Plasma prolactin in the periparturient sow

A homologous radioimmunoassay was used for measurement of porcine prolactin in blood plasma collected from sows during the periparturient period. The assay was able to detect prolactin over a range of 0.5 to 7.0 ng/assay tube. There was no significant cross reaction with growth hormone, luteinizing hormone, or follicle stimulating hormone at amounts up to 10⁵ ng/assay tube while porcine ACTH gave 30% binding at 10⁴ ng. Prolactin was not detected in plamsa from a hypophysectomized pig or 2 ergocryptine-treated sows when 100 μ l plasma were assayed. Prolactin concentration in plasma was then measured in 14 periparturient sows within a period extending from 7 days before farrowing to 7 days after farrowing. Samples were collected at 15 min intervals between 1330 and 1630 h each day. However, prolactin assays were done only on the even-numbered samples (30 min interval). Plasma prolactin concentrations (ng/ml, $\text{X}\text{± SEM}$) were 23.7 ± 2.0 on days ?7 to ?5 prepartum, began to rise by day ?3 prepartum (42.5 ± 5.9), and peaked at 127.5 ± 17.6 on day 1 prepartum. By day 3 postpartum, prolactin concentrations in plasma had decreased to 80.5 ± 12.6 and further declined to 51.6 ± 4.6 on day 7 postpartum. The mean prolactin concentration in plasma for all pigs on days ?1 to +2 was 116.8 ± 13.8. This mean concentration for days ?1 to +2 was different (P < 0.025) from the mean prolactin concentration for the period both prior and subsequent to these days (?8 to ?2 and +3 to +8 days).     

Uptake and effects of ovarian steroids in the early pig embryo: In vitro and in vivo studies

The role of ovarian steroids in the preimplantation pig embryo was studied in vivo and in vitro. Twenty gilts were treated three times daily on days 1 to 4 after insemination with either 25, 100, 250, or 1000 mg progesterone in oil, and 17 gilts were injected with corresponding amounts of sesame oil (controls). All gilts were slaughtered 5 days after insemination and the embryos were recovered. Oviduct and plasma progesterone content were significantly (P<0.001) higher in gilts treated with 750 mg of exogenous progesterone per day. After 750 mg progesterone, oviduct progesterone content was twice as high as control levels, while after 3000 mg progesterone per day the levels in oviduct and uterus exceeded those of controls by five and seven times, respectively. In gilts treated with 750 mg progesterone per day, plasma progesterone levels were 177.4 ± 22.1 ng/ml ($\text{x}\text{±}\text{SD}$) on day 3 and 186.4 ± 69.2 ng/ml on day 5 and resembled values found in superovulated pigs with more than 40 ovulations. Excessive plasma progesterone values of 1014.6 ± 840.4 ng/ml on day 3 and 473.2 ± 197.2 ng/ml on day 5 were found after treatment with 3000 mg of progesterone per day. Treatment with up to 750 mg of exogenous progesterone per day, did not affect embryonic development, but 3000 mg per day resulted in a significantly (P<0.001) higher percentage of retarded and degenerate embryos compared to controls (71.8% versus 3.2%).In addition, the amount and specificity of uptake of ³H-labelled progesterone and estradiol-17 beta by pig blastocysts recovered from superovulated gilts were investigated after 6 hrs in vitro culture. The uptake of ³H-progesterone was 131.9 ± 56.9 counts per million (cpm) per 10 blastocysts, corresponding to 1.1 fmoles progesterone. The uptake was non-specific for it was only slightly reduced in the presence of a 100-fold excess of unlabelled progesterone (20.1%) or estradiol-17 beta (27.0%). The uptake of ³H-estradiol-17 beta was 133.9 ± 74.12 cpm per 10 blastocysts, corresponding to 1.3 fmoles estradiol-17 beta. The uptake was significantly (P<0.01) reduced by 67.7% in the presence of a 100-fold excess of unlabelled estradiol-17 beta. Apparent specific binding was 0.87 fmoles estradiol-17 beta per 10 blastocysts or 72.5 fmoles estradiol-17 beta per mg protein. The uptake was only slightly reduced in the presence of a 100-fold excess of unlabelled progesterone (23.3%). This significant inhibition could be determined after 2 hrs in vitro culture. There was no competitive inhibition after 20 min. of culture.Uptake by unfertilized ova and degenerate embryos recovered on day 5 was significantly smaller (51.8 ± 10.3 cpm per 10 eggs; P<0.001) than by blastocysts recovered on the same day. No competitive inhibition could then be determined. In vivo, preimplantation pig embryos seem to be rather insensitive to high progesterone levels. Excessive amounts of progesterone probably can be metabolized to a great extent. Progesterone seems to be taken up rather non-specifically by the pig embryo. The uptake and binding of estradiol-17 beta seems to be more specific. Studies are in progress to investigate the physiological role of estradiol-17 beta uptake in early embryonic development.     

Uterine vein prostaglandin levels in late pregnant dogs

We studied the uterine venous plasma concentrations of prostaglandins E₂, F_2α, 15 keto 13,14 dihydro E₂ and 15 keto 13,14 dihydro F_2α in late pregnant dogs in order to evaluate the rates of production and metabolism of prostaglandin E₂ and F_2α in pregnancy $\text{in vivo}$. We used a very specific and sensitive gas chromatography-mass spectrometry assay to measure these prostaglandins. The uterine venous concentrations of prostaglandin E₂ and 15 keto 13,14 dihydro E₂ were 1.35±.27 ng/ml and 1.89±.37 ng/ml, respectively; however, we could not find any prostaglandin F_2α and very little of its plasma metabolite in uterine venous plasma. Since uterine microsomes can generate prostaglandin F_2α and E₂ from endoperoxides, prostaglandin F_2α production $\text{in vivo}$ must be regulated through an enzymatic step after endoperoxide formation. Prostaglandin E₂ is produced by pregnant canine uterus in quantities high enough to have a biological effect in late pregnancy; however, prostaglandin F_2α does not appear to play a role at this stage of pregnancy.     

Luteinizing hormone,total estrogens and progesterone secretion during lactation and after weaning in sows

Two experiments were conducted to determine changes in serum concentrations of LH, total free estrogens and progesterone before and after weaning in sows. Blood was collected either via indwelling anterior vena cava cannula or by venipuncture and serum hormones were measured by radioimmunoassay. In Exp. I, blood was collected at 15-min intervals for 4 hr on day 7 and day 21 postpartum from three sows on each day. In addition, individual samples were collected from 10 sows on days 4 and 14 postpartum and from 11 sows on days 1, 3 and 5 after weaning (day 23 postpartum). Serum LH ranged from .2 to .8 ng/ml during lactation and averaged 1.1 ± .7, 1.1 ± .7 and 2.7 ± .7 on days 1, 3 and 5 after weaning, respectively. Progesterone was low (< 1 ng/ml) during lactation and averaged 1.9 ± .3, .6 ± .3 and 1.2 ± .3 on days 1, 3 and 5 after weaning. Estrogens were variable during lactation, averaged 121 ± 36 pg/ml on day 1 after weaning and decreased thereafter. Estrus began on day 3 after weaning in 1 sow and on day 5 in the remaining 10 sows.In Exp. II, blood was collected from seven sows at 12 to 24 hr intervals from 2 days before until 5 days after weaning (day 26 postpartum). Mean serum LH was .7 ± .1 ng/ml during 48 hr before weaning and remained unchanged after weaning until day 3 when LH increased to 6.1 ± .8 ng/ml. Serum LH concentrations then declined to 1.3 ± .8 and .9 ± .8 ng/ml on days 4 and 5 after weaning. Total estrogens averaged 31 ± 4 pg/ml during 48 hr prior to weaning and 32 ± 4, 43 ± 17, 28 ± 1, 30 ± 2, 16 ± 2 and 18 ± 2 on days 0 to 5 after weaning. Progesterone increased from 1.0 ± .3 ng/ml 24 hr before weaning to 3.0 ± .3 at weaning and then remained low (< 1 ng/ml) until after ovulation when progesterone increased. Estrus began on day 4 after weaning in all seven sows.Results from these two experiments indicate that in sows: (1) LH is suppressed during early lactation (day 7), gradually increases during late lactation (day 21) and then reaches peak concentrations after weaning near the onset of estrus, (2) estrogens increase between weaning and estrus and decline thereafter, and (3) progesterone rises transiently at weaning and then increases after estrus and ovulation.     

The effects of prostacyclin (PGI2) and 6-keto-PGF1α on bovine plasma progesterone and LH concentrations

Injections of 1 mg PGI₂ directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI₂-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF_1α were without effect on plasma progesterone levels. The luteotrophic effect of PGI₂ was not due to alterations in circulating LH concentrations. An $\text{in vitro}$ experiment assessed the effects of either PGI₂ alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 μg PGI₂, 10 μg PGI₂, and 10 μg PGI₂ plus 5 ng LH averaged 99 ± 42, 353 ± 70, 152 ± 35, 252 ± 45, and 287 ± 66 ng/ml (n=4), respectively. Thus PGI₂ has luteotrophic effects on the bovine CL both $\text{in vivo}$ and $\text{in vitro}$.     

Synchronization of estrus in cyclic beef heifers with the prostaglandin analog alfaprostol

Seventy-eight Hereford-Angus crossbred heifers were injected intramuscularly twice with 6 mg of alfaprostol^b in 6 ml of propylene glycol. On each representative day of a 20-day estrous cycle (estrus = Day 0), either three or four heifers received their first injection. The second injection was given 12 days after the first, regardless of the response to the first injection. Thirty-nine heifers were not treated. The first alfaprostol injection reduced serum progesterone to less than 1 ng/ml in all heifers injected after Day 4. A total of 79.5% ($\text{62}\text{78}$) of the heifers exhibited estrus by five days after the first injection. Average interval from injection to estrus was 63 hours. The second injection occurred on Days 6 through 16 for all but one heifer, with 75.6% ($\text{59}\text{78}$) falling on Days 8 through 11 of the estrous cycle. Estrus was detected in 93.6% ($\text{73}\text{78}$) of the heifers within five days after the second injection, with an average interval to estrus of 66 hours.Day of cycle at second injection did not affect the interval to estrus. Conception occurred in 79.4% ($\text{58}\text{73}$) of the heifers inseminated in the five days after the second injection. Occurrence of estrus and conception was no different in treated heifers after five days of the insemination period than in nontreated heifers after 21 days of the insemination period, where 94.9% ($\text{37}\text{39}$) were observed in estrus and 83.8% ($\text{31}\text{37}$) conceived. Overall percent conception for a 55-day insemination period was 89.7 ($\text{70}\text{78}$) for treated and 87.2 ($\text{34}\text{39}$) for nontreated heifers. Day of cycle at first or second injection did not affect conception after the second injection. Some signs of estrus were observed in 11 of the 16 heifers injected before Day 5.A second trial to determine if alfaprostol induced luteolysis early in the cycle was conducted. Twenty purebred Angus, Hereford, or Simmental heifers received either one or two injections of alfaprostol on either Day 1, 2, 3, or 4. Only five heifers showed any signs of estrus, and the three that were inseminated did not conceive. Subsequent cycle length indicated that luteolysis occurred in only one heifer.Data suggest that alfaprostol is an effective luteolytic agent in cyclic beef heifers after Day 4 and that two injections 12 days apart will effectively synchronize estrus in heifers when distributed throughout the cycle at the first injection without affecting conception rate.     

The measurement of 18-hydroxy-11-deoxycorticosterone in human plasma by radioimmunoassay

A radioimmunoassay method for the measurement of plasma levels of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) has been developed. The antiserum against 18-OH-DOC was produced in rabbits immunized against 18-OH-DOC-3-oxime-bovine serum albumin. Plasma (1–2 ml) was extracted with dichloromethane and chromatographed on paper. The purified extracts were incubated with antiserum at a $\text{1}\text{22,000}$ dilution for $\text{1}\text{2}$ hour at 37°C and for 2 hours at 4°C. Saturated ammonium sulfate was used to separate free from bound 18-OH-DOC. 1, 2-³H-18-OH-DOC was added to all samples to correct for losses and to determine the percent free. Pyridine (0.1%) was added to solvents to maintain the stability of 18-OH-DOC. Recovery after extraction was 58 ± 8 (S.D.)%. The accuracy and precision of the method were acceptable, and a sensitivity of 2 pg per sample enabled the measurement of very low levels of 18-OH-DOC. High specificity was demonstrated by a low blank value (0 ± 0.2 pg) and by demonstrating that alternative paper chromatography separation systems gave results not differing significantly from those obtained by the present method. The mean 8AM plasma 18-OH-DOC level was 8.5 ± 1.2 ng per 100 ml in 18 normotensive control subjects. There was a marked response of plasma 18-OH-DOC to ACTH stimulation and dexamethasone suppression and a significant increase after 3 hours upright posture.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.     

Fertility of ram semen after storage at 5°C

In five experiments, fertilization, early (18–19-day) pregnancy, and lambing were examined after insemination with semen stored at 5°C in tris-fructose-egg yolk diluent.After deposition into uterine horns by surgical insemination of semen stored for 0 (control), 2, 4, 6, 8, 9 or 10 days, fertilized eggs were recovered in $\text{32}\text{34}\text{,}\text{16}\text{16}\text{,}\text{21}\text{22}\text{,}\text{15}\text{20}$, $\text{9}\text{17}\text{,}\text{2}\text{18}$ and $\text{1}\text{15}$ ewes; the 18–19-day pregnancy rates determined by progesterone assay were $\text{32}\text{48}\text{,}\text{15}\text{28}\text{,}\text{11}\text{20}\text{,}\text{12}\text{20}\text{,}\text{9}\text{20}$, $\text{2}\text{20}$ and $\text{1}\text{21}$ for the respective storage periods. There was a linear decrease in fertilization rates beyond 4 days of storage and in early pregnancy rates after 6 days of storage (P<0.001). The decline with time of storage in the fertilization rate was not associated with an increase in early embryonic loss. Surgical insemination with semen stored for 0, 4, 6, 8, 9 and 10 days resulted in 53, 35, 40, 25, 5, and 0% lambing.Single cervical (normal) insemination of a total of 281 ewes with 0, 1, 2 or 3-day-old semen, using within each semen treatment 90 × 10⁶ and 180 × 10⁶ spermatozoa, yielded mean lambing rates of 60.0, 34.3, 33.8, and 17.1%; and after using 150 × 10⁶ and 300 × 10⁶ spermatozoa in a total of 393 ewes the mean lambing rates for the above semen treatments were 69.0, 46.4, 36.1, and 24.2% (linear, P < 0.001). In both tests the lambing results were better after insemination of the higher number of spermatozoa, but the slope of decline in fertility with age of semen was not affected by the sperm dose.When single and double cervical inseminations were performed in a total of 411 ewes, with 150 × 10⁶ and 300 × 10⁶ spermatozoa per inseminate, the lambing rates for semen stored for 0, 1, 2 and 3 days were 57.7, 30.4, 26.8, and 4.7% after single insemination, and 66.7, 56.8, 46.4, and 41.5% after double inseminations. The sperm dose within method of insemination and semen treatment had no effect. The lambing rate was better after double than single insemination (P<0.001), but the slope of decline in fertility with age of semen was not significantly affected by number of inseminations.In the final experiment, involving 408 ewes, 300 μg of prostaglandin F₂α added to the inseminate did not improve the fertility of fresh semen or semen stored for 1 day.     

Semen backflow after insemination and its effect on fertilisation results in sows

The aim of the present study was to investigate the volume of and number of spermatozoa in semen backflow during and after insemination, and the effect of backflow on fertilisation results assessed at day 5 of pregnancy. Multiparous sows (n=140) were artificially inseminated with either (1, 3 or 6)×10⁹ mixed spermatozoa from three boars in a constant volume of 80 ml. Backflow of semen was measured three times: during insemination (M1); during the first half hour after insemination (M2); and from 0.5 h until about 2.5 h after insemination (M3). Transrectal ultrasonography was performed at intervals of 4 h to determine the time of ovulation. Sows were sacrificed at 120±0.4 h after ovulation to assess the results of fertilisation. Every sow had some backflow and the variation in volume, and number of spermatozoa within the backflow was high. The average semen backflow within 2.5 h after insemination was 70±3.4% of the volume and 25±1.4% of the spermatozoa of the inseminated dosage. The concentration of the backflow (% of the inseminated dosage) decreased with time after insemination from 65% at M1 to 40% and 26% at M2 and M3, respectively. The correlations between volume and number of spermatozoa were high: r=0.97, r=0.73 and r=0.81 in M1, M2 and M3, respectively. More than 5% of the inseminated spermatozoa in backflow during insemination affected fertilisation negatively in those sows inseminated with 1×10⁹ spermatozoa (P<0.05). Backflow after insemination had no effect on fertilisation results (P>0.05). Timing of insemination relative to ovulation and oestrus were not related to backflow during or after insemination (P>0.05). Of the sows which had backflow, those of parity 1 tended to have the highest proportion of sows with more than 5 ml backflow (47%; n=8 of 17) compared with sows from parity 2 and higher (24%; n=14 of 59) (P=0.075). It was concluded that excessive backflow of semen during insemination had a negative effect on fertilisation results when sows where inseminated with only 1×10⁹ spermatozoa. Causes of variation in backflow between sows were not clearly identifiable.     

Plasma relaxin concentrations in the pig during the periparturient period: Association with prolactin,estrogen and progesterone concentrations

Concentrations of relaxin, prolactin, unchromatographed estradiol 17_β (E_2β) and progesterone (P₄) were measured in serial samples of inferior vena caval blood, in three pigs, during late pregnancy, and parturition. Maximal relaxin concentrations occurred 60 to 24h before parturition, and ranged from 60 to 286ng/ml. Prolactin concentrations increased from 12.5ng/ml, 48 to 36 hours before parturition, to between 79 to 184ng/ml. At the time of the relaxin surge, E_2β levels were high, and P₄ concentrations were decreasing, thus raising the $\text{E}{}_{\mathrm{2\beta }}\text{P}{}_4$ ratio. A surge in prolactin concentrations followed upon a decline of P₄ to less than 10ng/ml, coinciding with the increase in relaxin concentrations in 2 gilts, and following the surge in relaxin in the third. Udder development occurred near the time of increased relaxin concentrations. ‘Milk let down’ followed maximal relaxin and prolactin concentrations in two gilts, and the increase in prolactin, rather than the increase in relaxin concentration, in the third.     

Direct effect of estradiol-17β on progesterone accumulation by ovarian granulosa cells from rhesus monkeys

We have previously demonstrated that estradiol-17β (E₂) administered in vivo induces atresia of the dominant ovarian follicle (DF). Whether this effect is exerted directly at the ovarian level or by central mediation has not been confirmed. The present study was designed to assess whether E₂ in amounts similar to those found in monkey follicular fluid (FF) directly alters in vitro progesterone (P) accumulation by granulosa cells (GC) aspirated from follicles in cycling rhesus monkeys. Follicular contents were aspirated from three to five animals on each of days 8–13 of the cycle. GC were plated at a density of 50,000 viable GC/0.5 ml medium; GC were incubated with 0 or 2–2,000 ng/ml E₂, and cultures were maintained for 72 h. P accumulation by GC collected on day 8 and treated with 2 ng/ml E₂ was augmented 37.5 ± 5.5% (X ± S. E. M.; P<.05) over controls but was diminished significantly at 20 ng/ml ( ?55 ± 18% with respect to controls), 200 ng/ml ( ?73.7 ± 13.2%), and 2,000 ng/ml ( ?77.3 ± 18.4%). A similar dose-response relationship was noted on other cycle days. At a concentration of 2,000 ng/ml, E₂ significantly reduced P 91.5 ± 8.5% (day 10), 81.5 ± 18.5% (day 11), 84.3 ± 4.7% (day 12), and 53.7 ± 15.8% (day 13). We conclude that E₂ at concentrations found in FF can inhibit P output by monkey GC through a direct action.     

The influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells in vitro

$\text{In vitro}$ experiments were performed on mouse spleen cells to establish possible influences of some naturally occurring prostaglandins on DNA synthesis and DNA excision repair. The prostaglandins A₁, B₁, E₁, E₂ and F₂ were tested in concentrations of lopg, 5ng and 2.5μg per ml cell suspension. DNA synthesis was significantly increased by pgF_2α in all the three concentrations tested, while the other tested prostaglandins were essentially ineffective. DNA excision repair was significantly inhibited by PgF₁ and PgF₂ at 5ng/'ml and at 2.5μg/ml but increased by PgF_2α in the two lower concentrations. The rejoining of DNA-strand breaks after γ-irradiation was slightly reduced by PgF₁, PgE₂ and PgF_2α at 2.5μg/ml.     

Maternal estrogen and progesterone levels after hypophysectomy in early pregnancy and in term fetuses or newborn monkeys

Pregnant rhesus monkeys ($\text{Macaca}$$\text{mulatta}$) were hypophysectomized at 8–10 weeks gestation to determine effects on plasma levels of estrone (E₁), estradiol-17β (E₂) and progesterone (P). The first group of monkeys was subsequently fetectomized At 107–114 days. After hypophysectomy there was an initial decrease in maternal peripheral plasma E₂ followed by a rise to preoperative levels within 4–5 weeks. Plasma levels of e₁ and P were not markedly altered. After fetectomy, peripheral estrogen concentrations, especially E₂, declined markedly. In the second experimental series, we have examined the effects of maternal hypophysectomy on levels of E₁, E₂ and P either (1) in both mother and newborn baby or (2) in mother, term fetus and umbilical vein. Groups of hypophysectomized and intact pregnant monkeys (3 each) were delivered by cesarean section at the expected time of parturition. Other hypophysectomized and intact monkeys (2 each) delivered normally. E₂ levels were elevated significantly in plasma of hypophysectomized monkeys at the time of cesarean delivery and in newborn babies of hypophysectomized mothers shortly after parturition. Except for these differences, the maternal hypophysis apparently is not a major factor in the control of E₁, E₂ and P concentrations in pregnant rhesus monkeys.     

Detection of leukotriene C4-like immunoreactivity in tear fluid from subjects challenged with specific allergen

Tear fluid was obtained from allergic subjects from control eyes and eyes challenged with specific allergen and levels of leukotriene C₄ (LTC₄)-immunoreactivity determined by radioimmunoassay. Formal identification of the leukotrienes released was not possible but the levels of LTC₄-immunoreactive material in allergen-challenged tear fluid (4.9 ± 2.3 ng/ml, n = 9) were significantly higher (p < 0.01) than those in control tear fluid (0.07 ± 0.06 ng/ml, n = 9). These results provide evidence that leukotrienes, which account for the biological activity of slow reacting substance of anaphylaxis, may be released in allergic reactions $\text{in vivo}$ in man.     

The falling drop method for deuterium oxide: Factors influencing accuracy

Factors affecting the accuracy of the falling drop method for D₂O were considered: selection of temperature of the constant-temperature bath, permissible temperature fluctuations, and D₂O concentration. Bath temperatures used, 27.24 to 27.32°, and constancy within ±0.002° had no influence on the regression relating drop velocity to concentration, v = a + bc, or sampling error (S.D._v). The latter increases significantly with concentration from 0.05 to 0.23 ml % D₂O, is significantly smaller than experimental error (S.D._e), and is inappropriate for estimating intra-assay limits. More appropriate is the S.D._c derived from the variance of the terms of the regression equation. Assay results using this error term can be expected to vary from about ±4.8% at 0.05 ml % to about ±2% at 0.23 ml %. These results compare favorably with those reported for the mass spectrograph used to determine the mass ratio of $\text{HDO}\text{H}{}_2\text{O}$ vapor. A more conservative estimate is obtained by using $\text{λ}\text{=}\text{S.D.}{}_{\mathrm{e}}\text{b}$, which in this work indicated within-assay variability of ±12.4% at 0.05 ml % and ±2.7% at 0.23 ml % D₂O. The term S.D._e, corresponding to 0.0062 ml % in these experiments, provides the best means for assay comparisons.Satisfactory recoveries of D₂O added to urine averaging 99% of known values were obtained after shell-freezing, without distillation of standards and without permanganate oxidation of samples. No increase in error beyond that anticipated from standards alone was observed when urine was the vehicle.     

Vaginal temperature is not related to the time of ovulation in sows

The relationship between vaginal temperature and ovulation time was studied in sows. The vaginal temperature was measured continuously between Day 4 and Day 10 after Altrenogest-treatment in 10 sows. Oestrus was checked with a vasectomized boar at 8-h intervals, and during oestrus, ovulation time was checked with transrectal ultrasonography at 2-h intervals between 07:00 h and 23:00 h. Two sows ovulated between 23:00 h and 07:00 h, and these sows were taken out of the experiment. In the eight remaining sows, a clear day/night rhythm in vaginal temperature was found: between 03:00 h and 09:00 h, vaginal temperature (LSM ± sem, corrected for sow) was on average 38.2 ± 0.01°C; between 15:00 h and 21:00 h, vaginal temperature was on average 38.5 ± 0.01°C (P < 0.001). Between 4 days before ovulation and 2 days after ovulation, no changes in temperature could be found that were related to ovulation time in any of the sows. Therefore, in sows, changes in vaginal temperature cannot be used as a predictor for ovulation time, and consequently cannot be used to predict the best time for insemination.     

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E1, E2 and I21

Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the $\text{in}$$\text{vitro}$ effects of luteinizing hormone (LH) and prostaglandins (PG) E₁, E₂ and I₂. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE₂, PGE₂, or PGI₂ (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P < 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E₁ and E₂ had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI₂ was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE₂ upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE₂ caused an increase (P < 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E₁, E₂ and I₂ are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.