Early pregnancy detection of Iraqi riverine buffalo (Bubalus bubalis) using the BioPRYN enzyme-linked immunosorbent assay for PSPB and the progesterone assay.

This study was undertaken to detect pregnancy in Iraqi riverine buffalo (Bubalus bubalis) using three different methods (rectal palpation, plasma progesterone concentration and detection of the presence of pregnancy-specific protein B (PSPB) with the BioPRYN(®) enzyme-linked immunosorbent assay (ELISA) test. The aim of the study was to identify the most sensitive, early and accurate method for detecting pregnancy. Twenty-two female riverine buffalo that were 6.0 ± 0.93 years old were used. Four blood samples per buffalo were taken via jugular venipuncture at days 22-24, 32-34, 42-44 and 58-61 post-mating (PM) to measure the progesterone concentration (ng/ml) and to detect the presence of plasma PSPB. The rectal palpation method was employed to evaluate all buffalo on days 42-44 and 58-61 PM. The BioPRYN(®) test differed (p<0.01) from the other tests with earlier accuracy for detecting pregnant and non-pregnant buffalo. Eighty-eight percent of pregnant and 76.9% of non-pregnant buffalo were distinguished early (days 22-24 PM) using BioPRYN(®) and plasma PSPB-ELISA level (2.09 ± 0.12 ng/ml) in relation to 66.7% and 53.9% detected using the progesterone assay at similar days (4.30 ± 0.40 ng/ml). In conclusion, these results described, for the first time, the early and accurate pregnancy detection of water riverine buffalo using BioPRYN(®) technology and provided the plasma levels of PSPB using an ELISA test. These findings will improve the reproductive and productive efficiency of Iraqi riverine buffalo by adapting the recent management and reproductive strategies in Iraq and in the world.

Partial purification and characterization of somatomedin from sheep serum.

A rapid, high yield preparative technique for the isolation of sheep somatomedin is reported. Purification of biologically active somatomedin from the 60% ammonium sulfate precipitate of sheep serum was accomplished using three gentle fractionation steps. Biological activity during purification was monitored using the rat adipocyte nonsuppressible insulin-like activity (NSILA) assay. A stepwise pH elution (pH 2.85, 3.5, 4.5, and 6.0) from SP-Sephadex resulted in the elimination of more than 99% of the serum proteins and a 500-fold enhancement of biological activity. The active fraction eluted at pH 6.0 and was further fractionated on Sephadex G-50 (fine) chromatography at pH 2.85. This resulted in about a 10,000-fold enhancement of activity over serum activity. The most active fractions from Sephadex chromatography were further separated on reverse phase HPLC in 0.1% trifluoroacetic acid using a linear gradient of 24-60% acetonitrile. The biological activity of the final preparation was enhanced 61,000- to 182,000-fold over that of serum (mean, 93,000-fold) when assayed in the NSILA assay. Protein yield was estimated to be 467 micrograms/liter serum. In addition to the NSILA activity, the final preparation demonstrated dose-dependent sulfation factor activity in the embryonic chick pelvic leaflet bioassay. Sheep somatomedin was active at physiological levels in both bioassays. Analysis of the somatomedin preparation by sodium dodecyl sulfate-electrophoresis at pH 8.8 showed that it was homogeneous by this criterion. The activity eluted from Sephadex G-50 was estimated to have a molecular size of 6900. Two Coomassie blue-stained bands were present in the final sheep somatomedin preparation after polyacrylamide gel electrophoresis at pH 3.2. Our purification process is a rapid, high yield technique which yields a polypeptide fraction enriched in NSILA and somatomedin-like activity. The molecular size and biological activity in the NSILA and sulfation factor assays suggest that our sheep NSILA is analogous to somatomedins purified from other species of animals.

Pregnancy-specific protein B induces release of an alpha chemokine in bovine endometrium.

Pregnancy-specific protein B (PSPB), is secreted from binucleate trophoblast of the bovine conceptus as early as day 15 of pregnancy. The objective of this experiment was to determine if PSPB induced uterine proteins. PSPB was purified from day 120 cotyledons using antibody-based affinity chromatography. Endometrium from day 14 nonpregnant cows (n = 3) was prepared for explant (3H-Leu added) culture. Radiolabeled proteins released into medium were dialyzed, separated using 1D-PAGE, and detected using fluorography and densitometry. PSPB (0, 0.5, 5, 25 & 50 nM) caused a concentration-dependent increase in the release of a radiolabeled 8-kDa uterine protein. Western blots revealed that the 8-kDa protein cross-reacted with antibody against granulocyte chemotactic protein-2 (GCP-2). PSPB also induced release of GCP-2 by bovine endometrial (BEND) cells in primary culture. The induction of GCP-2 by PSPB was blocked by addition of antiserum against PSPB (1:4 molar ratio). This is the first indication that PSPB has a hormonal role in inducing GCP-2, an alpha chemokine that also is induced by interferon-tau during early pregnancy. This chemotactic cytokine may be integral to mediating adhesion, inflammation and angiogenesis associated with early implantation.

Growth rate and secretion of pituitary hormones in relation to age and chronic treatment with thyrotropin-releasing hormone in prepubertal dairy heifers.

Holstein heifers were treated with synthetic thyrotropin-releasing hormone (TRH) or saline twice daily from one week through 6 mo of age. Plasma concentrations of prolactin (PRL) and thyrotropin (TSH) were elevated (P less than .01) within 30 min after the first TRH injection (1 week of age). At 1 and 3 mo of treatment, PRL and TSH increased in response to TRH, although the TSH response was reduced (P less than .01) as compared to the first day of treatment. Although plasma growth hormone (GH) appeared to be elevated following the first TRH injection, this effect was not statistically significant (P less than .05), nor was it significantly influenced by treatment following subsequent TRH injections. None of the 3 hormones, PRL, TSH or GH, was elevated following the final TRH injection at 6 mo of age. In contrast, plasma concentrations of PRL and TSH were increased in a control heifer injected with TRH at 6 mo. These data indicate that hormonal responsiveness to TRH stimulation decreases with continued twice daily treatment at doses of TRH used in the present studies. Examination of weight gains indicated that chronic treatment with TRH was associated with increased growth rate through 6 mo of age (10.6% increased average daily gains P less than .10), which was exhibited in a steeper slope (P less than .05) of the growth curve in the TRH group. Feed intake was slightly greater in TRH heifers, although feed efficiency (kg feed/kg gain) was not different between the two groups. Plasma concentrations of PRL increased (P less than .01) with age (r = +0.938) in control heifers while plasma TSH and GH were not significantly related to age. This observation establishes a positive correlative relationship between PRL secretion and the approach of puberty in the dairly heirfer. It was also noted that elevation of PRL secretion by TRH treatment was associated with significant advancement of age at first observed estrus (9.4 vs. - 10.5 mo) suggesting that a functional relationship between PRL secretion and puberty may exist in dairy heifers.

Effect of the aromatase inhibitor CGS-16949A on pregnancy and secretion of progesterone, estradiol-17beta, prostaglandins E and F2alpha (PGE; PGF2alpha) and pregnancy specific protein B (PSPB) in 90-day ovariectomized pregnant ewes.

The aromatase inhibitor CGS-16949A was used to determine whether CGS-16949A altered secretion of progesterone, estradiol-17beta, PGE (PGE1 + PGE2), PGF2alpha and PSPB. Ninety day pregnant ewes were ovariectomized and received vehicle, PGF2alpha, CGS-16949A or PGF2alpha+CGS-16949A. None of the ewes treated with PGF2alpha, CGS-16949A or PGF2alpha+CGS-16949A aborted (P > or = 0.05) during the 108-h experimental period. Treatment with CGS-16949A lowered (P < or = 0.05) progesterone in jugular venous plasma but concentrations of progesterone were not affected (P > or = 0.05) by treatment with PGF2alpha. Concentrations of estradiol-17beta and PSPB in jugular venous plasma and PGE in inferior vena cava plasma were decreased (P < or = 0.05) by treatment with CGS-16949A. Concentrations of PGF2alpha in inferior vena cava plasma were not affected (P > or = 0.05) by treatment with CGS-16949A. Decreases in estradiol-17beta occurred before decreases in PSPB, which was then followed by decreases in PGE (P < or = 0.05). It is concluded that these data support the hypothesis that estradiol-17beta regulates placental secretion of PSPB; PSPB regulates placental secretion of PGE; and PGE regulates placental secretion of progesterone during mid-pregnancy in ewes.

Effect of mifepristone on pregnancy, pregnancy-specific protein B (PSPB), progesterone, estradiol-17beta, prostaglandin F2alpha (PGF2alpha) and prostaglandin E (PGE) in ovariectomized 90-day pregnant ewes.

The objective of this experiment was to determine the effect of mifepristone, a progesterone receptor antagonist, on pregnancy and secretion of steroids, pregnancy-specific protein B (PSPB) and prostaglandins at mid-pregnancy in ewes. Ninety-day pregnant ewes were ovariectomized (OVX) and treatments were initiated 72 h post-OVX. Ewes received (1) vehicle, (2) prostaglandin F2alpha (PGF2alpha, 8 mg/58 kg/bw, i.m.) 84 h post-OVX, (3) mifepristone (50 mg intrajugular at 72, 84, 96, and 108 h post-OVX), (4) mifepristone (50mg) + PGF2alpha, (5) mifepristone (100 mg intrajugular at 72, 84, 96, and 108 h), and (6) mifepristone (100 mg) + PGF2alpha. Ewes treated with vehicle or PGF2alpha alone did not abort (P > or = 0.05). But, 60, 80, 60, and 100% of ewes treated with mifepristone (50 mg), mifepristone (50 mg) + PGF2alpha, mifepristone (100 mg), and mifepristone (100 mg) + PGF2alpha, respectively, aborted (P < or = 0.05). Profiles of progesterone, estradiol-17beta, prostaglandin E (PGE), or PSPB did not differ (P > or = 0.05) among treatment groups. Profiles of PGF2alpha of treatment groups receiving mifepristone with or without PGF2alpha differed (P < 0.05) from vehicle or PGF2alpha alone-treated ewes. It is concluded that progesterone actions are necessary to suppress uterine/placental secretion of PGF2alpha and that maintenance of critical progesterone: estradiol-17beta and PGE:PGF2alpha ratios are necessary for maintenance of pregnancy.

Effect of luteinizing hormone (LH), PGE2, 8-EPI-PGE1, 8-EPI-PGE2, trichosanthin, and pregnancy specific protein B (PSPB) on secretion of progesterone in vitro by corpora lutea (CL) from nonpregnant and pregnant cows.

Secretion of progesterone by Day 14 bovine corpora lutea (CL) of the estrous cycle and Day 200 CL of pregnancy was evaluated in vitro to determine what regulates secretion of progesterone by CL of pregnancy. Weights of Day 200 CL of pregnancy (4356 +/- 223 g) were heavier when compared to Day 14 CL of the estrous cycle of Brahman cows (3643 +/- 128 g; p < or = 0.05); however, both Day 14 and Day 200 minced CL slices secreted similar basal amounts of progesterone per unit mass (p > or = 0.05). Secretion of progesterone in vitro by Day 14 CL of the estrous cycle was increased at 4 and 8 h (p < or = 0.05) by 10 or 100 ng/mL luteinizing hormone (LH) and did not differ between doses (p > or = 0.05). Progesterone secretion in vitro by Day 200 CL of pregnancy was not increased (p > or = 0.05) by LH at 4 or 8 h. However, progesterone secretion in vitro by Day 14 CL of the estrous cycle or Day 200 CL of pregnancy was increased (p < or = 0.05) at 4 h by 10 or 100 ng/mL PGE2, which did not differ by dose or reproductive status (p > or = 0.05). At 8 h, Day 14 CL of the estrous cycle secretion of progesterone in vitro was increased (p < or = 0.05) by both doses of PGE2 but only at 8 h by 100 ng/mL from Day 200 CL of pregnancy (p < or = 0.05). Secretion of progesterone in vitro was not affected (p > or = 0.05) by 10 or 100 ng/mL 8-Epi-PGE1 or 8-Epi-PGE2 at 4 or 8 h from Day 14 CL of the estrous cycle or Day 200 of pregnancy. Trichosanthin increased (p < or = 0.05) secretion of progesterone in vitro by 10 ng/mL at 4 h and at 8 h by Day 14 CL of the estrous cycle or at 8 h by Day 200 CL of pregnancy but trichosanthin at 100 ng/mL did not affect (p > or = 0.05) secretion of progesterone in vitro by Day 14 CL of the estrous cycle or Day 200 CL of pregnancy at 4 or 8 h. Pregnancy specific protein B (PSPB) increased (p < or = 0.05) secretion of progesterone in vitro at 4 and 8 h by Day 14 CL of the estrous cycle and did not differ between incubation times (p > or = 0.05). PSPB increased secretion of progesterone at 4 h but not at 8 h (p > or = 0.05) by Day 200 CL of pregnancy. These data suggest that PGE2 or PSPB but not LH, 8-Epi-PGE1 or 8-Epi-PGE2 regulates luteal secretion of progesterone by bovine CL at mid-pregnancy. In addition, it is suggested that weights of bovine CL of pregnancy increase to compensate for a lack of placental secretion of progesterone.

Effects of luteinizing hormone (LH), PGE2, 8-Epi-PGE1, 8-Epi-PGF2 alpha, trichosanthin and pregnancy specific protein B (PSPB) on secretion of prostaglandin (PG) E (PGE) or F2 alpha (PGF2 alpha) in vitro by corpora lutea (CL) from nonpregnant and pregnant cows.

Both Day 14 corpora lutea (CL) of the estrous cycle and Day 200 CL of pregnancy secrete detectable prostaglandin E (PGE) and prostaglandin F2 alpha (PGF2 alpha) in vitro. Corpora lutea from Day 200 pregnant cows secrete more PGE and PGF alpha in vitro than Day 14 CL of the estrous cycle when incubated in control medium without treatments (p < or = 0.05). In addition, secretion of both PGE and PGF2 alpha in vitro by both Day 200 CL of pregnancy and Day 14 of the estrous cycle increase (p < or = 0.05) with time in culture in the absence of treatments. The PGE:PGF2 alpha ratio secreted at 4 h in the absence of treatments by Day 14 CL of the estrous cycle was 1.2 and at 8 h was 1.0 and did not differ (p > or = 0.05), while the PGE:PGF2 alpha ratio secreted by 200 day CL of pregnancy in the absence of treatments at 4 h was 0.8 and at 8 h decreased (p < or = 0.05) to 0.4. The PGE:PGF2 alpha ratio at 8 h by 200 day CL of pregnancy was lower (p < or = 0.05) than in the Day 14 CL of the estrous cycle at 4 or 8 h. Secretion of PGE or PGF2 alpha was affected by luteinizing hormone, PGE2, 8-Epi-PGE1, 8-Epi-PGE2, trichosanthin, and pregnancy specific protein B (PSPB) and was time and dose dependent (p < or = 0.05). In summary, the altered ratio of PGE:PGF2 alpha may explain the decreased secretion of progesterone at 8 h by Day 200 CL of pregnancy reported previously from the same samples. In addition, caution should be exercised in interpretation of progesterone secretion data with bovine CL studies in vitro. Also, PSPB may play an indirect role through PGE to regulate bovine luteal secretion of progesterone.

PGE2 induces its own secretion in vitro by bovine 270-day placenta but not by 200-day placenta.

Two separate experiments were conducted to determine whether prostaglandin (PG) E2 stimulates the secretion of progesterone by 270- or 200-day Brahman placentas in vitro. Secretion of progesterone, PGF2alpha, pregnancy specific protein B, or estradiol-17beta by 270-day Brahman placentas was not affected (p > or = 0.05) by PGE2, during the 4-h incubation period at the doses tested. Indomethacin or meclofenamic acid decreased (p < or = 0.05) 270-day Brahman placental secretion of PGE and PGF2alpha by 98 and 60%, respectively. However, PGE2 induced (p < or = 0.05) its own secretion, but not the secretion of PGF2alpha (p > or = 0.05), by 270-day Brahman placentas, even in the presence of indomethacin or meclofenamic acid at a dose of 100 ng/mL. Also, secretion of 8-Epi-PGE2 by Day 270 Brahman placentas was increased (p < or = 0.05) by PGE2. Secretion of progesterone, estradiol-17beta, or pregnancy specific protein B by 200-day Brahman placentas was not affected by PGE2, 8-Epi-PGE2, PGF2alpha, estradiol-17beta, or trichosanthin during the 4- or 8-h incubation period (p > or = 0.05). Secretion of estradiol-17beta at 8 h was lower (p < or = 0.05) in all treatment groups and did not differ (p > or = 0.05) among the 8-h incubation treatment groups. Secretion of PGE by 200-day Brahman placentas was reduced (p < 0.05) by indomethacin 72 and 82% and by meclofenamic acid 72 and 96%, respectively, at 4 and 8 h when compared to controls. Secretion of PGF2alpha was reduced (p < or = 0.05) 71 and 86% by indomethacin or 89 and 89% by meclofenamic acid at 4 and 8 h, respectively, and did not differ (p > or = 0.05) between 4 and 8 h of incubation. PGE2 did not (p > or = 0.05) induce secretion of PGE above what was added in any treatment group. PGE in culture media was increased (p < or = 0.05) by 8-Epi-PGE2, pregnancy specific protein B, and the 100 ng/mL PGF2alpha dose (p < or = 0.05), but not by PGE2, progesterone, estradiol-17beta, 8-Epi-PGF2alpha, or trichosanthin. Secretion of PGF2alpha by 200-day Brahman placentas was not affected (p > or = 0.05) by 8-Epi-PGE2, progesterone, or estradiol-17beta, but PGF2alpha secretion was increased (p < or = 0.05) by trichosanthin or PGE2, even in the presence of indomethacin or meclofenamic acid. It is concluded that PGE does not affect secretion of progesterone by 200- or 270-day bovine placentas, but, pregnancy specific protein B may regulate placental secretion of PGE. Also, indomethacin and meclofenamic may affect enzymes converting PGH to PGE rather than acting only on cyclooxygenase because indomethacin and meclofenamic acid lowered PGE secretion by 270-day Brahman placentas more than they lowered PGF2alpha. In addition, it is concluded that PGE2 can induce bovine placental secretion of PGE, but this is dependent upon the stage of gestation.

Effect of PGF2alpha, indomethacin, tamoxifen, or estradiol-17beta on incidence of abortion, progesterone, and pregnancy-specific protein B (PSPB) secretion in 88- to 90-day pregnant sheep.

One objective of this experiment was to evaluate our hypotheses that estradiol-17beta regulates secretion of pregnancy specific protein B (PSPB) and that secretion of progesterone during pregnancy is regulated by a prostanoid by examining the effects of prostaglandin F2alpha (PGF2alpha), a luteolyic agent; indomethacin, a prostanoid synthesis inhibitor; tamoxifen, an estrogen receptor antagonist; estradiol 17-beta; and interaction of these factors on the incidence of abortion and progesterone and PSPB secretion. Another objective was to determine if there is a luteal source of PSPB. Weights of corpora lutea were decreased (P < or = 0.05) by PGF2alpha, indomethacin, PGF2alpha + tamoxifen, PGF2alpha + indomethacin, and PGF2alpha + estradiol-17beta but not (P > or = 0.05) by tamoxifen or estradiol-17beta alone. No ewe treated with PGF2alpha alone aborted (P > or = 0.05). Forty percent of ewes treated with PGF2alpha + estradiol-17beta aborted (P < or = 0.05), but ewes were not aborted by any other treatment within the 72-h sampling period. Profiles of progesterone in jugular venous blood differed (P < or = 0.05) among control, indomethacin-, tamoxifen-, and PGF2alpha + indomethacin-treated ewes. Progesterone in jugular venous blood of control ewes decreased (P < or = 0.05) by 24 h, followed by a quadratic increase (P < or = 0.05) from 24 to 62 h. Progesterone in jugular venous blood of indomethacin-, PGF2alpha-, PGF2alpha- + tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + estradiol-17beta-, and tamoxifen-treated ewes was reduced (P < or = 0.05) by 18 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Progesterone in vena cava and in uterine venous blood was reduced (P < or = 0.05) at 72 h in PGF2alpha-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, PGF2alpha + tamoxifen-, and PGF2alpha + estradiol-17beta-treated ewes. Weights of placentomes did not differ among treatment groups (P > or = 0.05). Profiles of PSPB in inferior vena cava blood differed (P < or = 0.05) among control, estradiol-17beta-, indomethacin-, tamoxifen-, PGF2alpha + indomethacin-, and PGF2alpha + tamoxifen-treated 88- to 90-day pregnant ewes. Concentrations of PSPB in inferior vena cava blood were increased (P < or = 0.05) in indomethacin-, estradiol-17beta-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated 88- to 90-day pregnant ewes within 6 h and did not vary (P > or = 0.05) for the remainder of the 72-h sampling period. Concentrations of PSPB in uterine venous blood of indomethacin-, tamoxifen-, PGF2alpha + tamoxifen-, and PGF2alpha + indomethacin-treated ewes were greater (P < or = 0.05) at 72 h than at 0 h. PSPB in ovarian venous blood did not differ (P > or = 0.05) adjacent or opposite to the ovary with the corpus luteum. It is concluded from these data that estrogen regulates placental secretion of PSPB and that a prostanoid, presumably prostaglandin E, regulates placental secretion of progesterone during 88-90 days of gestation in sheep and that there is no luteal source of PSPB.

Secretion of progesterone, estradiol-17beta, PGE, PGF2alpha, and pregnancy-specific protein B by 90-day intact and ovariectomized pregnant ewes.

Ninety-day pregnant ewes were either laparotomized, ovaries left in situ or bilaterally ovariectomized, and a jugular venous catheter and an inferior vena cava catheter via the saphenous vein were installed. Seven days later, placenta slices were collected and incubated in vitro for 4 h. Secretions of progesterone, PGE, estradiol-17beta and pregnancy-specific protein B (PSPB) in vitro by placenta from ovariectomized ewes were increased (P < or = 0.05) by 2.7-, 3.6-, 2.2-, and 2.4-fold, respectively, when compared to placenta slices from intact 90-day pregnant ewes. Secretion of PGF2alpha in vitro was unchanged (P > or = 0.05). Ovariectomy decreased (P < or = 0.05) jugular venous progesterone for 78 h followed by a quadratic increase (P < or = 0.05), whereas progesterone remained unchanged (P > or = 0.05) in intact ewes over the 162-h sampling period. Ovariectomy increased (P < or = 0.05) PGE in inferior vena cava plasma over the last half of the 162-h sampling period, whereas concentration of PGF2alpha did not change (P > or = 0.05). Increases in PGE occurred before the increase in progesterone. Concentrations of PSPB in inferior vena cava plasma of ovariectomized pregnant ewes increased (P < or = 0.05) during the last half of the 162-h sampling period, but not in intact ewes (P > or = 0.05). PSPB increased before PGE and progesterone. Concentrations of estradiol-17beta in jugular venous plasma of ovariectomized pregnant ewes increased (P < or = 0.05) during the last half of the sampling period, but not in intact ewes (P > or = 0.05). Increases in estradiol-17beta occurred before increases in PSPB. It is concluded that these data support the hypothesis that estradiol-17beta may control placental secretion of PSPB; PSPB may regulate placental secretion of PGE; and PGE may regulate placental secretion of progesterone.

Morphologic, endocrine and thermographic measurements of testicles in comparison with semen characteristics in mature Holstein-Friesian breeding bulls.

Twenty Holstein-Friesian breeding bulls (62-79 months of age) were examined 3 times, at 30-day intervals. Scrotal thermograms for assessment of scrotal surface temperature (SST) and blood samples for plasma testosterone concentrations were taken just before and then 45 and 90 min, respectively, after treatment with GnRH (50 micrograms, Gonavet, i.m. per bull). Following GnRH treatment, there generally were significant increases in mean values of both top SST (range, -0.1 to 1.4 degrees C) and bottom SST (range, 0.3 to 1.8 degrees C). Scrotal circumference was highly repeatable but SST and video-measurements of scrotal dimensions were less repeatable, because apparently they were affected by ambient temperature. Plasma testosterone concentrations before GnRH treatment were more repeatable than those after GnRH treatment. Correlations between examinations of 0.67 to 0.81 and -0.14 to 0.47, respectively, but the converse was true for SST measurements. Semen was collected with an artificial vagina 3 times per week for 12 weeks starting 2 weeks before the first examination. The total number of spermatozoa per ejaculate was highly repeatable and the percentage of motile and live spermatozoa were relatively consistent. Separate regressions for each variable and for each examination were conducted for these 3 semen characteristics as dependent variables. For the number of spermatozoa per ejaculate and for the percentage of motile spermatozoa, significant independent variables were plasma testosterone concentrations and difference between top and bottom SST, respectively. The slopes of these equations were nearly all negative and the R2 was from 0.15 to 0.42. For prediction of the percentage of live spermatozoa, both SST gradient and plasma testosterone concentrations were significant independent variables. For these regressions, the slopes were negative and the regression coefficients were generally lower than for the other 2 dependent variables (range, 0.16 to 0.25). Treatment with GnRH and assessment of SST and plasma testosterone concentrations have some correlation with the semen production in the mature bull.

Diagnosis of pregnancy in moose using a bovine assay for pregnancy-specific protein B.

Blood samples were collected from 26 moose (Alces alces ) and evaluated for the presence of an antigen that cross-reacted with antisera to bovine pregnancy-specific protein B (P-SPB). The objective of this study was to determine if the P-SPB radioimmunoassay (RIA) was a reliable indicator of pregnancy in these animals. In the first year of the study calf production the following summer was used as the index of previous pregnancy. In the second year all females were subjected to palpation per rectum after chemical immobilization. Seven of the 10 cows sampled in the first year were also sampled in the second year. All animals determined pregnant by rectal palpation were positive for P-SPB; however, P-SPB was not detected in males.

Effect of fetal mass, number and stage of gestation on pregnancy-specific protein B concentrations in the bovine.

In this study we characterized the peripheral plasma pregnancy-specific protein-B (PSPB) profile throughout gestation and examined the effect of stage of gestation, fetal mass and number on this profile in Holstein cows after non surgical embryo transfer. Cows (n = 12) were divided into 2 groups: Group 1 = single embryo recipient cows (n = 5), Group 2 = twin-embryo recipient cows (n = 7). Blood was collected approximately every third day from Day 0 (Day 0 = first day of standing estrus), then daily for the last 10 d of gestation, and sampling was stopped 1 d post partum. Two twin-embryo recipient cows had abnormal pregnancies; therefore, their data were excluded from the group. The time trend concentrations of plasma PSPB were significantly affected by the stage of gestation (P < 0.001) and fetal number (P < 0.001). In both groups PSPB increased gradually, with the mean levels being significantly higher (P < 0.01) in the twin-bearing group from Day 50 onwards (0.7 +/- 0.2 vs 9.2 +/- 4.5 ng/ml, singleton and twin-bearing cows, respectively) except for Day 10 pre-partum. By mid-gestation (Day 140), mean PSPB levels increased in the singleton (P < 0.001) cows by thirty-fold (21.2 +/- 3.2 ng/ml) as opposed to a ten-fold (98.4 +/- 13.2 ng/ml) increase in the twin-bearing (P < 0.001) group. The mean PSPB concentrations between Days 30 to 20 prepartum dramatically increased by about 700 to 200% in singleton (128.8 +/- 46.3 to 745.6 +/- 66.7 ng/ml) and twin-bearing cows (375.6 +/- 130.4 to 861.5 +/- 127.9 ng/ml), respectively. The PSPB levels between Day 10 prepartum to parturition were significantly higher (P < 0.001) in the twin-bearing group than in the singleton group (745.6 +/- 66.7 to 1627.4 +/- 238.9 ng/ml vs 861.5 +/- 127.9 to 3103.0 +/- 643.0 ng/ml in singleton and twin-bearing groups, respectively). Calf birthweight was correlated (P < 0.01) to peripheral PSPB concentration in singleton cows; however, this relationship decreased with the subsequent increase in fetal number. Cows giving birth prematurely to stillborn calves or to a schistosomus reflexus calf exhibited abnormal PSPB profiles. These results indicate that peripheral PSPB levels are correlated to the stage of gestation and fetal number. In addition, the peripheral pattern of PSPB is a valuable guage for predicting fetoplacental viability.

Detection of pregnancy in sheep by radioimmunoassay of sera for pregnancy-specific protein B.

A radioimmunoassay (RIA) for bovine pregnancy-specific protein B (bPSPB) has been shown to be a reliable test for pregnancy in cows. Pregnant ewes have a blood antigen that cross-reacts in this RIA. Two studies were conducted to determine the accuracy of detection of pregnancy in sheep using the bPSPB RIA. In Study 1, 33 ewe lambs were bred over a 70-d period in late fall. At 26, 56, and 83 d after the end of the breeding period, blood samples were collected for assay in the bPSPB RIA, and the Pregmatic 3 ultrasonic device was used to detect pregnancy. Pregmatic 3 detected pregnancy in 14, 27 and 28 ewes and nonpregnancy in 19, 6 and 3 ewes at Days 26, 56 and 83 past the breeding period, respectively. The bPSPB assay detected pregnancy in 32, 31 and 30 ewes and nonpregnancy in 1, 2 and 2 ewes at Days 26, 56 and 83 past breeding, respectively, Thirty ewes lambed and three did not. In Study 2, 180 multiparous ewes were bred over a 60-d period in summer. At 35 d after the end of the breeding period, blood samples were collected for assay in the RIA, and a real-time ultrasonic scan was done to detect pregnancy. Real-time ultrasonic testing detected pregnancy in 163 ewes and nonpregnancy in 17 ewes; whereas, the RIA detected pregnancy in 161 ewes and nonpregnancy in 19 ewes. One hundred fifty-nine ewes lambed and 21 did not. The bPSPB RIA detected pregnancy earlier and more accurately than the Pregmatic 3 ultrasonic device and was equally as accurate as the real-time scanning instrument. These studies demonstrate an accurate serological test for a pregnancy-specific antigen in sheep.

Pregnancy-specific protein B and progesterone in monitoring viability of the embryo in early pregnancy in the cow after experimental infection with Actinomyces pyogenes.

Actinomyces pyogenes can cause embryonic death and abortion during the early stages of pregnancy in cows. Bovine pregnancy-specific protein B (PSPB) is produced in response to a viable embryo and as such it could be a potential marker for embryronic survival. The plasma concentration of PSPB was monitored in cows following an intrauterine infection with A. pyogenes and during the subsequent abortion and recovery from infection. Plasma progesterone concentrations were also monitored, and the results were compared withthose for animals in which abortion had been induced by prostaglandin F2alpha treatment. In abortions induced both by infection and by cloprostenol, the plasma concentration of PSPB fell steadily from the day of treatment, with a half-life of 7 days. In the cloprostenol-induced abortions, progesterone levels fell dramatically to <0.5ng/ml within 24 hours of treatment, while following inoculation with A. pyogenes , progesterone concentration remained elevated for 20 to 40 days and fell to <0.5ng/ml after evacuation of pus from the uterus. Sequential monitoring of PSPB, which identifies embryonic death when a continuing fall in plasma concentration is demonstrated, is a better indicator of embryonic death following bacterial infection with A. pyogenes than plasma progesterone concentration, which falls only when infection is resolved.

Early pregnancy detection and the hormonal characterization of embryonic-fetal mortality in fallow deer (Dama dama).

The objectives of this investigation were to 1) determine serum concentrations of progesterone (P4), estrone sulfate (E1S) and pregnancy-specific protein B (PSPB) from estrus synchronization through mid-gestation in the fallow doe (Dama dama) and 2) characterize the hormonal profiles of does whose embryos or fetuses died in utero. Ten fallow does were synchronized for 14 d with an intravaginal P4-releasing device (CIDR) and were naturally mated after CIDR removal. Blood samples were collected at CIDR insertion, CIDR removal and at intervals through Day 203 post-CIDR removal for analysis of P4, E1S and PSPB by radioimmunoassay (RIA). Ultrasonography was performed on Days 49 and 69 post-CIDR removal. Serum P4 at the time of CIDR insertion was 4.8 +/- 0.6 ng/ml, and at CIDR withdrawal it was 6.2 +/- 0.3 ng/ml. Concentrations of E1S and PSPB were nondetectable at CIDR insertion. Serum E1S was highest at Day 93, and PSPB was first detectable in pregnant does at Days 27 to 30 post-CIDR withdrawal. Ultrasonography on Day 49 revealed that 6 does were pregnant, 2 were not pregnant and 2 others were diagnosed originally as early pregnant. At Day 69, ultrasonography revealed that 6 does (60%) were pregnant and 4 (40%) were not. A comparison of the ultrasonographic and hormonal data indicated that the 2 does diagnosed as early pregnant on Day 49 had conceived but had lost the pregnancy. A third doe which was pregnant on Day 69 lost the fetus later in gestation. Hormonal profiles of does whose embryo or fetus had died were characterized by erratic P4 and E1S profiles, with PSPB becoming undetectable in the 3 does by Days 49, 65 and 80 post-CIDR removal. These data 1) demonstrate the timing for the collection of serum samples for determining early pregnancy in fallow does using 3 hormonal methods and 2) characterize the hormonal profiles of 3 fallow does with embryonic-fetal loss.

Computer analysis of video and ultrasonographic images for evaluation of bull testes.

The objectives of this study were to determine relationships between scrotal size (SC; estimated from a video image) and testicular size, and between ultrasonographic echotexture of the testis and seminiferous tubule area in bulls. Video images of the scrotum of 49 Holstein-Friesian (H-F) bulls were recorded and digitized. Scrotal width and length were measured with custom software. After slaughter, scrotums (containing testes) were excised, SC and testicular height, width and volume were measured, and the testes were examined ultrasonographically. Correlations between SC and testicular width or volume (r = 0.86, P < 0.001 and r = 0.84, P < 0.001, respectively) were much higher than those between scrotal width and testicular width or volume (r = 0.23, P < 0.11 and r = 0.28, P < 0.06). Histological examination of the testes was performed in 31 of the bulls. Ultrasonographic echotexture of the testes (determined with custom software) was highly correlated (r = -0.5, P < 0.005) with seminiferous tubule area. Although SC was superior to video imaging for estimating testicular size, ultrasonographic imaging of the testes has considerable potential for the evaluation of testicular function in bulls.

Methods for pregnancy determination and the effects of body condition on pregnancy status in Rocky mountain elk (Cervus elephus nelsoni ).

The objective of this study was to determine the effectiveness of transrectal ultrasonography and serum progesterone (P(4)), estrone sulfate (E(1)S) and pregnancy-specific protein B (PSPB), without prior knowledge of reproductive status, in detecting pregnancy in elk cows. In addition, body weight and body condition score (BCS) were determined to assess whether body condition affects pregnancy status in elk cows. Twenty-five elk cows were sampled during the early rut (Period 1) and after the rut (Period 2), an interval of 120 d. Age, weight, BCS and blood samples, for P(4), E(1)S and PSPB determinations, were taken at Periods 1 and 2. Ultrasonography was performed at Period 2. The younger elk cows weighed less (P<0.05) than older cows. However, pregnancy status was not affected (P> 0.10) by age or weight of the cow. Elk cows that calved had higher (P<0.02) BCS at Periods 1 and 2 than cows that remained open. Serum P(4) and E(1)S were higher (P<0.0001) in pregnant cows at Period 2 than in open cows. Progesterone was 85.8% accurate in detecting pregnant versus open cows at Period 1, while E(1)S and PSPB were not effective. Elk cows at Period 1 were <20 d pregnant with the exception of 1 cow at 46 d. Ultrasonography was 92% accurate, P(4) was 95% accurate, and E(1)S and PSPB were both 100% accurate in determining pregnant versus open cows at Period 2. Pregnant cows at Period 2 were all > 100 d pregnant. Ultrasonography, serum E(1)S and PSPB all may provide a reliable means for pregnancy diagnosis in elk cows at > 100 d of gestation, while serum P(4) may be effective when multiple samples are compared during or after the rut, or when used in combination with the other diagnostic methods described. Further research is needed to determine the optimum time period after breeding in elk cows for accurate pregnancy detection through hormonal analysis.

Embryonic loss from 30 to 60 days post breeding and the effect of palpation per rectum on pregnancy.

This study was conducted over a 12-mo period to determine the rate of bovine embryo death between 30 and 60 d of gestation. In addition, palpation per rectum as a means of pregnancy detection was evaluated as a possible cause of embryo death. Estrus was synchronized in Holstein heifers (n = 1358), weighing > or = 385 kg, with a single intramuscular injection of 25 mg prostaglandin F(2alpha). Estrus was primarily detected by the absence of paint marks on the tailhead. The heifers were artificially inseminated with semen from 5 Holstein sires. Blood was collected between 30 and 45 d after breeding, and sera were evaluated for the presence of bovine pregnancy-specific protein B (bPSPB) by RIA to determine pregnancy. Palpation for fetal membrane slip was conducted by an experienced technician in approximately one-half of the inseminated heifers. To determine embryonic survival, a second blood sample was collected at approximately 60 d from 862 heifers that were determined to be pregnant at the first blood sampling. Embryonic loss averaged 5.3% during the interval between the initial detection of pregnancy at 30 to 45 d and the subsequent detection of pregnancy at 60 d of gestation. Embryo loss in heifers that were palpated was 6.5% compared with that of 4.3% in the control heifers (X(2): P = 0.145). These findings establish that there was substantial loss of embryos between 30 and 60 d post breeding but that embryo loss was not affected by palpation per rectum.