The impact of diet and arginine supplementation on pancreatic mass, digestive enzyme activity, and insulin-containing cell cluster morphology during the estrous cycle in sheep.

To determine the effect of feed intake and arginine treatment during different stages of the estrous cycle on pancreatic mass, digestive enzyme activity, and histological measurements, ewes (n = 120) were randomly allocated to 1 of 3 dietary groups; control (CON; 2.14-Mcal metabolizable energy/kg), underfed (UF; 0.6 × CON), or overfed (OF; 2 × CON) over 2 yr. Estrus was synchronized using a controlled internal drug release device for 14 d. At controlled internal drug release withdrawal, ewes from each dietary group were assigned to 1 of 2 treatments; Arg (L-Arg HCl, 155-µmol/kg BW) or Sal (approximately 10-mL saline). Treatments were administered 3 times daily via jugular catheter and continued until slaughter on d (day) 5 and 10 of the second estrus cycle (early luteal phase, n = 41 and mid-luteal phase, n = 39; yr 1) and d 15 of the first estrus cycle (late luteal phase, n = 40; yr 2). A blood sample collected from jugular catheters for serum insulin analysis before slaughter. The pancreas was then removed, trimmed of mesentery and fat, weighed, and a sample snap-frozen until enzyme analysis. Additional pancreatic samples were fixed in 10% formalin solution for histological examination of size and distribution of insulin-containing cell clusters. Data were analyzed as a completely randomized design with a factorial arrangement of treatments. Diet, treatment, and diet × treatment were blocked by yr and included in the model with initial BW used as a covariate. Day of the estrous cycle was initially included in the model but later removed as no effects (P > 0.10) were observed for any pancreatic variables tested. Overfed ewes had the greatest (P < 0.001) change in BW, final BW, change in BCS, and final BCS. A diet × treatment interaction was observed for change in BW and final BW (P ≤ 0.004). Overfed and CON had increased (P < 0.001) pancreas weight (g) compared with UF ewes. Protein concentration (g/pancreas) was the lowest (P < 0.001) in UF ewes, whereas protein content (mg/kg BW) was greater (P = 0.03) in UF than OF ewes. Activity of α-amylase (U/g, kU/pancreas, U/kg of BW, and U/g protein) and trypsin (U/pancreas) was greater (P ≤ 0.003) in OF than UF ewes. Serum insulin was the greatest (P < 0.001) in OF ewes. No effects were observed for pancreatic insulin-containing cell clusters. This study demonstrated that plane of nutrition affected several measurements of pancreatic function; however, the dosage of Arg used did not influence pancreatic function.

Effects of realimentation after nutrient restriction during mid- to late gestation on pancreatic digestive enzymes, serum insulin and glucose levels, and insulin-containing cell cluster morphology.

This study examined effects of stage of gestation and nutrient restriction with subsequent realimentation on maternal and foetal bovine pancreatic function. Dietary treatments were assigned on day 30 of pregnancy and included: control (CON; 100% requirements; n = 18) and restricted (R; 60% requirements; n = 30). On day 85, cows were slaughtered (CON, n = 6; R, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12), or realimented to control (RC; n = 11). On day 140, cows were slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5) or realimented to control (RRC, n = 6). On day 254, the remaining cows were slaughtered and serum samples were collected from the maternal jugular vein and umbilical cord to determine insulin and glucose concentrations. Pancreases from cows and foetuses were removed, weighed, and subsampled for enzyme and histological analysis. As gestation progressed, maternal pancreatic α-amylase activity decreased and serum insulin concentrations increased (p ≤ 0.03). Foetal pancreatic trypsin activity increased (p < 0.001) with advancing gestation. Foetal pancreases subjected to realimentation (CCC vs. RCC and RRC) had increased protein and α-amylase activity at day 254 (p ≤ 0.02), while trypsin (U/g protein; p = 0.02) demonstrated the opposite effect. No treatment effects were observed for maternal or foetal pancreatic insulin-containing cell clusters. Foetal serum insulin and glucose levels were reduced with advancing gestation (p ≤ 0.03). The largest maternal insulin-containing cell cluster was not influenced by advancing gestation, while foetal clusters grew throughout (p = 0.01). These effects indicate that maternal digestive enzymes are influenced by nutrient restriction and there is a potential for programming of increased foetal digestive enzyme production resulting from previous maternal nutrient restriction.

Influence of level of dried distillers grains with solubles on feedlot performance, carcass characteristics, serum testosterone concentrations, and spermatozoa motility and concentration of growing rams.

The objective of this study was to evaluate the effects of dried distillers grains with solubles (DDGS) on ram lamb feedlot performance, carcass characteristics, serum testosterone concentration, and semen quality. One hundred twenty ram lambs (40.4 ± 9.1 kg; Suffolk × western white face) were used in a completely randomized design to determine the effects of DDGS on feedlot performance and carcass characteristics. Rams were allotted into one of three dietary treatments (n = 4 pens/treatment; 10 rams/pen): 1) 0DDGS: 85% corn and 15% commercial market lamb pellet, 2) 15DDGS: 15% DDGS substituted for corn (DM basis), and 3) 30DDGS: 30% DDGS substituted for corn (DM basis). Rams were weighed on consecutive days at the beginning (d 0 and 1) and end (d 96 and 97 and d 116 and 117) of the trial. Scrotal circumference was measured on all rams on d 84, 96, and 116. Semen and blood samples were collected on a subset of 48 rams (4 rams/pen; 16 rams/treatment; n = 4) to evaluate semen quality. Blood samples were collected every 14 d throughout the study. Semen samples were collected on d 84, 98, and 112. Rams were fed to market weight, shipped to a commercial abattoir, and harvested for carcass data collection. Initial BW, final BW, change in scrotal circumference, days on feed, carcass characteristics, serum testosterone concentrations, and spermatozoa motility score were not different (P ≥ 0.23) due to dietary treatment. However, DMI increased linearly (P < 0.001) as DDGS increased in the ration, resulting in a linear increase (P = 0.02) in ADG. Additionally, spermatozoa concentration decreased linearly (P = 0.05) as DDGS concentration increased in the ration. Increasing DDGS in the diet did not have a negative impact on ram feedlot performance or carcass characteristics; however, spermatozoa production may have been negatively affected, necessitating the need for additional research on the impact of DDGS on ram development.

Effects of plane of nutrition on in vitro fertilization and early embryonic development in sheep.

Nutrition has been shown to influence several reproductive functions, including hormone production, oocyte competence and fertilization, and early embryonic development. To determine the effects of maternal diet on in vitro fertilization (IVF) and early embryonic development, ewes (n = 18; 47.0 +/- 1.5 kg of initial BW) were divided into control and underfed (60% of control) nutritional planes for 8 wk before oocyte collection. Pelleted diets containing 2.4 Mcal of ME/kg and 13% CP (DM basis) were fed once daily. During the first 4-wk acclimation phase, control and underfed ewes were fed 1,000 and 600 g/d, respectively. From wk 4 to 8, control (adequate) ewes were fed to maintain BW and offered 720 g/d, whereas underfed ewes received 432 g/d (60% restricted). Synchronization of estrus was performed using progestagen sponges for 14 d. Follicular development was induced by twice daily injections of FSH on d 13 (5 units/injection) and 14 (4 units/injection) of the estrous cycle. Oocytes were collected from all visible follicles on d 15 of the estrous cycle. After IVF, the proportion of developing embryos was evaluated throughout an 8-d culture period. Under-nutrition decreased (P < 0.006) the rate of cleavage, number of blastocysts per ewe, and rate of blastocyst formation (from 79 to 64%; from 3.3 to 0.8; and from 31 to 8%, respectively). However, the number of visible follicles, total number of oocytes, number of healthy oocytes, percentage of healthy oocytes, number of cleaved oocytes, and morula formation per ewe were similar for control and underfed ewes. These data indicate that undernutrition of donor ewes, resulting in lower BW and BCS, has a negative effect on oocyte quality, which results in lower rates of cleavage and blastocyst formation.

Recruitment of TRADD, FADD, and caspase 8 to double-stranded RNA-triggered death inducing signaling complexes (dsRNA-DISCs).

Rapid elimination of virus-infected cells by apoptosis is an efficient anti-viral strategy. Double-stranded RNA (dsRNA), a viral product, is potently and rapidly apoptogenic in susceptible cells. Caspase 8 plays an important role in the dsRNA-induced apoptosis; however, the mechanisms of caspase 8 activation in response to dsRNA are unknown. We demonstrate here that, in HeLa cells, the dsRNA-triggered activation of caspase 8 is independent of ongoing proteins synthesis (and is, therefore, independent of changes in pro- and anti-apoptotic gene expression) and involves the formation of multiprotein dsRNA-triggered death inducing signaling complexes (dsRNA-DISCs). DsRNA-DISCs contain FADD, TRADD, and caspase 8; however, several experimental approaches suggest that death ligands and death receptors (such as Fas/Apo1 and DR4/Apo2) are not involved in the formation of dsRNA-DISCs.

A modified presynchronization protocol improves fertility to timed artificial insemination in lactating dairy cows.

To compare 2 hormonal protocols for submission of lactating dairy cows for timed artificial insemination (TAI), nonpregnant lactating Holstein cows (n = 269) >60 d in milk were randomly assigned to each of 2 treatments to receive TAI (TAI = d 0). Cows assigned to the first treatment (Ovsynch, n = 134) received 50 microg of GnRH (d -10), 25 mg of PGF2alpha (d -3), and 50 microg of GnRH (d -1) beginning at a random stage of the estrous cycle. Cows assigned to the second treatment (Presynch, n = 135) received Ovsynch but with the addition of 2 PGF2alpha (25 mg) injections administered 14 d apart beginning 28 d (d -38 and -24) before initiation of Ovsynch. All cows received TAI 16 to 18 h after the second GnRH injection. Ovulatory response after each GnRH injection for a subset of cows (n = 109) and pregnancy status 42 d after TAI for all cows were assessed using transrectal ultrasonography. Based on serum progesterone (P4) profiles determined for a subset of cows (n = 109), P4 concentrations decreased for Presynch cows after the first 2 PGF2alpha injections, and Presynch cows had greater P4 concentrations at the PGF2alpha injection on d -3 compared with Ovsynch cows. Although the proportion of cows ovulating after the first and second GnRH injections did not differ statistically between treatments (41.1 and 69.6% vs. 35.9 and 81.1% for Ovsynch vs. Presynch, respectively), pregnancy rate per artificial insemination (PR/AI) at 42 d post TAI was greater for Presynch than for Ovsynch cows (49.6 vs. 37.3%). Parity, DIM, and body condition score (BCS) at TAI did not affect PR/AI to TAI. These data support use of this presynchronization protocol to increase PR/ AI of lactating dairy cows receiving TAI compared with Ovsynch.

Effects of epidermal growth factor on early embryonic development after in vitro fertilization of oocytes collected from ewes treated with follicle stimulating hormone.

Epidermal growth factor (EGF) has been shown to enhance the in vitro rate of blastocyst formation in several species. Follicular development was induced in ewes (n=15) by twice daily administration of FSH-P on Days 13 and 14 of the estrous cycle. Cumulus oocyte complexes (COCs) were collected from all visible follicles (n=25+/-2.4/ewe) on Day 15. COCs from each ewe were cultured separately for 24h in maturation medium (containing 10% serum, LH, FSH and estradiol) with (8.2+/-0.9 per ewe) or without (7.8+/-0.8 per ewe) EGF (10 ng/ml). Oocytes were then denuded by hyaluronidase treatment, and healthy oocytes were cultured in the presence of frozen-thawed semen in synthetic oviductal fluid (SOF) medium containing 2% sheep serum. After 18-20 h, zygotes were transferred to SOF medium without glucose and cultured for about 36 h until they reached the 4-8 cell stage. Embryos were transferred to SOF medium with glucose for further development. Medium was changed every other day until blastocyst formation on Day 8 of culture (Day 1=day of fertilization). The rate of embryonic development was evaluated throughout the culture period. After maturation, cumulus cells were more expanded in the presence than in the absence of EGF. The rates of fertilization (overall 75.7+/-3.9%) and morula formation (overall 40.6+/-7.1%) were similar (P>0.05) for COCs cultured with or without EGF. However, EGF increased (P<0.01) the number of blastocysts (1.4+/-0.1 versus 0.6+/-0.2 per ewe) and tended to increase (P<0.1) the rate of blastocyst formation (21.0+/-6.6% versus 13.4+/-4.3% per ewe). These data demonstrate that EGF increases blastocyst formation in FSH-treated ewes. Therefore, EGF is recommended as a supplement to maturation medium to enhance embryonic development in vitro in FSH-treated sheep.

Effects of follicle stimulating hormone (FSH) on follicular development, oocyte retrieval, and in vitro fertilization (IVF) in ewes during breeding season and seasonal anestrus.

Administration of FSH increases the number of developing follicles, and affects oocyte health and cleavage rate. To determine the optimal level of FSH treatment, studies were conducted during the normal breeding season and seasonal anestrus. In Experiment 1, ewes were implanted with SyncroMate-B (SMB; norgestomet) for 14 days during the breeding season. Beginning on day 12 or 13 after SMB implantation, ewes were treated with saline (control; n=10), or treated with FSH for two days (2D; n=9) or three days (3D; n=10). In Experiment 2, conducted during seasonal anestrus, ewes were implanted with SMB for 14 days (n=23) or were not implanted (n=26). The SMB-implanted and nonimplanted ewes were assigned to one of three treatments as in Experiment 1: control (n=13), 2D (n=21) or 3D (n=15). In Experiments 1 and 2, ewes were laparotomized to count the number of follicles < or = 3 mm and > 3 mm and to retrieve oocytes. Healthy oocytes from each treatment were used for IVF. In Experiment 3, ewes (n=6) were implanted twice with SMB for 14 days during seasonal anestrus. Ewes were injected with FSH for 2 days, and the oocytes were collected and fertilized as in Experiments 1 and 2. In Experiment 1, FSH-treatment increased (P < 0.05) the number of follicles > 3 mm, the number of oocytes retrieved from follicles < or = 3 mm and > 3 mm, the proportion of healthy oocytes, and the number of oocytes used for IVF. Oocytes from control and 2D ewes had greater (P < 0.01) cleavage rates than 3D ewes (68% and 71% vs. 42%). In Experiment 2, implanted and nonimplanted ewes had similar (P > 0.05) numbers of follicles, total oocytes, and healthy oocytes; therefore, data were combined. The FSH treatment increased (P < 0.01) the number of follicles > 3 mm, and the number of oocytes recovered from follicles > 3 mm. The recovery rate of oocytes and the percentage of healthy oocytes were similar for control and FSH-treated ewes. The cleavage rate in Experiment 2 ranged from 4 to 16%. In Experiment 3, the cleavage rate for ewes treated twice with SMB was 27% which tended to be greater (P < 0.07) than for the 2D ewes that received one SMB implant in Experiment 2. These data indicate that FSH increased the number of developing follicles and the number of healthy oocytes retrieved from ewes during the breeding season and seasonal anestrus. However, cleavage rates during seasonal anestrus were lower than during the normal breeding season in both FSH-treated and control ewes. Treatment of ewes for 2 days with FSH resulted in a greater cleavage rate than treatment of ewes for 3 days.

Effect of crambe meal on performance, reproduction, and thyroid hormone levels in gestating and lactating beef cows.

Crambe meal was compared to a combination of sunflower and soybean meal as a protein supplement for mature beef cows in two experiments. In Exp. 1, cows (n = 80, average BW 651+/-14.4 kg) were fed crambe meal at 9.86% of dry matter intake (DMI) during the last trimester of gestation. No differences (P < .05) were detected due to treatment for cow weight, condition score, thyroid hormones, calf birth weight, or calving interval. In Exp. 2, cows (n = 100, average BW 566+/-6.82 kg) were fed crambe meal at 7.44% of DMI during the last trimester of gestation and at 8.33% of DMI during early lactation (53+/-6 d of lactation). Gains were greater during gestation (P = .09) and throughout the supplementation period (P = .06), and days to first estrus were reduced (P < .01) for cows fed crambe meal. During lactation, serum triiodothyronine (T3) concentrations did not decline as much (P = .03) in cows fed crambe meal as in cows fed sunflower-soybean meal-based supplements. No differences (P > .10) were apparent for condition score, birth weight, calf growth rate, weaning weight, thyroid hormones during gestation, or calving interval. These data indicate that crambe meal fed at the levels used in this experiment can be used as a protein supplement for beef cows without negatively affecting cows' performance.

Undegraded intake protein supplementation: II. Effects on plasma hormone and metabolite concentrations in periparturient beef cows fed low-quality hay during gestation and lactation.

Hereford x Angus cows (n = 36; initial wt 568+/-59 kg) were used to evaluate the effects of undegradable intake protein (UIP) supplementation on plasma hormone and metabolite concentrations. Treatments were control (unsupplemented) or one of three protein supplements. Supplements were fed at 1.3 kg DM/d and included UIP at low, medium, or high levels (53, 223, or 412 g UIP/kg supplement DM, respectively). Supplements were formulated to be isocaloric (1.77 Mcal NEm/kg) and to contain equal amounts of degradable intake protein (DIP; 211 g DIP/kg supplement DM). Prairie hay (5.8% CP) was offered for ad libitum consumption. Jugular blood samples were collected daily from each cow during six 7-d collection periods (corresponding to mo 7, 8, and 9 of gestation and to mo 1, 2, and 3 of lactation). Plasma glucose concentrations were similar between control and supplemented cows during mo 2 and 3 of lactation; however, the low UIP treatment group had consistently higher plasma glucose (P< or =.02) than cows fed medium or high UIP supplements during gestation and the last month of lactation. During gestation, cows fed the high UIP supplement had higher (P< or =.08) plasma glucose than cows fed the medium UIP supplement. During gestation, plasma insulin concentration was increased (P = .01) by supplementation; insulin also increased (P<.01; mo 8 and 9) as supplemental UIP increased. During lactation, plasma insulin was greater (P = .01) in supplemented than in control cows. During mo 2 and 3 of lactation, insulin was lower (P< or =.04) in cows fed low UIP supplement compared with cows fed medium or high UIP supplements. Growth hormone concentration was higher (P< or =.03) in control cows than in supplemented cows in all periods measured except mo 7 of gestation. Plasma nonesterified fatty acid concentrations were higher (P< or =.03) in control cows than in supplemented cows in all periods measured except the 1st mo of lactation. These data are interpreted to suggest that protein supplementation and level of UIP can alter plasma concentrations of hormones and metabolites in gestating and lactating beef cows consuming low-quality hay.

Injection of MK-801 affects ocular dominance shifts more than visual activity.

Kittens were given intramuscular injections of the N-methyl--aspartate (NMDA) antagonist MK-801 twice daily (morning and midday) during the peak of the period of susceptibility for ocular dominance changes. They were then exposed to light with one eye closed for 4 h after each injection. The ocular dominance of these kittens was shifted significantly less than that of kittens injected with saline and exposed to light over the same period at the same age. After recording a sample of cells for an ocular dominance histogram, the kittens were injected with the same dose of MK-801 that was used during rearing to observe its effect on the activity of single cells in the visual cortex. In the majority of cells (7/13) there was no significant change in activity. Positive evidence for a reduction in activity was seen in only a minority (3/13) of cells. In a separate series of experiments, dose-response curves were measured for cells in the visual cortex in response to iontophoresis of NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and the effect of an injection of MK-801 on these curves was measured. MK-801, at doses similar to those used in the ocular dominance experiments, had a significant effect on the dose-response curves for NMDA, but little effect on the dose-response curves for AMPA, or the visual responses of the cells. We conclude that ocular dominance shifts can be reduced significantly by a treatment that has little effect on the level of activity of cells in the visual cortex but does specifically affect the responses of the cells to NMDA as opposed to the responses to AMPA.

Nondestructive tablet hardness testing by near-infrared spectroscopy: a new and robust spectral best-fit algorithm.

A new algorithm using common statistics was proposed for nondestructive near-infrared (near-IR) spectroscopic tablet hardness testing over a range of tablet potencies. The spectral features that allow near-IR tablet hardness testing were evaluated. Cimetidine tablets of 1-20% potency and 1-7 kp hardness were used for the development and testing of a new spectral best-fit algorithm for tablet hardness prediction. Actual tablet hardness values determined via a destructive diametral crushing test were used for construction of calibration models using principal component analysis/principal component regression (PCA/PCR) or the new algorithm. Both methods allowed the prediction of tablet hardness over the range of potencies studied. The spectral best-fit method compared favorably to the multivariate PCA/PCR method, but was easier to develop. The new approach offers advantages over wavelength-based regression models because the calculation of a spectral slope averages out the influence of individual spectral absorbance bands. The ability to generalize the hardness calibration over a range of potencies confirms the robust nature of the method.

Time-course of the uterine response to estradiol-17beta in ovariectomized ewes: uterine growth and microvascular development.

The time-course of uterine growth, cell proliferation, and microvascular development was evaluated during the first 72 h after implanting estradiol-17beta (E2) into ovariectomized (OVX) ewes. Uterine fresh weight increased 2.3-fold by 24 h and increased further (3.3-fold) by 48 h. The majority (approximately 75%) of this growth response was associated with tissue growth rather than a change in the tissue dry weight:fresh weight ratio. Both uterine cell number (DNA content) and cell size (RNA:DNA ratio) increased from 0 to 24 h (1.8-fold and 1.7-fold, respectively). Cell proliferation also increased dramatically between 8 h and 24 h after E2 implantation. Endometrial microvascular volume density (percentage of tissue volume occupied by microvessels) increased approximately 1.8-fold by 24 h and then remained constant or declined slightly through 72 h. The total endometrial microvascular volume, however, increased approximately 5-fold from 0 to 24 h and increased further by 72 h. Thus, treatment of OVX ewes with E2 caused a dramatic increase in uterine fresh and dry weights by 24 h, due primarily to hyperplasia and hypertrophy, with only a relatively small change in tissue dry weight:fresh weight ratio. This dramatic uterine growth was associated with a profound increase in endometrial microvascular volume.

Time-course of the uterine response to estradiol-17beta in ovariectomized ewes: expression of angiogenic factors.

Uterine expression of angiogenic factors (vascular endothelial growth factor [VEGF] and basic fibroblast growth factor [bFGF]) was evaluated in ovariectomized ewes at 0, 2, 4, 8, 24, 48, or 72 h after estradiol (E2) treatment. Endometrial VEGF mRNA increased more than 5-fold from 0 to 4 h, remained elevated at 8 h, and then declined through 72 h after E2 treatment. In contrast, endometrial bFGF mRNA remained constant from 0 to 4 h, increased 2.2-fold from 4 to 8 h, remained elevated at 24 h, and then declined through 72 h. Immunostaining for VEGF was present in myometrial and endometrial microvessels (arterioles, venules, and/or capillaries) and also in myometrial smooth muscle; the pattern of VEGF immunostaining followed that of mRNA expression, being elevated at 4 and 8 h after E2 treatment. Immunostaining for bFGF was present exclusively in uterine glands; the pattern of bFGF immunostaining also followed that of its mRNA, being elevated at 8 and 24 h after E2. On the basis of these observations, we suggest that VEGF and bFGF are probably important factors responsible for the dramatic uterine microvascular response that occurs 8 to 24 h after E2 treatment in ovariectomized ewes.

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: effects of prostaglandin F2 alpha, protein kinase C and calcium.

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The present studies were performed to evaluate the effects of luteotropic and luteolytic hormones, and also intracellular regulators, on contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. Bovine corpora lutea (CL) from the early, mid and late luteal phases of the estrous cycle were dispersed with collagenase and incubated with no treatment, LH, PGF or LH + PGF (Experiment 1), or with no treatment, or agonists or antagonists of protein kinase C (TPA or H-7) or calcium (A23187 or EGTA; Experiment 2). After incubation, media were collected for determination of progester-one concentrations. Then the rate of GJIC was evaluated for small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells by using the fluorescence recovery after photobleaching technique and laser cytometry. Luteal cells from each stage of the estrous cycle exhibited GJIC, but the rate of GJIC was least (P < 0.05) for luteal cells from the late luteal phase. LH increased (P < 0.05) GJIC between small luteal cells from the mid and late but not the early luteal phase. PGF increased (P < 0.05) GJIC between small luteal cells from the mid luteal phase and diminished (P < 0.05) LH-stimulatory effects on GJIC between small luteal cells from the late luteal phase. Throughout the estrous cycle, TPA decreased (P < 0.05) the rate of GJIC between large and small, and between small luteal cells, and A23187 decreased (P < 0.05) the rate of GJIC between large and small luteal cells. LH and LH + PGF, but not PGF alone increased (P < 0.05) progesterone secretion by luteal cells from the mid and late luteal phases. Agonists or antagonists of PKC or calcium did not affect progesterone secretion by luteal cells. These data demonstrate that both luteal cell types communicate with small luteal cells, and the rate of communication depends on the stage of luteal development. LH and PGF affect GJIC between small luteal cells during the fully differentiated (mid-luteal) and regressing (late luteal) stages of the estrous cycle. In contrast, at all stages of luteal development, activation of PKC decreases GJIC between small and between large and small luteal cells, whereas calcium ionophore decreases GJIC only between large and small luteal cells. Luteotropic and luteolytic hormones, and intracellular regulators, may be involved in regulation of cellular interactions within bovine CL which likely is an important mechanism for coordination of luteal function.

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: effects of cyclic adenosine 3',5'-monophosphate.

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The objective of the present study was to evaluate the role of cAMP in regulation of contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. In experiment 1, corpora lutea (n = 5) from the mid-luteal phase of the estrous cycle were dissociated with collagenase, and cells were preincubated in a medium with serum. Then the medium was changed to serum-free media containing a cAMP agonist (dbcAMP; 1 mM) or antagonist (Rp-cAMPS; 0, 3, 10, 30, or 100 microM). In experiment 2, corpora lutea from the early (n = 7), mid- (n = 6), and late (n = 6) luteal phases of the estrous cycle were dissociated and preincubated as in experiment 1, and luteal cells were then incubated with no treatment, LH (100 ng/ml), dbcAMP (1 mM), forskolin (1 microM), Rp-cAMPS (100 microM), or LH+Rp-cAMPS. After incubation of luteal cells with treatments for 18-24 h, media were collected for determination of progesterone and cAMP concentrations. Then the rate of GJIC was evaluated for selected cells (small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells) by using the fluorescence recovery after photo-bleaching technique and laser cytometry. In experiment 1, dbcAMP increased (p < 0.01) but Rp-cAMPS (p < 0.05) decreased GJIC between small luteal cells and between large and small luteal cells. In addition, dbcAMP stimulated (p < 0.01) but Rp-cAMPS did not affect progesterone secretion. In experiment 2, treatments affected (p < 0.05) GJIC and progesterone production of luteal cells from the mid- and late but not from the early luteal phase of the estrous cycle. GJIC between small luteal cells was increased (p < 0.01) by LH, dbcAMP, and forskolin. GJIC between large and small luteal cells was increased (p < 0.05) by dbcAMP and forskolin. Rp-cAMPS decreased (p < 0.01) GJIC between small luteal cells (mid-luteal phase) and between large and small luteal cells (mid- and late luteal phases). In addition, Rp-cAMPS inhibited (p < 0.05) the stimulatory effects of LH on GJIC between small luteal cells from the mid- and late luteal phases of the estrous cycle. For luteal cells from the mid- and late luteal phases, progesterone production was increased (p < 0.05) by LH, dbcAMP, forskolin, and LH+Rp-cAMPS, but was not affected by Rp-cAMPS. Across all stages of the estrous cycle, cyclic AMP accumulation in media was greater (p < 0.05) in LH- and forskolin-treated cultures than in control cultures and was greater (p < 0.01) in forskolin-treated than in LH-treated cultures. These data demonstrate that small and large luteal cells communicate with each other and that the rate of GJIC is modulated by LH and cAMP, as has been shown previously for other cell types. Thus, cAMP appears to be involved in the regulation of GJIC within the bovine corpus luteum, which probably is an important mechanism for coordinating luteal cell function.

Near-infrared spectroscopic monitoring of the film coating process.

PURPOSE: The purpose of this study was to investigate the potential of near-infrared (near-IR) spectroscopy for non-destructive at-line determination of the amount of polymer coat applied to tablet cores in a Wurster column. METHODS: The effects of coating composition on the near-IR spectroscopic determination of ethylcellulose (Aquacoat ECD-30) or hydroxypropylmethylcellulose (HPMC)-based (Spectrablend) coating were evaluated, as were the performance of several chemometric techniques. RESULTS: Tablets were coated with up to 30% ethylcellulose or 22% HPMC, and samples were pulled at regular intervals during each coating run. Near-IR reflectance spectra of the intact tablets were then collected. The spectra were preprocessed by multiplicative scatter correction (MSC) or second derivative (D2) calculations, and calibrations developed using either principal components (PCs) or multiple spectral wavelengths. The near-IR method provided predictions of film applied with standard errors of 1.07% w/w or less. CONCLUSIONS: Near-IR spectroscopy can be profitably employed in a rapid and non-destructive determination of the amount of polymer film applied to tablets, and offers a simple means to monitor the film coating process.

Transient immunoglobulin-like molecules are present in the subplate zone and cerebral cortex during postnatal development.

A monoclonal antibody (mAb SP-1) labels subplate neurons of the cat visual cortex but does not stain the remnants of the subplate neuronal population that comprise the interstitial cells of adult cortical white matter. mAb SP-1 was shown previously to recognize a cytosolic polypeptide of 56 kDa (Naegele et al., 1991). We have now characterized the distribution of SP-1 immunoreactive neurons in the visual cortex and carried out additional biochemical studies at a range of postnatal ages in various tissues. Brain, liver and serum were found to contain the previously identified 56 kDa polypeptide. This polypeptide was also recognized by a cat immunoglobulin antiserum. The epitope recognized by mAb SP-1 was present on cat IgG Fc fragment but not cat IgG Fab fragment. By 4 weeks postnatal, levels of the 56 kDa antigen decreased in cortex and an additional higher molecular weight SP-1 reactive polypeptide of 75 kDa was detected. In the mature cortex, both polypeptides were absent from cytosolic fractions. Immunocytochemical staining comparing the distributions of SP-1 (SP-1+) and anti-IgG (Ig+) immunoreactive neurons showed complete colocalization in subplate neurons beneath primary visual cortex. By 4 weeks, some pyramidal neurons in cortical areas 17 and 18 were weakly positive for SP-1 but negative for IgG. At subsequent ages, the immunoreactive staining became progressively fainter until it was no longer detectable in white or gray matter of adult cat visual cortex.

Determination of film-coated tablet parameters by near-infrared spectroscopy.

Near-infrared (near-IR) spectroscopy was used in the determination of three parameters of theophylline tablets film-coated with ethylcellulose. Spectra of individual intact tablets were collected on two near-IR spectrometers: a grating-based spectrometer, and an acousto-optic tunable filter spectrometer. Calibrations were developed for the prediction of the time to 50% dissolution (t50%) of theophylline for tablets of varying coat thickness, for the determination of the thickness of the ethylcellulose coat applied, and for the prediction of the hardness of coated tablets. Principal component analysis was performed on the spectra prior to calibration development. The standard errors of calibration (SEC) and prediction (SEP) for determination of dissolution rates were 2.8 and 6.6 min, respectively. The SEC for the coating thickness calibration was 0.0002 inches, with an SEP of 0.00024 inches, and the SEC and SEP for the determination of tablet hardness were 0.54 and 0.62 kilopons, respectively.