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Biomedical subjects

R F Seamark

Publications and source records attributed to R F Seamark.

At least 73 records · Page 4Linked to original sources
PubMed1989

Uterine presensitization and embryo survival and growth in Booroola Merino x South Australian Merino ewes.

The fertility enhancing effects of semen were examined following the intra-uterine insemination of killed spermatozoa plus seminal plasma 17 d prior to insemination with viable spermatozoa. Three experiments were conducted: two on 1.5-yr old and 2.5 to 5.5 yr-old Booroola Merino x South Australian Merino ewes in 1986 and one on 1.5 yr-old ewes in 1987. Differences between treatment and control groups for the percentage of ewes exhibiting estrus by Days 21 and 35 following fertile insemination, the percentage of ewes with viable embryos at Day 35, the number and weight of viable embryos per ewe, the nubmer of caruncular implantation sites and the progesterone level were not statistically significant (P>0.05). There were no statistically significant treatment by experiment interactions for any of the variables examined. Inflammation and edema of the endometrial tissue was not observed following the presensitization treatment.

Journal Article↗
PubMed1989

Time of ovulation in the South Australian Merino ewe following synchronization of estrus. 1. Variation within and between flocks.

Observations were made by repeated laparoscopy to determine the time of ovulation in seven flocks of South Australian Merino ewes treated with pregnant mare serum gonadotropin (PMSG, 400 IU per ewe) and an intravaginal pessary containing 60 mg medroxy-progesterone acetate (MAP). Ovulation most often commenced within 57 h of pessary removal and was completed in all flocks within 81 h. There was, however, significant variation in the time of onset of ovulation; it did not commence until 69 h in one of three observations of Flock 1 and in two of three observations of Flock 2. On the other hand, in a flock of two-year-old nulliparous ewes (Flock 7), ovulation commenced significantly (P < 0.001) earlier than in most observations of other flocks, with 18 39 ewes ovulating within 57 h of pessary removal. The effect of the progestagen (MAP vs flugestone acetate) and the gonadotropin preparation (Pregnecol vs Folligon) on the time of ovulation were observed in separate studies. The characteristic time of ovulation was not influenced in either comparison. However, ovulation occurred significantly (P < 0.001) earlier in ewes treated with progesterone released from a controlled internal drug release dispenser (CIDR). The implications of these findings to artificial insemination and embryo transfer are discussed.

Journal Article↗
PubMed1989

Time of ovulation in the South Australian Merino ewe following synchronization of estrus. 2. Efficacy of GnRH treatment and its relevance to insemination programs utilizing frozen-thawed semen.

In a study of the time of ovulation following synchronization of estrus in the ewe, the effect of time of treatment with GnRH (24 vs 36 h after pessary removal) and dosage (6.25 to 100 ug per ewe) were examined. All treatments synchronized the time of ovulation irrespective of when untreated ewes commenced to ovulate. As part of an evaluation of GnRH treatment in artificial insemination programs, an assessment was made of the quality of eggs obtained from control ewes and ewes treated with GnRH at either 24 or 36 h after pessary removal. Treatment at 24 h increased the number of retarded embryos (P < 0.01) and unfertilized ova (P < 0.01) collected per ewe, reduced the number of embryos collected per ewe (P < 0.01), and reduced (P < 0.05) the percentage of pregnant ewes compared with other groups. However, there were no differences between control ewes and ewes treated with GnRH at 36 h. GnRH treatment at 36 h was consequently examined as a means of improving conception rates following the intrauterine insemination of frozen-thawed semen. Insemination of GnRH-treated ewes 8 to 12 h before the median time of ovulation resulted in a nonsignificant increase (range 5.7 to 7.3%) in the percentage of ewes of mature age which became pregnant. Insemination 0 to 4 h before the median time of ovulation resulted in a nonsignificant decrease in the percentage of pregnant ewes. GnRH treatment did not influence the number of fetuses per ewe. Reasons for the failure of this treatment to significantly improve ewe fertility are discussed.

Journal Article↗
PubMed1989

Culture of sheep zygotes in synthetic oviduct fluid medium with different concentrations of sodium bicarbonate and HEPES.

An investigation was conducted of the viability of sheep zygotes in vivo subsequent to culture in synthetic oviduct fluid medium (SOFM) in which sodium bicarbonate was partly or wholly replaced with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid); concentrations of 0, 6.25, 12.5, 18.75 and 25 mM HEPES were studied. The addition of HEPES lowered the pH during the culture period, and was associated with a reduction in the percentage of zygotes that developed to blastocysts (range 60.4 to 85.2%); this reduction was significant (P < 0.05) at concentrations of 18.75 and 25 mM. There was also a significant (P < 0.05) reduction in the percentage of zygotes that commenced hatching (range 47.2 to 74.1%) and a significant (P < 0.05) delay in the time of blastocyst formation (range 4 to 7 d) in medium containing 25 mM HEPES. Viability, as assessed by elongation of the trophoblast after transfer to recipient ewes, was similar for zygotes cultured for 3 d either in medium with 12.5 mM HEPES or in medium without HEPES (90.3 and 93.9%, respectively). Extending the culture period to 5 d was associated with a general decline in viability (P < 0.05), with 67.7% of zygotes cultured with HEPES elongating compared with 45.5% for those cultured without HEPES. This study confirms that sheep zygotes can be successfully cultured in SOFM for up to 3 d without loss of viability. The addition of HEPES provides additional buffering capacity but zygote development may be compromised.

Journal Article↗
PubMed1989

Addition of steroids to embryo-uterine monolayer co-culture enhances embryo survival and implantation in vitro.

Co-culture with a mixed cell monolayer established from trypsinized uterine tissue increased the numbers of embryos developing in a minimum essential medium beyond the hatching blastocyst stage in the 5-day period of culture to 56.0% (343/613) compared with 30.2% (93/308) for embryos cultured in medium alone. The inclusion of progesterone (3.2 x 10(-6), 3.2 x 10(-5), or 3.2 x 10(-4) M) or oestradiol (3.7 x 10(-5) M) to the co-cultures increased the mean percentage of embryos developing to the advanced stages to 72.7-78.4%. The addition of progesterone (3.2 x 10(-6) M) together with oestradiol (3.7 x 10(-5) M) resulted in additional improvement to a mean of 86.5% (787/910, P less than 0.001) but the combination was without significant effect on embryos cultured in media alone. Blastocyst viability was not impaired by co-culture as assessed in embryo survival following surgical transfer to pseudopregnant recipients. This study confirms the feasibility of establishing co-culture systems to facilitate investigation of pre-implantation events in vitro and highlights a role of steroid hormones in enhancing the capacity of uterine cells to support pre-implantation-stage embryos.

Animals↗
PubMed1989

Transgenesis: the challenge for the reproductive biologist.

The last decade has witnessed dramatic developments in molecular biology to a point where it is now possible for genetic engineers to consider programmes aimed at isolating and cloning specific genes from any genomic source for introduction into the germ line of other organisms. The impact of this technology on livestock breeding is potentially revolutionary.

Animals↗
PubMed1989

Evidence of high concentrations of melatonin in lateral ventricular cerebrospinal fluid of sheep.

In this study we examined the cerebrospinal fluid (CSF) for regional differences in the concentration of melatonin. Cerebrospinal fluid samples were collected at hourly intervals from either the lateral ventricle or the cisterna magna of nine Merino ewes and were compared against jugular plasma. The study revealed that the CSF of the cisterna magna and the lateral ventricle had temporal patterns of melatonin that were similar to those found in jugular plasma. However, the concentrations of melatonin within the CSF obtained from the lateral ventricle were one order of magnitude higher than those of the jugular plasma, as verified by gas chromatography and mass spectrometry, while the melatonin concentrations within the CSF obtained from the cisterna magna were comparable to those of the jugular plasma. The data from this study suggest that there may be regional differences in the concentration of melatonin within the CSF and indicate that this medium is an important route of transport for melatonin from the pineal gland to putative target tissues.

Animals↗
PubMed1988

Effects of prior exposure to prolonged continuous light on the pattern of melatonin secretion in sheep held under continuous darkness.

To gain insight into the nature of the neural centre regulating melatonin production by the pineal gland we have measured hourly changes in plasma melatonin in 16 ewes held for 42-56 h under continuous darkness following a 28-day period of exposure to continuous light. Plasma melatonin was undetectable while animals were in constant light but increased to normal night time levels (120-2,200 pmol/litre) within 10 min of the onset of darkness in 14 out of the 16 ewes regardless of whether this occurred at 1,200 h (group 1) or 2,400 h (group 2). In four ewes melatonin secretion was maintained for approximately 36 h, indicating that the pineal gland remained responsive to a static stimulatory signal presumably emanating from the supra chiasmatic nucleus. In all ewes, melatonin secretion ceased at approximately 12 and or 36 h after the onset of darkness and did not require a separate zeitgeber. Although the increase and the decline of plasma melatonin levels were synchronized within groups 1 and 2, there were sufficient differences in the pattern of melatonin secretion between the groups to suggest that the regulatory mechanism had an intrinsic component. A hypothesis involving a diurnal change in sensitivity to circulating melatonin is advanced to explain the observed differences in the synchrony of melatonin secretion and the prolonged episodes of melatonin secretion.

Animals↗
PubMed1988

Introduction of a porcine growth hormone fusion gene into transgenic pigs promotes growth.

Six transgenic pigs have been produced by microinjecting a human metallothionein promoter/porcine growth hormone gene construct into the pronuclei of fertilized eggs which were transferred to synchronized recipient sows. The resulting transgenic animals contained between 0.5 and 15 copies of the gene construct per cell, and at least one of the animals expressed the introduced gene and grew at an increased rate compared to both transgenic and non-transgenic littermates. Some of the transgenic animals that did not appear to grow at increased rates were found to contain rearranged gene sequences. Two of the transgenic pigs have been shown to pass on the introduced genes to their offspring.

Animals↗
PubMed1988

Flow and composition of lymph from the ovary and uterus of cows during pregnancy.

Ovarian or uterine lymph was collected continuously for periods of up to 25 days from 16 cows cannulated at stages of pregnancy ranging from 96 to 278 days post coitum. Blood samples were taken acutely from the ovarian and uterine veins during surgery and periodically from the jugular vein during the course of lymph collection. The flow rate and cell content of lymph was measured and blood and lymph plasma samples were analysed for progesterone, pregnenolone, pregnenolone sulphate, androstenedione, testosterone, oestrone, oestrone sulphate, oestradiol-17 beta, prostaglandin (PG) F-2 alpha, total protein and albumin. There was a high flow rate of protein-rich lymph from luteal ovaries with rates up to 101.7 ml/h occurring in individual lymphatics over short periods. Peripheral ovarian and uterine lymph contained a low concentration of cells (mean less than 10(5) cells/ml) comprising about 82-87% lymphocytes, 11-14% macrophages and monocytes and 2-4% other cells. At all stages of pregnancy, the concentration of progestagens and androgens was higher in ovarian lymph than in uterine lymph or blood plasma. The differences were greatest for progesterone and androstenedione which occurred at 200-fold and 60-fold greater concentration respectively in ovarian lymph than in jugular plasma. When serial 10 min samples were collected over a 12-h period, the concentration and output of progesterone in ovarian lymph varied in a phasic manner, ranging from 3.5 to 7.6 microM and from 31.7 to 293.1 nmol/h respectively. There was a positive correlation between the output of progesterone in lymph and the progesterone concentration in jugular blood samples taken every 20 min. During most of pregnancy there was little difference between the concentration of oestrogens in ovarian lymph, ovarian venous plasma and jugular plasma but, during the 3-5 days before calving, these hormones occurred at slightly higher concentration in ovarian lymph. Apart from pregnenolone and androstenedione, all steroids occurred at lower concentrations in uterine lymph than in jugular plasma. Shortly before parturition there was an abrupt increase in the concentration of PGF-2 alpha in uterine lymph. Lymph reflects more accurately the milieu of tissue cells than efferent blood and further analysis of differences in the concentration of substances in lymph relative to the output in the ovarian and uterine arterial and venous blood may lead to the identification of factors important in local regulatory mechanisms in the reproductive tract.

Androgens↗
PubMed1988

Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse.

Zona cutting and zona drilling of the mouse oocyte significantly increased the fertilization rate (3.8-90%) at low sperm concentrations (less than 200,000/ml) compared with zona-intact controls (0-45%). More oocytes were fertilized after zona drilling. Zona cutting was associated with a low loss of oocytes (less than 1%), no increase in polyspermy and normal development in vitro and in vivo after fertilization. There was a 4% oocyte loss rate after zona drilling, mostly due to extrusion of the oocyte from the zona during the procedure. Hatching of blastocysts occurred about 12 h earlier for zona-drilled than for zona-cut and zona-intact control oocytes. Zona drilling was associated with a higher, but not statistically significant, rate of polyspermy at all sperm concentrations tested. The proportion of zygotes developing to the blastocyst stage was not different between the techniques (zona cut, 77%; zona drilled, 66%; control, 71%). Similarly, no difference was found in the percentage of embryos implanting after blastocyst transfer to the uterine horns of pseudopregnant female mice (zona cut, 67%; zona drilled, 68%; control, 77%). Transmission electron microscopy demonstrated the induced defects in the zona with no damage to the oocyte or oolemma. Parthenogenetic activation was not seen after either of the micromanipulative techniques. Both techniques have promise for application to the human.

Animals↗
PubMed1988

Increases in ovulation rate in lupin-fed ewes are initiated by increases in protein digested post-ruminally.

Parous Merino ewes were maintained outdoors in feedlots during the beginning of the spontaneous breeding season and fed a maintenance ration of wheaten hay. For 14 days, ewes in each of 4 groups (N = 40/group) were given supplements of lupin grain or formaldehyde-treated casein and/or wheat starch. These were calculated to supply equivalent amounts of protein post-ruminally and/or digestible energy. Supplementation with lupin grain significantly increased ovulation rate by 37% by increasing the proportion of ewes with two ovulations. Similar increases in ovulation rate were achieved by increasing the supply of digestible protein post-ruminally in the casein and casein + starch-supplement groups. Increasing the intake of digestible energy separately in the starch-supplement group did not increase ovulation rate. It is concluded that increases in ovulation rate in ewes fed a lupin supplement are the result of significant increases in the amount of protein digested post-ruminally.

Animals↗
PubMed1987

Variation in the timing of multiple ovulations following gonadotropin releasing hormone treatment and its relevance to collecting pronuclear embryos of sheep.

Timing of superovulation was examined by repeated laparoscopy in two Merino flocks treated with either pregnant mare serum gonadotropin (PMSG) plus gonadotropin releasing hormone (GnRH) or follicle stimulating hormone (FSH) plus GnRH. Observations were made in May (late breeding season), August (early anoestrous season), November (late anoestrous season), and February (midbreeding season). Data examined were time to first ovulation, time to all ovulations, and time from first to last ovulation. A GnRH-induced synchrony in the timing of superovulation occurred in Flock 1 irrespective of the month of observation. Approximately 80% of ovulations were recorded within 3 h with the median ovulation occurring 47 to 49 h after progestagen treatment. A similar synchrony was observed in Flock 2 in November and February. However, in May and August, the timing was asynchronous with some ewes superovulating as early as 10 or more hours before the median time obtained in November and February. An examination of this phenomenon indicated that 1) it also occurred when GnRH was not included in the treatment protocol, 2) it occurred irrespective of when ewes were exposed to vasectomized rams, and 3) it was more common in anovular ewes induced to superovulate than in spontaneously cyclic ewes. We concluded that treatment protocols developed for the collection of pronuclear embryos in Merino ewes during the breeding season can be less reliable when used out of season, thus increasing the possibility of collecting two- to four-cell embryos.

Journal Article↗
PubMed1987

Changes in peripheral progestagen levels in early pregnant gilts following injection of human chorionic gonadotrophin.

The capacity of human chorionic gonadotrophin (hCG) to raise plasma progestagen levels during the first 8 days of gestation in gilts was examined. The effective half-times of hCG in gilts treated with 500 and 5000 i.u. hCG were 29.1 and 26.3 h respectively (P greater than 0.05). Neither 500 nor 5000 i.u. hCG caused rises in peripheral concentrations of progesterone or pregnenolone sulphate, and plasma pregnenolone concentrations declined (P less than 0.05) following hCG treatment. Apart from diminished total corpus luteal weights in gilts treated with 500 i.u. hCG (P less than 0.05) and lower peripheral progesterone levels in gilts treated with 5000 i.u. hCG (P less than 0.05), ovarian and plasma steroid characteristics of hCG-treated animals between 23 and 25 days of gestation were similar to control values. Furthermore, treatment with hCG did not affect embryo survival during the first 4 weeks of pregnancy, and plasma oestrone/oestrone sulphate levels provided no evidence for differences between control and treated animals in trophoblastic outgrowth. These results challenge the rationale for the treatment of early pregnant sows with hCG in order to reduce the levels of embryonic wastage in early pregnancy.

Animals↗
PubMed1986

Measurement by radioimmunoassay of prostaglandins as their methyl oximes.

Antisera have been raised to the following prostaglandins as their methyl oximes; PGE2, PGD2, 13-14-dihydro-15-oxo PGE2, 13,14-dihydro-15-oxo PGF2 alpha, 6-oxo PGF1 alpha, 6-oxo PGE1 and thromboxane B2. These antisera have good specificity and sensitivity and their use allows the immediate treatment of biological fluids with oximating solution which prevents sample decomposition during storage. A methyl oximating reagent is described which gives greater than 95% conversion of PGs to their methyl oximes by treating samples at 20 degrees C overnight. The use of this reagent allows easy and reliable sample derivatisation prior to assay with the above antisera. Since the antisera (with the exception of that for thromboxane B2) do not recognise the underivatised PG or the oxime (=NOH) form, oxime formation can be carried out in parallel with methyl oxime formation and the oximated portion of the sample can act as a "reference" for the methyl oximated portion, which will allow non specific interference to be recognised.

Antibody Specificity↗
PubMed1986

Production of steroids and release of prostaglandins by spherical pig blastocysts in vitro.

Levels of pregnenolone and progesterone in spherical pig blastocysts (near 4 and 15 microM respectively) exceeded respective levels in histotroph by about 400-fold. When blastocysts were cultured for 5 days in a synthetic medium containing pregnenolone sulfate (1 microM), daily rates of release of pregnenolone, progesterone, androstenedione, testosterone, oestrone and oestradiol were determined to be near 320, 45, 26, 27, 0.8 and 9.2 fmol per blastocyst respectively. Daily outputs of progesterone and testosterone (fmol per blastocyst) diminished (P less than 0.05) to 1.3 and undetectable levels (less than 2) respectively in the presence of Trilostane (94 microM). Increasing the content of pregnenolone sulfate in the culture medium (to 4.5 microM) resulted in higher daily rates of release of pregnenolone and progesterone (to near 1740 and 380 fmol per blastocyst respectively), verifying activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase, and of arylsulfatase, in tissues of intact spherical pig blastocysts. Prostaglandin E2 was the predominant prostaglandin (PG) released by cultured blastocysts (about 1 fmol per blastocyst per hour), hourly rates of release of PGH2 (derived) and PGF2 alpha being near 0.1 and less than 0.06 fmol per blastocyst respectively. The data establish a capacity for spherical pig blastocysts to release a range of steroids and PGs of possible significance to embryonic growth and development in vivo.

Androstenedione↗
PubMed1986

Timing of multiple ovulations in the ewe after treatment with FSH or PMSG with and without GnRH.

Ovulation rate, median time to first ovulation, median time of all ovulations and median time from first to last ovulation were studied by repeated laparoscopy in Merino ewes. Treatments with FSH or PMSG significantly affected ovulation rate (8.4 +/- 0.81 and 7.3 +/- 1.21 respectively, P less than 0.05) and in median time of all ovulations (60 and 54 h respectively after progestagen sponge removal, P less than 0.05). Differences in the median time to first ovulation (60 and 48 h) and median time from first to last ovulation (6 and 6 h) for the respective treatments were not significant. The synchrony of ovulation after both treatments was adversely affected by (1) the occurrence of premature ovulations before the onset of superovulation, (2) variability in the time of commencement of superovulation, and (3) variability in the time from first to last ovulation. Administration of GnRH synchronized the timing of ovulation with both gonadotrophin treatments. This synchrony was due to a reduction in the period during which superovulation began and in the interval from first to last ovulation. The median time of all ovulations was significantly less with FSH + GnRH than with PMSG + GnRH (45 and 48 h after progestagen sponge removal, respectively, P less than 0.05). Administration of GnRH at 16, 20 or 24 h after progestagen sponge removal significantly affected all traits examined except ovulation rate. Administration at 20 and 24 h produced an equally good synchrony of ovulation which was better than that obtained at 16 h. We suggest that the use of GnRH in embryo collection programmes appears justified and is likely to improve embryo yields due to improved rates of fertilization.

Animals↗