Periodontal probing and its relation to degree of inflammation and bleeding tendency.
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Animals↗
Biomedical subjects
G B Anderson
Publications and source records attributed to G B Anderson.
Identification of a male-specific histocompatibility protein on preimplantation porcine embryos.
An indirect immunofluorescence assay was used to detect the presence of male-specific protein(s) on various stages of preimplantation porcine embryos. Embryos were collected at slaughter from the reproductive tracts of day-2.5, -4, -5, -6, and -8 (day 0 = first day of estrus) sows and gilts. Embryos were placed in medium containing an anti-male primary antibody, washed, and transferred to culture drops containing a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Embryos were classified as either fluorescent (H-Y positive) or nonfluorescent (H-Y negative), transferred to coded drops, and karyotyped to examine sex chromosomes. A total of 91 eight-cell to blastocyst stage embryos were evaluated; of these, 46% were classified as fluorescent and 54% as nonfluorescent. Of readable metaphase spreads (65%) from these embryos, 81% (48 of 59, P less than 0.005) were correctly sexed by immunological detection of the male-specific antigen. Although 13% (2/15) of four-cell embryos evaluated were classified as fluorescent, the accuracy with which embryos at this stage were sexed by detection of H-Y antigen was not different from 50%. Fifty percent of eight-cell embryos were classified as H-Y positive with 78% of embryos correctly sexed. It was concluded that the eight-cell embryo is the earliest stage of development for which there is evidence for expression of H-Y antigen. Detection of the male-specific protein was difficult at the expanded blastocyst stage.
Animals↗
Detection of histocompatibility-Y antigen: identification of sex of pre-implantation ovine embryos.
Indirect immunofluorescence was used to detect the presence of a male-specific protein (H-Y antigen) on pre-implantation ovine embryos. Eight-cell to blastocyst stage embryos were surgically collected from superovulated ewes, classified as fluorescent (H-Y positive) or non-fluorescent (H-Y negative) and either transferred to recipient ewes or karyotyped to confirm embryonic sex. H-Y antigen was detected on eight-cell through blastocyst stage embryos. Overall, 88% (50/57) of the embryos (eight-cell to early blastocyst stage) classified as H-Y positive or H-Y negative were male and female, respectively. Survival after transfer of embryos subjected to the H-Y antigen assay was high (63%), which supports the use of this procedure in conjunction with embryo transfer in sheep to produce pregnancies in which sex of the fetus is known.
Animals↗
Expression of a male-specific factor on various stages of preimplantation bovine embryos.
Four-cell to blastocyst stage bovine embryos were collected from superovulated donors and cultured for 90 min in Ham's F-10 medium (HF-10) containing 10% (V/V) absorbed anti-histocompatibility (H)-Y antiserum. Embryos were then washed 3 times and placed in HF-10 supplemented with 10% (V/V) fluorescein isothiocynate (FITC)-conjugated goat anti-mouse gamma globulin. After an additional wash, embryos were placed in fresh drops of HF-10, individually evaluated at 200 X magnification, and classified as either fluorescent (H-Y-positive) or nonfluorescent (H-Y-negative). Embryos were then placed in drops of HF-10 containing 14% vinblastin and cultured for 4-6 h. Embryos were coded and individually karotyped, and the sex chromosomes were identified. H-Y antigen was detected as early as the eight-cell stage, but not at the four-cell stage. Seventy-nine percent of fluorescent embryos and 89% of nonfluorescent embryos were XY and XX, respectively. Another experiment was carried out in which H-Y antigen was detected on intact inner cell masses (ICM) isolated by immunosurgery from expanded blastocysts that also had been assayed for H-Y antigen. Eighty-eight and 92%, respectively, of ICM classified as fluorescent or nonfluorescent had been scored the same as intact blastocysts. It is concluded from these data that H-Y antigen can be detected on eight-cell to blastocyst stage bovine embryos. There appears to be a localization of detectable antigen in the area of the ICM at the expanded blastocyst stage. Detection of H-Y antigen is an effective, noninvasive method for identification of the sex of preimplantation bovine embryos.
Animals↗
A comparative study of ovine placental lactogen and prolactin secretion patterns in genetically dissimilar fetal co-twins.
Simultaneous fetal and maternal ovine placental lactogen (oPL) and prolactin (oPRL) measurements were determined from blood samples collected from six mixed breed (MB; gestation = 141.5 +/- .7 d) pregnancies and single Rambouillet (n = 2; Ra gestation = 148 and 149 d) and Finnish Landrace (n = 2; Finn gestation = 141 and 143 d) pregnancies for the purpose of examining a possible relationship between fetal-maternal oPL and oPRL levels in the prepartal period. The MB pregnancies were produced by embryo transfer and consisted of genetically different fetuses co-existing in a common uterine environment. Despite the apparent prematurity of Ra co-twins in MB pregnancies, similar oPL and oPRL patterns during the final 14 d of gestation were observed for both Ra and Finn co-twins. Fetal oPL levels decreased at parturition (range 23 to 90 ng/ml) to approximately 60% of values recorded 8 d prepartum (range 100 to 150 ng/ml) in both fetal siblings. Increases in fetal oPRL concentrations, first observed at approximately 4 d prepartum in all fetuses, reached peak concentrations (40 to 60 ng/ml) at term. Results of this study suggest a similarity in regulation of fetal oPL and oPRL secretion, despite genetic differences for gestation period, in fetuses in the M.B. pregnancy.
Animals↗
Production of ovine chimeras by inner cell mass transplantation.
Ovine chimeras were produced by micro-injection of isolated inner cell masses (ICM) into recipient blastocysts. Inner cell masses were isolated by immunosurgery. A total of 57 chimeric embryos was produced, 52 of which were transferred to recipient ewes. Thirty-seven live lambs were born, of which 15 were determined to be chimeric on the basis of blood type analysis. One lamb, although not a blood chimera, exhibited overt signs of chimerism. An additional six lambs were determined to have developed solely from the injected ICM. The rate of chimerism in live lambs was 43% (16/37) while the survival rate of injected ICM was 59% (22/37). The method presented allows the production of relatively large proportion of viable, chimeric embryos without the use of an intermediate recipient.
Animals↗
Production of sheep-goat chimeras by inner cell mass transplantation.
Embryos were surgically flushed from goats and sheep on d 6 and 7, respectively, following the first day of estrus (d 0). After enzymatic removal of the zonae pellucidae, inner cell masses were isolated from caprine blastocysts by immunosurgery. The intact inner cell masses were injected into ovine blastocysts with the aid of a micromanipulator. Twenty-two manipulated blastocysts were surgically transferred into 12 ovine recipients. Nine ewes gave birth to a total of 13 young (59% embryo survival). Ten were classified by serum electrophoretic assays or karyotypes as lambs, one as a kid, and two as interspecific chimeras.
Animals↗
Use of chimaeras to study development.
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Animals↗
Identification of sex in mammalian embryos.
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Animals↗
The effect of recipient age on the success of embryo transfer in mice.
Eight-hundred-sixteen morulae and blastocysts were transferred to the uteri of 102 recipients of various ages on Day 3 or 4 of pseudopregnancy (Day 1 is the day of the vaginal plug). Day 3 recipients had significantly higher pregnancy rates and embryo survival rates than Day 4 recipients. Recipient age had little effect on pregnancy rates, but had a significant effect on embryo survival in Day 3 recipients. Day 3 recipients of 11-13 weeks of age had the highest pregnancy rate (100%) and embryo survival (75%). The results suggest that recipient age should be considered an important factor in embryo transfer experiments.
Age Factors↗
Manipulation of the mammalian embryo.
Technological advances in manipulation of mammalian embryos outside the maternal environment have resulted in opportunities for study of preimplantation embryo development, identification of developmental phenomena that are unique to mammals, and further improvement of technology. Mammalian embryos may be cultured in vitro at 37 C for up to several days or they may be stored at -196 C indefinitely. The mammalian embryo possesses the unique capacity to regulate its development and differentiate into a normal individual after being stimulated to incorporate foreign cells or after a portion of its cells are removed. This regulatory ability has proven useful in research dealing with the production of chimeras. It allows genetic copies of an embryo to be produced by dissociation of an early cleavage-stage embryo into its component blastomeres or by bisection of a morula. Production of large sets of identical animals may be possible in the future by serial transplantation of nuclei from one embryo into enucleated ova. Progress has been made in producing unique genetic combinations by manipulation of the pronuclei of fertilized ova. It is also possible in some cases to identify the sex of a living cleavage-stage embryo. Some of these manipulations have been carried out primarily in laboratory mice, but as animal scientists identify beneficial uses in farm animals, these procedures are being extended to embryos of the large domestic species.
Animal Husbandry↗
Studies on the endocrinology of parturition: relative steroidogenesis in coexisting genetically dissimilar ovine fetuses, concomitant with the temporal patterns of maternal C18 and C19 steroids and prostaglandin F2 alpha release.
Ovine embryos collected from two breeds of ewes possessing different gestational periods [Finnish Landrace (Finn), 145.5 +/- 1.4 days; Rambouillet (Ra), 150.7 +/- 1.3 days] were transferred to common, randomly selected recipients. Fetal plasma samples were collected from mixed-breed (MB, one Finn and one Ra; n = 6) and single-breed (SB, single Finn, n = 3; single Ra, n = 3) chronic fetal preparations during the last trimester. In the MB pregnancy, the Ra sibling had temporal patterns of androstenedione similar to those observed in the Finn co-twin; however, these particular changes were greatly accelerated in comparison to the same day of gestation in SB Ra fetuses. Similarly, temporal patterns of change in unconjugated estrone, estrone sulfate, and 17 beta-estradiol were accelerated by as much as 4.6 to 5 days in Ra fetuses of MB pregnancies when compared to Ra fetuses in the SB pregnancies (P less than 0.001), with no difference noted between Ra and Finn co-twins. Maternal progesterone concentrations changed from 8 to 2 ng/ml, androstenedione from 200 to 400 pg/ml, estrone from 65 to 250 pg/ml, and 15-keto-13,14-dihydroprostaglandin F2 alpha from 200 pg/ml to 6000 pg/ml over the last 4 or 5 days of gestation. Chronic fetal manipulation accelerated parturition, with mean gestational lengths in MB, SB Finn, and SB Ra pregnancies being 141.5 +/- 0.7, 141.7 +/- 0.7, and 149.3 +/- 0.9 days, respectively. In this study fetal endocrine changes were largely controlled in the MB pregnancy by the fetal co-twin exhibiting a shorter gestational period.
Androstenedione↗
Investigation of sperm cytotoxicity as an indicator of ability of antisera to detect male-specific antigen on preimplantation mouse embryos.
H-Y antisera were produced in C57BL/6 female mice by repeated intraperitoneal injections of syngeneic male spleen cells. Epididymal spermatozoa were incubated in the presence of H-Y antisera and guinea-pig serum as a complement source. Levels of ATP remaining after treatment were used to calculate the amount of specific killing. Sera of different cytotoxic titres were used in an indirect immunofluorescent assay with a fluorescein isothiocyanate-conjugated IgG fraction of goat anti-mouse IgG (Fc fragment specific) as second antibody. Embryos were classified as fluorescent or nonfluorescent, transferred to pseudopregnant recipients, and allowed to develop to term. Of 12 sera tested for sperm cytotoxicity, 5 were different from a nonimmunized control serum (P less than 0.05). Percentage specific killing in each of these sera was 7.8 +/- 4.2, 11.7 +/- 3.0, 26.0 +/- 2.2, 27.7 +/- 3.7 and 39.2 +/- 4.8, respectively (mean +/- s.e.m. with three replicates). The 5 sera and an additional one (4.9 +/- 1.3% specific killing) were used in the embryo sexing experiment. The accuracy with which these sera correctly identified sex of preimplantation embryos was 60, 46, 74, 73, 74 and 48%, respectively. Correlation coefficients were 0.86 (P less than 0.05) for specific sperm cytotoxicity and percentage of nonfluorescent embryos that were female and 0.78 (n.s.) for specific sperm cytotoxicity and percentage of fluorescent embryos that were male. Therefore, although the sperm cytotoxicity test is useful for screening antisera for the study of H-Y antigen expression on preimplantation embryos, nonfluorescent embryos are more accurately classified as females than are fluorescent embryos as male.
Adenosine Triphosphate↗
Reproductive performance of chimeric mice produced from genetic lines that differ in litter size.
A population of chimeras was made by aggregating 8- and 16-cell embryos from two mouse strains: a randomly bred line (C) and a selected line characterized by large litters (JU), with litter sizes of 7.7 and 13.5, respectively. The two genotypes were developmentally "balanced", as judged by the high frequency (90%) of chimeras with an intermediate or high degree of coat-color chimerism, a chimeric sex ratio of 2.2:1 males:females, and a high percentage of chimeras (31% of males, 71% of females) with germ cells of both strains. Litter size characteristics, including ovulation rate, implantation rate, rates of pre- and postimplantation embryo survival and number born were studied in the female chimeras and compared with the performance of both parent lines and to the genetic cross of the two lines. Values for JU females exceeded those for C females for all parameters studied except postimplantation embryo survival, which was the same for both lines in second litters and was lower for JU's third litters. For most traits, means for genetic crossbreds and chimeras were similar, regardless of whether the means were at or above the midparent average. In contrast, for ovulation rate and body weight, genetic crossbreds and chimeras clearly differed, with chimeric females being similar to the JU line and genetic crossbred females exhibiting additive inheritance. Because of phenotypic differences between experimental chimeras and crossbreds produced from the same two lines, chimeras may provide a useful model for studying the physiologic basis for expression of genetic differences in quantitative traits.
Animals↗
Lethality of a tritiated amino acid in early mouse embryos.
2- to 4-cell and morula- to blastocyst-stage mouse embryos were cultured for 1 h in tritiated leucine at two specific activities and their subsequent development followed in vitro and in vivo (after transfer to recipients), respectively. 2- to 4-cell embryos that incorporated an average of 42 d.p.m. per embryo were impaired in their ability to develop to the morula and blastocyst stage. Recipients receiving morulae and blastocysts that had incorporated an average of 384 d.p.m. per embryo failed to produce young. Reduction of the specific activity improved the viability of embryos both in vitro and in vivo but development was still less than that of unlabelled embryos. Protein degradation curves were different for both 2- to 4-cell and morulato blastocyst-stage embryos labelled at the two different specific activities. Most studies using tritiated amino acids have employed higher specific activities than those used here and they may have to be reevaluated due to the possibility of radiation-induced artifacts.
Animals↗
Nonsurgical collection of blastocysts from dairy goats.
In two trials, eight attempts were made to collect fertilized ova from dairy goats by nonsurgical methods. In both trials the cervix of each doe was dilated by inserting a Laminaria japonica tent device into the cervical canal prior to flushing. In Trial 1, an attempt was made to collect embryos from four nonsuperovulated does by flushing phosphate-buffered saline (PBS) through a rigid pipette. Little fluid and no embryos were recovered from the does. All four donors were in estrus two days after the procedure. In the second trial, FSH-superovulated does were collected on day 5 following estrus. The donors were anesthetized, and a modified Foley catheter was passed through the cervical canal. In three does, a 24 ga. two-way Foley was stiffened with a size 10 (French) polypropylene catheter which penetrated the Foley, extending 7 cm beyond the tip. Recovery of flushing medium with this device was minimal, and laparotomy of one doe revealed a punctured uterus. Replacement of this device with a different catheter, through which a polypropylene catheter (size 5 Fr.) penetrated only 1 to 2 cm, resulted in satisfactory return of infused PBS, and recovery of two blastocysts and one degenerated ovum from this doe. Use of the same device on a second doe without laparotomy resulted in collection of seven blastocysts and three degenerated ova. Of three observed donors that received Laminaria tents (including one which was not flushed) two were in estrus three days after the procedure, while unused synchronized recipients showed normal cycle lengths. Surgical transfer of two blastocysts from each donor to each of two synchronized recipients resulted in the birth of twin kids from one recipient doe. The study demonstrates the feasibility of embryo collection from dairy goats by nonsurgical means.
Journal Article↗
Temporal patterns of delta 4 C-21 steroids in coexisting, genetically dissimilar twin lamb fetuses throughout late gestation.
We have examined the relationships among fetal maturity, adrenal steroidogenesis, and initiation of parturition using embryo transfer procedures. Ovine embryos, genetically coded for relatively short [145.5 +/- 1.4 days; Finnish Landrace (Finn)] and long [150.7 +/- 1.3 days; Rambouillet (Ra)] gestational periods, were transferred to a common recipient, thereby facilitating comparative endocrine studies in genetically dissimilar fetuses with a common intrauterine environment. Maternal and fetal plasma samples were obtained daily from chronic fetal lamb preparations in six mixed breed (MB), three Finn, and three Ra single breed (SB) pregnancies during the last trimester and assayed for cortisol, desoxycortisol, and progesterone by RIA. Mean +/- SEM gestational lengths of the forenamed pregnancies were 141.5 +/- 0.7, 141.7 +/- 0.7, and 149.3 +/- 0.9 days, respectively. Time-trend analysis of C-21 steroid data showed an increase in Finn and Ra SB fetal plasma cortisol of 1.0 ng/ml X day at 9.03 +/- 1.0 and 9.58 +/- 1.10 days prepartum, respectively. Significant (P less than or equal to 0.05) differences in the number of days of incremental change (T*) were noted between Finn (10.56 +/- 0.67) and Ra (4.82 +/- 1.08) siblings in MB pregnancies at increment rates of 1.0 ng/ml X day. Similarly, significant (P less than or equal to 0.05) differences in T* values were observed between Finn (8.31 +/- 0.38 days) and RA (4.63 +/- 1.0 days) siblings at 2.0 ng/ml X day. While Finn and Ra SB fetal desoxycortisol T* values were similar at 0.5 and 0.1 ng/ml X day, significant differences (P less than or equal to 0.05) were noted in Finn and Ra cofetuses in the MB pregnancy. Prepartal changes in maternal and fetal progesterone concentrations were correlated, and a direct parallelism was noted between fetal sibling progesterone concentrations. These results are consistent with the view that the fetal genome has a principal role in the timing of parturition.
17-Hydroxycorticosteroids↗
The influence of fetal genome on cortisol biokinetics and gestational duration.
The biokinetics of cortisol metabolism were assessed simultaneously in chronically catheterized pregnant ewes and their fetuses in two breeds of sheep (Finnish Land-race, (Finn) and Rambouillet, (Ra)) with distinctly different gestational durations. The purpose of these studies was to examine the genetic differences in fetal development and its influence in regulating the duration of gestation. Catheterized fetuses were born at 147.5 +/- 0.7 (Ra) and 141.7 +/- 0.9 days (Finn), respectively. Uncatheterized purebred Ra and Finn ewes had gestational periods of 150.7 +/- 1.3 and 145.5 +/- 1.4 days, respectively. Cortisol blood production rates in Finn fetuses at approximately 141 days gestation were 5.71 +/- 1.08 mg/day (mean +/- SD) and comparable to those in Ra fetuses (6.16 +/- 1.32 mg/day) at 146 days gestation. These differences could not be attributed to corresponding changes in maternal cortisol production rates which varied from 11.66 mg/day to 17.91 mg/day in all ewes. This study provides additional data describing fetal cortisol metabolism in utero as well as further describing the role of fetal genome in initiating adrenal metabolic activity and regulating gestational length in sheep.
Animals↗