated in the absence of any conditioning tissues served as control. Artefactual immunosuppression was detected in incubated control material that could be attributed, in part, to the mixing of different tissue culture media, the type of plastic-ware employed for incubation and supplementation of media with additional L-glutamine. It was observed that day-2 to day-9 bovine embryos, cultured in medium able to support the lymphocyte proliferation assay, did not release immunosuppressive substances. Medium conditioned by day-10 to day-12 embryos produced variable immunosuppression while that conditioned by trophoblastic vesicles derived from day-14 to day-19 embryos was consistently highly suppressive. Since bovine embryo transfer is normally conducted at 6-8 days of gestation, it is unlikely that measuring the immunosuppressive products released from bovine embryos will be of value for predicting their viability.

Animals↗
PubMed1988

Nucleolus organizer regions and nucleoli in preattachment bovine embryos.

Embryos (1-cell to elongated blastocyst stage) were recovered from superovulated heifers at surgery (Days 2-4; oestrus = Day 0), after slaughter (Day 4), or by transcervical flushing (Days 6, 7 and 14). The 175 embryos were cultured for 4, 8, 24 or 48 h, fixed on slides and sequentially stained with Giemsa and silver nitrate. Twenty-three 2-cell to blastocyst-stage embryos were fixed, embedded and examined by transmission electron microscopy. Argentophilic nucleolus organizer regions (Ag-NORs), indicative of transcriptionally active rRNA genes, were observed in embryos in which short- or long-term culture began at or after the late 8-cell stage. The nucleoli of embryonic cells also showed increased affinity for silver from the 8-cell stage onward. Differences in the number of Ag-NORs observed after the 8-cell stage reached statistical significance only when Day-5 and Day-7 embryos cultured for 4 h were compared. Ultrastructurally, the nucleoli were seen to develop from small, dense, fibrillar masses at the 2-cell stage, to ring-shaped structures (signifying a low level of activity) at the 8-cell stage. At the 16-cell stage the nucleoli became reticulated, suggesting an increase in activity, and by the morula and blastocyst stages they were characteristic of fully active nucleoli. It is concluded that a significant transcriptional activity of the rRNA genes in the embryos of cattle begins around the 8-cell stage.

Animals↗
PubMed1987

Equine zona pellucida and capsule: some physicochemical and antigenic properties.

The capsule which surrounds the pre-attachment equine embryo has been compared with the zona pellucida (zp) that it replaces, as well as with the rabbit blastocyst coverings, by means of physicochemical and immunological methods. Trypsin solution at pH varying between 7.5 and 9.0 completely solubilized the capsule, as did Na borohydride. However, solutions of pH 2.0 or 12.0, urea, high temperature (65 degrees C, 60 min or 80 degrees C, 30 min), mercaptoethanol and dithiothreitol were able to solubilize the zp but not the capsule at the concentrations used. Indirect immunofluorescence on cryostat sections and whole mounts of fresh or frozen-thawed material showed that 1) common antigens are shared by equine, porcine and bovine zp; 2) day 7 to day 15.5 capsule reacted with anti-capsule-serum but not with anti-zp-serum except for a few patches on the surface of the capsule; 3) anti-capsule-serum, but not anti-zp-serum, reacted with the capsular material recovered along with a broken day 27.5 conceptus; 4) anti-capsule-serum does not react with rabbit blastocyst coverings; 5) anti-capsule antibodies can be absorbed from the anti-capsule-serum by uterine proteins from either pregnant or non-pregnant mares; and 6) the capsule does not contain mouse laminin-like material.

Animals↗
PubMed1987

The meiotic stage of preovulatory oocytes in mares.

Confusion exists as to whether the oocytes of the domestic horse are ovulated at the first meiotic metaphase (MI) or the second (MII). In this study eight oocytes were collected from the preovulatory follicles of 16 mares 36 h after human chorionic gonadotropin CG treatment. Six of the eight oocytes were judged to be at MII by the presence of the first polar body and this judgement was confirmed by semithin sectioning in one. Of the two that had no polar body, one was found to be at MII after fixation for chromosomal analysis and the meiotic stage of the other remained undetermined. Since all seven oocytes yielding conclusive evidence were at MII, it was concluded that horse oocytes, like those of most mammals studied, are ovulated after completion of the first meiotic division and formation of the first polar body.

Animals↗
PubMed1987

Anatomy of the reproductive tract of the female muskox (Ovibos moschatus).

Reproductive tracts from 23 female muskoxen were collected from the Canadian high arctic during annual native muskox hunts. Twenty tracts were collected during the late breeding season and the last third of pregnancy or anoestrus, with 3 additional specimens taken just before the breeding season in August. The non-gravid muskox uterus was very similar to that of sheep and goats except for two features found in parous muskoxen. The first was endometrial pigmentation found only in the caruncles and associated with a dense collection of lipofuscin granules in the endometrial stroma. The second was a prominent 5 mm deep band of muscular tissue protruding from the antimesometrial border of the uterine horns throughout most of their length. The pregnant uterus and the fetal membranes of the muskox resembled homologous structures in domestic ruminants. However, there was no morphological evidence of a corpus luteum during late pregnancy, apart from a luteal scar in the ovary ipsilateral to the pregnant horn. Of the 4 females collected at the end of the breeding season, 2 lactating females were apparently not cyclic while 2 others had more than one CL, suggesting that they had undergone at least 2 cycles without conceiving or remaining pregnant.

Animals↗
PubMed1987

Analysis of X-chromosome inactivation in horse embryos.

To define the time of X-chromosome inactivation in the horse, 122 conceptuses were collected transcervically between Days 6 and 28 (ovulation = Day 0) and subjected to cytogenetic analysis: 59 of the embryos were divided and in 41 of these separate cytogenetic analysis of the embryonic disc and remaining tissues was possible. Conceptuses were measured and photographed before capsule removal, culture in the presence of 5-bromodeoxyuridine and subsequent fixation for cytogenetic analysis. On average, 15 slides were prepared per conceptus. C-banding was used to determine the sex of each conceptus and, in the females, the effects of 5-bromodeoxyuridine incorporation were revealed by u.v. irradiation which produced R-bands and allowed identification of the inactive X-chromosome. Inactivation of the X chromosome began gradually in the trophoblastic (+/- attached endoderm and mesoderm) cells around Day 7.5 and in the embryonic disc around Day 11.5. Cells with an inactive X-chromosome predominated in the trophoblast by Day 10.5 and in the disc by Day 12.5.

Animals↗
PubMed1987

Steroid secretion by different cell types of the horse conceptus.

Horse conceptuses were recovered non-surgically at Day 12-Day 15 and were dissociated with collagenase. Separation of the cells on a 31.8% Percoll gradient gave two bands of cells and indirect evidence suggests that the low density cells (LDC) are endoderm and the higher density cells (HDC) are trophectoderm. Each band was incubated for 24 h in Minimum Essential Medium and concentrations of oestradiol and progesterone in the medium were measured by radioimmunoassay (RIA). The LDC secreted predominately progesterone (log oestradiol/progesterone = -0.994 +/- 0.141; N = 15) whereas the HDC secreted mainly oestradiol (log oestradiol/progesterone = 0.522 +/- 0.135). When incubated for a further 24 h in fresh medium the oestradiol production decreased by 85% and 84% whereas the progesterone production decreased by 57% and 50% for LDC and HDC respectively. When cells from each band were incubated for 24 h with [3H]pregnenolone, separation of the radiolabelled metabolites by reversed-phase high-pressure liquid chromatography revealed a different steroid profile for each cell type. Therefore, in the intact embryo, an interaction between cells may be important for oestrogen synthesis and thus the maintenance of pregnancy.

Androstenedione↗