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.     

Protein production by cultures established from Day-14-16 sheep and pig conceptuses

Primary cell cultures were established from cells derived from dissociated Day-14 and -16 sheep and pig blastocysts. The appearance of cells in culture from both species was similar. Cultures contained a variety of cells with distinct morphologies, some were small and compact and formed clumps and multiple layers while others were large, flat and formed a monolayer. Within 4 h of culturing small floating fluid-filled spheres of cells were observed in the medium; some of these increased in size to greater than 1 cm diameter over 1-2 weeks. In addition, fluid-filled domes of cells arose from the underlying monolayer. Contractile cells became evident after about 8 days and some became organized into large patches of contracting tissue. Two-dimensional polyacrylamide gel electrophoresis and fluorography were performed on proteins released into the medium by confluent monolayers, floating spheres and floating cells that failed to attach during the first 24 h. All cultures produced as major products proteins with electrophoretic mobilities identical to certain fetal plasma proteins. In general, cultures did not produce proteins characteristic of short-term cultures of whole conceptuses harvested at Days 14-16. In cultures established from sheep blastocysts only the cells that failed to attach produced ovine trophoblast protein-1, a major polypeptide produced by the trophectoderm of the sheep conceptus between Days 13 and 21 of pregnancy.     

Ultrasonic detection of the conceptus and characterization of intrauterine fluid on days 10 to 22 in heifers

Nonbred and pregnant heifers were examined ultrasonically on alternate days from Days 10 to 22 in three experiments. The goal was to define criteria that could be used for identification of the early conceptus. The location and size of circular and elongated nonechongenic structure (apparently free intrauterine luminal fluid) were recorded (Experiment 1). On Day 12, there was a significantly greater number of circular nonechogenic structures in the ovarian half of the uterine horn ipsilateral to the corpus luteum in pregnant heifers than in nonbred heifers (means, 5.1 vs 3.5). Therefore, the embryonic vesicle was apparently detected on Day 12, but was not distinguished from circular nonechogenic structures representing free luminal fluid. The number of circular nonechogenic structures decreased after Day 14, with a corresponding increase in elongate nonechogenic structures associated with luteal regression (nonbred heifers) or elongation of the blastocyst (pregnant heifers). On Day 16, there were more elongated than circular nonechogenic structures in pregnant heifers than in nonbred heifers. In nonbred heifers, there was no evidence of a local utero-ovarian relationship between the amount of intrauterine luminal fluid and the location of either the corpus luteum or the next ovulatory follicle. Uterine luminal fluid in each horn increased several fold between Days 16 and 18 in nonbred heifers; there was significantly more lumina fluid in nonbred than in pregnant heifers on Days 18 and 20. In pregnant heifers, intrauterine luminal fluid area increased first in the ovarian end of the uterine horn ipsilateral to the corpus luteum; this was apparently due to detection of the elongating blastocyst. The length of the longest specular reflector on the endometrial surface of the uterine lumen increased over Days 10 to 22 in nonbred and pregnant heifers, but was not a useful criterion for detection of the conceptus (Experiment 2). The accuracy of a 7.5 MHz transducer for pregnancy diagnosis was not greater than a guess (50%) before Day 16 (Experiment 3). Results indicated that early pregnancy diagnosis in heifers was confounded by the presence of intrauterine fluid.     

Formation and characterization of vesicles from day-10 horse conceptuses

The objectives of this research were 1) to determine if cellular vesicles could be formed from Day-10 horse conceptuses similar to trophoblastic vesicles reported for other domestic species and 2) to characterize various aspects of their development in vitro and their ability to withstand the freeze-thaw process. Twenty-five conceptuses were recovered from lighthorse mares on Day 10 after ovulation. After two washes in 0.05% trypsin, each conceptus was placed in 0.25% trypsin until the capsule thinned. Mechanical dispersion with a glass pipette resulted in a combination of individual cells and cell clumps. When cultured in vitro, all preparations exhibited both partially and completely formed vesicles within 24 h. Cellular monolayers also developed within the first 24 h of culture and were predominant by 96 h. Within 13 to 20 d, all monolayers developed dense areas of cells that eventually released from the cell matrix and aggregated to form vesicles. Progesterone and total estrogen concentrations in media samples were lower (P < 0.05) for conceptuses that had required long trypsinization periods for dispersion. Pregnant mare serum gonadotropin was not detectable in any samples. Vesicles cultured in nontreated tissue culture flasks.doubled in size within 24 h but did not increase further by 48 h. Of 234 vesicles frozen after 48 h of culture, the postthaw viability, as measured by the ability to return to prefreeze characteristics, was 34, 21 and 11% after 24, 96 and 168 h in culture, respectively. We conclude that vesicles can be formed by enzymatic dispersion of Day-10 horse conceptuses. However, monolayers were the predominant result of both the initial dispersions and the long-term culture of vesicles. Vesicles showed a limited ability to grow in vitro.     

Prostaglandin production in the umbilical and uterine circulations in pregnant sheep at 129-136 days gestation.

Prostaglandins circulating in the maternal and foetal blood have been implicated in important physiological systems. These functions include foetal adrenal function, maintenance of patency of the ductus arteriosus, regulation of uterine and umbilical circulations, and labor and delivery type myometrial contractions. The placenta is a major site of prostaglandin production in pregnancy. Limited data are available which combine measurements of veno-arterial differences across the uterine and umbilical circulations with blood flow in these circulations to enable calculation of umbilical-placental and utero-placental production rates for the prostaglandins. In chronically instrumented pregnant ewes, between 129 and 136 days of gestation, prostaglandin F2 alpha(PGF2 alpha), 13, 14 dihydro-15-keto prostaglandin F2 alpha (PGFM), prostaglandin E2 (PGE2) were measured in the maternal carotid artery and uterine vein. Foetal PGE2, and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) (the major metabolite of prostacyclin) were measured in umbilical venous and foetal descending aorta arterial plasma. Umbilical and uterine blood flow were measured using the diffusion-equilibrium technique. Uterine blood flow was 1693 +/- 137 ml.min-1 (mean +/- SEM); uterine production rates were 480 +/- 88 ng.min-1 for PGF2 alpha, 517 +/- 144 ng.min-1 for PGFM, and 165 +/- 27 ng.min-1 for PGE2. Umbilical blood flow was 147 +/- 17 ml.min-1.kg-1 foetal body weight. Umbilical production rates into the foetal circulation were 11 +/- 2 ng.min-1.kg-1 for PGE2 and 6 +/- 2 ng. ng.min-1.kg-1 foetal body weight for PGI2.     

Accuracy of ultrasonography for pregnancy diagnosis on days 10 to 22 in heifers

Two operators independently conducted ultrasonic pregnancy examinations on nulliparous Holstein heifers on Days 10, 12, 14, 16, 18, 20 and 22, and assigned a diagnosis (pregnant or nonpregnant) and a score for degree of certainty in the diagnosis (1, 2 or 3 for low, intermediate or high, respectively). Pregnancy was retrospectively confirmed by the ultrasonographic detection of an embryo proper and embryonic heartbeat on Day 24 in 20 25 bred heifers; the five nonpregnant heifers were excluded from the analyses. Eleven nonbred heifers were included as an unequivocal source of nonpregnant heifers. Accuracy was not significantly greater than a guess (50%) before Day 18, but reached 100% on Days 20 and 22. Mean accuracy was higher (P<0.005) for nonpregnant (65 77 , 84%) than pregnant heifers (91.5 140 , 65%). For certainty score, there were main effects of day (P<0.0001), reproductive status (pregnant or nonpregnant, P<0.003), and an interaction of day and reproductive status (P<0.0001). The certainty score increased in all heifers among days and was higher (P<0.05) in pregnant than nonpregnant heifers on Days 16 to 20. For luteal area (area of corpus luteum, excluding area of fluid filled center, if present), there were significant main effects of day, reproductive status and a day by status interaction (P<0.0001 for each). Luteal area was approximately constant in pregnant heifers, but in nonpregnant heifers it was lower (P<0.05) on Days 16 to 22 than on Days 10 to 14. Uterine echotexture was scored on a scale of 1 to 3, characteristic of a diestrus, intermediate and estrus uterus, respectively. There were main effects of day and reproductive status (P<0.0001 for each) and an interaction of day and reproductive status (P<0.025). Uterine echotexture was approximately constant in pregnant heifers, but in nonpregnant heifers it was higher (P<0.05) on Days 16 to 22 than on Days 10 to 14. Pregnancy diagnosis on Days 10 to 14 was based on detection of the conceptus; however, detection of the conceptus was not accurate prior to visualization of the embryo proper (mean Day 22, range Days 20 to 24). In nonpregnant heifers, a correct diagnosis with high certainty was made when a small corpus luteum and uterine echotexture characteristic of estrus were detected. In the absence of these changes on Days 18 to 22, a diagnosis of pregnancy was made with high accuracy and intermediate or high certainty.     

Distribution of lectin receptors in postimplantation mouse embryos at 6-8 days gestation

We have examined the pattern of binding of eleven lectins--BSL-II, WGA, LPA, Con A, DBA, SBA, LTA, UEA-I, MPA, PNA, and RCA-I, with specificity for a range of saccharides, to postimplantation mouse embryos from 6 to 8 days of gestation. The lectins were used to stain sections of ethanol-fixed paraffin-embedded and formaldehyde-fixed gelatin-embedded embryonic material. Our observations reveal a complex pattern of lectin binding to both cell surfaces and cytoplasm. Many of the lectins bind particularly to the outer surface of visceral endoderm (e.g., DBA, WGA, SBA, and RCA-I) and to the surface of the proamniotic cavity (e.g., RCA-I, PNA, and WGA). In the newly formed mesenchyme of primitive-streak-stage embryos, galactose and N-Ac-neuraminic acid are present but lectins with specificity for other sugars either did not bind to the cells or bound only in small amounts.     

Imprinting analyses of the porcine GATM and PEG10 genes in placentas on days 75 and 90 of gestation

Imprinted genes are expressed monoallelically depending on their parental origin, and escape the Mendel's laws of heredity. They play important roles in the mammalian development, growth, and behavior. Placenta is a key tissue for the normal development and growth of fetus. It is also used to illuminate the evolution of genomic imprinting. In this study, we cloned the porcine GATM and PEG10 genes. Somatic cell hybrid panel (SCHP) and porcine radiation hybrid (IMpRH) panel were employed to locate GATM and PEG10 genes to SSC1q12-21 and SSC9p13-21, respectively. By sequencing PCR products, we detected several cSNPs in the two genes. The BseLI (GATM) and TaqI (PEG10) polymorphisms were used to investigate the allele frequencies in different pig breeds and the imprinting status in porcine placentas on days 75 and 90 of gestation. The results showed that for the GATM BseLI polymorphism, the Yorkshire and Duroc pigs had higher allele frequencies at the G allele, whereas the local pigs had higher allele frequencies at the A allele. Expression and sequencing analyses showed that both alleles were expressed for the GATM gene, indicating the GATM was not imprinted in the porcine placentas on days 75 and 90 of gestation. The allele frequencies of TaqI polymorphism for PEG10 gene were significantly different in native Chinese Erhualian breed comparing to Yorkshire. PEG10 was monoallelically expressed, showing the PEG10 gene may be imprinted in porcine placentas on days 75 and 90 of gestation.     

Pineal and ovarian response to 22- and 24-h days in the ewe

Melatonin secretion in ewes was entrained by 22-h light-dark cycles whether of long (16L:6D) or short (6L:16D) photoperiod. In photoperiods of 6L:16D, a phase-delay of melatonin secretion was evident, leading to a dark-phase duration shorter than that found in 8L:16D. Early onset of estrus was induced in anestrous ewes kept in 8L:16D, but not 6L:16D, from 22 July compared to controls in natural light. In photoperiods of 16L:6D, the melatonin profile corresponded precisely to the dark phase. Early offset of estrus was induced in estrous ewes kept in both 18L:6D and 16L:6D from 18 December compared to controls in natural light. Thus, when the duration of melatonin secretion was appropriate to the long photoperiod (16L:6D), but with a constantly changing phase position, a long-day reproductive response was found. Activity-rest cycles were not entrained by 16L:6D; thus the synchronization of melatonin and activity-rest cycles does not appear to be essential for the induction of a long-day reproductive response. These results support the hypothesis that the duration, not the circadian-phase position, of melatonin is critical to the induction of photoperiodic effects.     

Studies on orthocephalization. 10. Behaviour of the visceral part of the rat head during the first 14 days after gestation

The present paper considers the significance of interosseous flexions of the palatal complex in the process of orthocephalization of the rat skull between birth and 7 d p.n. The study is based on a sample of 90 rats divided into 4 age groups, i.e. 0, 4, 7, and 14 d. These rats have been X-rayed, and their photographs subsequently analysed. During the studied period, the constituents of the bony palate, i.e. the horizontal part of the palatine bone, the palatal process of maxilla and the palatal part of premaxilla, increase markedly in length, but with individual differences in growth rate. There is, in the period, a marked decrease in angulation between the cranial base and the palatal plane. This means that the rat skull becomes more orthocranial. There is also a straightening (orthopalatalization) of the palate, as the angle between maxilla and premaxilla becomes more obtuse, and a marked decrease in angulation between the palatine bone and the cranial base. The patterns of angular changes suggest that the process of orthocephalization in the period between birth and 14 d p.n. primarily is a result of an upwards rotation of the palatine bone relative to the cranial base, while interosseous deflections in the palate only play a minor role.     

Cell survival and proliferation are modified by insulin-like growth factor 2 between days 9 and 10 of mouse gestation

The size of mammalian species involves the interaction of multiple genetic modifiers that control the timing and extent of growth mechanisms. Disruption of the paternal allele of the imprinted embryonic gene coding for insulin-like growth factor 2 (IGF2, Igf2(+m/-p)), results in viable mice that are 60% the weight of wild-type littermates. Differences in weight are first detected at embryonic day (E) 11, and the growth deficit is maintained throughout life. We report the mechanisms that account for this unusual phenotype. In order to quantify growth, we used novel methods to generate single cell suspensions of post-implantation mouse embryos. We were then able to quantify cell number, cell proliferation and cell death between E8.5 and E11.5 using flow cytometry. Determination of total embryo cell number also allowed us to time litters by a method other than by plugging. Wild-type and Igf2(+m/-p) embryos accumulated similar total cell numbers up to E9.25, but cell number began to diverge by around E9.5, with significant differences by E11 (75% of wild type). A relative increase in pyknotic nuclei, sub-GI cytometry counts and caspase activity, all indicative of cell death, occurred in Igf2(+m/-p) embryos at E9.25, reverting to wild-type levels by E9.75. This was followed at E9.75 by a significant reduction in the proportion of cells in S phase, quantified by S-phase cytometry counts and BrdU labelling. No significant differences in cell size were detected. We conclude that the majority of the cell number differences between wild-type and Igf2(+m/-p) mice can be accounted for by modification of cell survival and proliferation during the period (E9 to E10) of post-implantation development.