Leptin secretion in horses: effects of dexamethasone, gender, and testosterone.

Five experiments were performed to evaluate the effects of dexamethasone (DEX), gender, and testosterone on plasma leptin concentrations in horses. In experiment 1, plasma leptin, insulin, glucose, and IGF-1 concentrations were increased (P < 0.01) in stallions following five daily injections of DEX (125 microg/kg BW). In experiment 2, leptin concentrations increased (P < 0.01) in mares, geldings, and stallions following a single injection of DEX, and the response was greater (P < 0.01) in mares and geldings than in stallions. The gender effect was confounded by differences in body condition scores and diet; however, based on stepwise regression analysis, both BCS and gender were significant sources of variation in the best fit model for pre-DEX leptin concentrations (R(2) = 0.65) and for maximum leptin response to DEX (R(2) = 0.75). In experiment 3, in which mares and stallions were pair-matched based on age and body condition and fed similar diets, mares again had higher (P < 0.01) leptin concentrations than stallions after DEX treatment as used in experiment 2. In experiment 4, there was no difference (P > 0.1) in plasma leptin response in mares following four single-injection doses of DEX from 15.6 to 125 microg/kg BW. In experiment 5, treatment of mares with testosterone propionate every other day for 5 days did not alter (P > 0.1) plasma leptin concentrations or the leptin response to DEX. In conclusion, multiple injections of DEX increase leptin concentrations in stallions, as does a single injection in mares (as low as 15.6 microg/kg BW), geldings and stallions. The greater leptin levels observed in mares and geldings relative to stallions were due partially to their greater body condition and partially to the presence of hyperleptinemic individuals; however, even after accounting for body condition and diet, mares still had greater leptin concentrations than stallions after DEX administration. Elevation of testosterone levels in mares for approximately 10 days did not alter leptin concentrations in mares.

Effect of transient early hyperthyroidism on onset of puberty in Suffolk ram lambs.

The thyroid function and sexual development of eight 6-week-old Suffolk ram lambs were studied. The lambs were divided into either control or treatment groups and housed indoors. From 6 to 12 weeks of age, four lambs in the treatment group received 15 mg kg(-1) body weight per day of 6-propyl 2-thiouracil orally to suppress normal thyroid function. During the same period, thyroxine and tri-iodothyronine were injected s.c. at the rate of 8 and 16 microg kg(-1) body weight per day, respectively, to induce a hyperthyroid state. Four control lambs received sham injection and oral excipient. Concentrations of thyroxine, tri-iodothyronine, FSH, testosterone and insulin-like growth factor I were determined in blood collected by indwelling jugular catheters once a week, every 20 min from 09:00 to 15:20 h. Scrotal circumference was recorded each week. Semen collection was attempted by electro-ejaculation between weeks 17 and 36. Lambs were castrated at week 36 and testicular histology was examined. During the treatment period only, the concentration of thyroid hormones was higher in treated lambs than in controls (P < 0.05). From week 6 to week 9 only, concentrations of FSH in treated lambs were lower than in controls (P < 0.05). Insulin-like growth factor I concentrations were lower in treated lambs than in controls from week 10 to week 13 (P < 0.05). Frequency of testosterone pulses was higher (P < 0.01) in the treated lambs but concentrations were similar in the control and treated lambs throughout the experiment. Scrotal circumference was greater in treated lambs from week 26 to week 36 (P < 0.05). Treated lambs produced viable spermatozoa earlier than did control lambs. At week 36, sperm concentration in treated lambs was higher than in controls (P < 0.01) but semen volumes were similar (P > 0.1). Diameter of the seminiferous tubules in treated lambs was larger than in controls (P < 0.05). In conclusion, transient neonatal hyperthyroidism decreased FSH and insulin-like growth factor I concentrations temporarily, increased testosterone pulses and sperm production and advanced puberty in Suffolk ram lambs.

The role of steroidogenic and nonsteroidogenic luteal cell interactions in regulating progesterone production.

The corpus luteum is an extremely important reproductive endocrine gland that influences numerous physiological processes and is therefore perhaps the most widely studied reproductive endocrine structure to date. The corpus luteum and its major product, progesterone, are central to any discussion of impaired reproductive performance. It is generally agreed that our best chances for reducing reproductive losses lie in a thorough understanding of the mechanisms controlling luteal function during reproductive cycles and pregnancy. Normal corpus luteum function is dependent on numerous regulatory factors, such as prostaglandins, oxytocin, steroids, growth factors, cytokines, etc. Recently, increased interest in studying luteal cell interactions (steroidogenic large and small luteal cells as well as nonsteroidogenic "accessory" cells of the corpus luteum) has produced new evidence of communication within the corpus luteum that influences its function. The exact means of communication among the luteal cells are not known; however, this interaction seems to involve both luteotropic and luteolytic actions and can occur through either contact-dependent and contact-independent mechanisms.

Prostaglandin and progesterone production by bovine luteal cells incubated in the presence or absence of the accessory cells of the corpus luteum and treated with interleukin-1beta, indomethacin and luteinizing hormone.

This experiment examined production of prostaglandin (PG) F2alpha, PGE2 and progesterone by bovine luteal cells incubated with or without the accessory cells and treated with recombinant bovine interleukin-1beta (10 ng), indomethacin (5 microg) or luteinizing hormone (50 ng). During pretreatment, progesterone production was similar in the luteal and luteal plus accessory cells, being greater in either of these than in accessory cells; PGF2alpha was greatest in luteal plus accessory, intermediate in accessory and lowest in the luteal cells; PGE2 was similar among all cell arrangements. Luteinizing hormone or luteinizing hormone plus indomethacin stimulated progesterone in the luteal and luteal plus accessory cells, this being similar in these two cell arrangements. Interleukin-1beta stimulated PGF2alpha in luteal and luteal plus accessory cells, and tended to elevate PGF2alpha in accessory cells. Interleukin-1beta stimulated PGE2 in all cell arrangements. Indomethacin inhibited the interleukin-1beta-stimulated PGF2alpha and PGE2 release. Indomethacin or indomethacin plus luteinizing hormone inhibited basal PGE2 in luteal and luteal plus accessory cells. These data suggest that progesterone production is not influenced by the presence of accessory cells of the corpus luteum, that accessory and luteal cells produce appreciable amounts of PG, and that recombinant bovine interleukin-1beta stimulates PGE2 and PGF2alpha in the luteal and accessory cells.

Bovine luteal cell production in vitro of prostaglandin E2, oxytocin and progesterone in response to pregnancy-specific protein B and prostaglandin F2 alpha.

A study was conducted to determine the effects of pregnancy-specific protein B (PSPB) and prostaglandin F2 alpha (PGF2 alpha) on bovine luteal cell progesterone, prostaglandin E2 (PGE2) and oxytocin production in vitro. Corpora lutea were enucleated from multiparous cows with normal oestrous cycles during the mid-luteal (days 10-12; n = 5) or late-luteal (days 17-18; n = 5) stage. Mixed large and small cells (1.5 x 10(5) cells per well) were incubated in 500 microliters modified Ham's F-12 medium. Cells were incubated for 18 h before treatments were added. Cells were treated with PSPB (0, 2.5, 5.0 micrograms) and PGF2 alpha (0, 100, 200 ng) in a 3 x 3 factorial arrangement. After treatments were added, media samples were collected at 6 and 12 h. During the 18 h pretreatment incubation, progesterone, PGE2 and oxytocin production was similar between the prospective treatment groups. Progesterone production was greater (P < 0.001) by mid-stage than by late-stage cells. In addition, progesterone decreased (P < 0.001) as incubation time increased. Progesterone production was not affected by PGF2 alpha, but PSPB increased (P < 0.02) progesterone at the 5.0 micrograms dose. Late-stage luteal cells produced more (P < 0.001) PGE2 than did mid-stage cells; PGE2 production decreased (P < 0.001) with increased incubation time. Luteal PGE2 production increased in response to PSPB treatment (P < 0.01) and PGF2 alpha treatment (P < 0.001). Luteal oxytocin production was greater (P < 0.01) by mid-stage compared with late-stage cells. Oxytocin production decreased (P < 0.001) with incubation time in mid-stage cells, but in late-stage cells oxytocin production was similar over time. Neither PSPB nor PGF2 alpha had an effect on oxytocin. These results indicate that PSPB does not affect luteal oxytocin, but does increase progesterone and PGE2 production. In addition, PGF2 alpha increases luteal PGE2, but does not affect progesterone or oxytocin production. These data do not show an interaction between PSPB and PGF2 alpha in regulating bovine luteal cell endocrine function.

Effect of pregnancy-specific protein B on luteal cell progesterone, prostaglandin, and oxytocin production during two stages of the bovine estrous cycle.

This experiment was designed to evaluate the effects of pregnancy-specific protein B (PSPB) on luteal cell progesterone (P4), PGF2 alpha, PGE2, and oxytocin secretion. Corpora lutea were collected during the mid (d 10 to 12; n = 5) or late luteal (d 17 to 18; n = 5) stage of the estrous cycle. Large and small cells (1.5 x 10(5)/well) were treated with PSPB (0, 2.5, or 5.0 micrograms) and LH (0, 50, or 100 ng) in a 3 x 3 factorial arrangement. Cells were incubated for 18 h before adding treatments; after treatments, medium was collected at 6 and 12 h. During the 18-h pretreatment period, P4, PGF2 alpha, PGE2, and oxytocin production was similar between the prospective treatment groups. The PSPB did not affect P4 production. Stage of the cycle (stage) x time interaction (P < .001) indicated that mid-stage luteal cells produced more P4 than late-stage cells; regardless of stage, P4 decreased with time. The time x LH interaction (P < .001) revealed that at 6 and 12 h the 50- and 100-ng doses of LH increased P4 to greater than the 0-ng dose. Production of PGF2 alpha by mid-stage cells was similar among the three PSPB treatments; however, PGF2 alpha production by late-stage cells increased (P < .01) in response to the 5.0-micrograms dose of PSPB. The LH did not affect PGF2 alpha production. Late-stage luteal cells produced more (P < .001) PGF2 alpha than mid-stage cells during the 18-h pretreatment period and at 6, but not 12, h.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin F2 alpha, progesterone and oxytocin production by cultured bovine luteal cells treated with prostaglandin E2 and pregnancy-specific protein B.

The objectives of this experiment were to study the effects of pregnancy-specific protein B (PSPB) and prostaglandin E2 (PGE2) on bovine luteal cell progesterone (P4), prostaglandin F2 alpha (PGF2 alpha) and oxytocin production. Corpora lutea were collected during the mid- (days 10-12; n = 5) or late-luteal (days 17-18; n = 5) stages of the estrous cycle. Luteal cells were dispersed and accessory cells removed. Luteal cells (1.5 x 10(5)) were incubated in a 3 x 3 factorial arrangement and treated with PSPB (0, 2.5, or 5.0 micrograms) and PGE2 (0, 100, or 200 ng) in 500 microL of Ham's F-12 medium. All cells were incubated for 18 h before adding treatments. Samples were then collected at 6 h and 12 h. During the 18 h pretreatment period, P4, PGF2 alpha, and oxytocin production was similar between the prospective treatment groups. The PSPB failed to increase P4 production. The PGE2 x time interaction showed that P4 increased in response to PGE2 treatment at 6 h (P < 0.001) and 12 h (P < 0.03). Also, the stage x time interaction indicated that mid-stage cells produced more (P < .001) P4 than late-stage cells during the pretreatment period at 6 h and 12 h. The PSPB did not alter PGF2 alpha production by mid-stage cells, but increased (P < .05) PGF2 alpha by late-stage cells. Also, PGE2 stimulated (P < 0.001) PGF2 alpha secretion by both mid- and late-stage cells; luteal cells treated with 200 ng of PGE2 produced more (P < 0.001) PGF2 alpha than 100 ng of PGE2. Oxytocin secretion was not changed by treatment with PGE2 or PSPB. Oxytocin production was greater (P < 0.001) by mid-stage than late-stage cells during the pretreatment period at 6 h and 12 h. Oxytocin production was similar between the 6 h and 12 h culture times within stage of the cycle. These data indicate that PSPB does not change bovine luteal cell P4 or oxytocin production, but elevates PGF2 alpha in late-stage cells. The PGE2 increases both P4 and PGF2 alpha, but does not alter oxytocin production. Lastly, PSPB and PGE2 do not interact to promote P4 PGF2 alpha, or oxytocin production by cultured bovine luteal cells.

Contact-associated interactions between large and small bovine luteal cells during the estrous cycle.

This experiment was designed to study the effects of cell-to-cell contact, arachidonic acid (10 microM; AA), oxytocin (10 microM), and luteinizing hormone (5 ng; LH) on bovine luteal cell function. Corpora lutea collected from Holstein cows between Days 10 and 12 (n = 4; midluteal stage) or 17 and 18 (n = 4; late-luteal stage) of the estrous cycle (Day 0 = estrus) were dispersed, and small and large cells were separated by unit gravity sedimentation and flow cytometry. Large and small luteal cells were either incubated together, allowing intercellular contact, or separately, without intercellular contact, with culture well inserts. Cells were incubated in a modified Ham's F-12-N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid medium. After an 18-hr preincubation period, treatments were introduced and cells were incubated for 240 hr. Media samples were collected and treatments were replaced at 48-hr intervals. Incubations were maintained at 37 degrees C in 5% CO2 in humidified air. Overall, progesterone secretion decreased with increased incubation time (P < 0.0001), regardless of treatment, stage of the cycle, or cell arrangement. During the 18-hr pretreatment period, large and small luteal cells with contact secreted more progesterone than did luteal cells without contact during both the mid- (P < 0.0001) and late-luteal stages (P < 0.06) of the estrous cycle. After treatments were initiated, both mid- and late-stage luteal cells treated with LH secreted more (P < 0.0001) progesterone than occurred with any other treatment; oxytocin, AA, and control treatments were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between large and small luteal cells collected during the mid- or late-luteal stages of the bovine oestrous cycle.

The effects of contact between large and small bovine luteal cells together with those of luteinizing hormone (LH) or arachidonic acid (AA) on progesterone production during the oestrous cycle were investigated. Corpora lutea were collected during the mid-luteal stage (Days 10-12; n = 4) and late-luteal stage (Days 17-18; n = 4) of the oestrous cycle. Large and small luteal cells were dispersed and separated and then incubated together or separately. Mid-luteal stage cells were treated with LH (0 or 5 ng) whereas late-luteal stage cells were treated with LH (0 or 5 ng) or AA (0 or 10 microM). Culture medium was collected and replaced 1, 3 and 6 h after starting treatments. Progesterone production decreased (P < 0.0001) with increased incubation time irrespective of cell arrangement, the stage of the oestrous cycle or treatment. During the 18 h before treatment, cells in the contact arrangement produced more progesterone (P < 0.003) than cells without contact in both mid- and late-luteal stages of the oestrous cycle; progesterone production within cell arrangements between prospective treatment groups was similar. After initiating treatments, mid-luteal stage cells in the control group without contact produced more progesterone (P < 0.01) than cells with contact. Mid-luteal stage cells treated with LH produced more (P < 0.0001) than control cells; progesterone production between cell arrangements within the LH treatment group was similar. In the late-luteal stage cells, both LH and AA increased (P < 0.01) progesterone production by comparison with control cells; LH and AA treatment groups produced similar results.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between large and small bovine luteal cells in a sequential perifusion co-culture system.

The objectives of this experiment were to study large and small luteal cell interactions and examine the effect of arachidonic acid (AA) on progesterone production by separated bovine luteal cells. Corpora lutea collected from Holstein cows (n = 5) on d 12 of the estrous cycle were dispersed, and small (SLC) and large (LLC) luteal cells were separated by unit gravity sedimentation and flow cytometry. Cells were incubated at 37 degrees C in separate perifusion chambers with a modified Ham's F-12-HEPES medium and aerated with 95% O2:5% CO2. The flow rate of medium was 100 microL/min, and fractions were collected at 30-min intervals for 4 h. Luteal cells were arranged in tandem so that medium from the first cell type would pass through the chamber containing the second cell type. Luteal cells were arranged so that medium flowed from 1) SLC to SLC, 2) LLC to LLC, 3) SLC to LLC, 4) LLC to SLC, 5) SLC to LLC, 6) LLC to SLC; medium for arrangements 5 and 6 contained 10 microM AA. Cells in arrangements 5 and 6 were perifused for 30 min before AA was added. Progesterone was measured with an enzymeimmunoassay. The LLC to LLC arrangement had a greater (P < .05) average progesterone secretion rate than all other cell arrangements, and the SLC to SLC arrangement had the least progesterone secretion rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous uterine infections are associated with elevated prostaglandin F(2)alpha metabolite concentrations in postpartum dairy cows.

Postpartum Holstein (n=21) and Jersey (n=4) cows were used to determine if uterine infections are associated with elevated plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F(2)alpha (PGFM). Based upon clinical examinations and bacterial content of intrauterine fluid samples, cows detected with uterine infections between 21 and 28 d post partum were used (infected; n=14). These cows were matched with herdmates that were free of infection (control; n=11). Beginning on the day the cows were assigned to the experiment (Day 1), blood samples were collected on alternate days for the next 14 to 15 d. Plasma samples were stored at -20 degrees C until assayed. From Day 1 until the end of the experiment, uterine fluid samples were collected transcervically twice weekly for aerobic bacterial culture. Endometrial biopsies were collected between Days 6 and 8 and Days 13 and 15. Control cows did not show signs of uterine infection throughout the trial, and bacterial cultures indicated that there were no significant bacterial populations in the uteri of the control cows. The uteri of infected cows harbored numerous microbes. Actinomyces pyogenes was most prominent. Various species of Streptococcus and Pasteurella were also prevalent in the infected cows. Escherichia coli was present in the uterus of both infected and control cows. Biopsies showed that infected cows had more (P<0.05) neutrophils, plasma cells and lymphocytes in the endometrium than did the control cows. As determined by plasma progesterone concentrations, 83% of the control and 50% of the infected cows had functional luteal tissue during the 2-wk sampling period. Plasma PGFM profiles were linear (P<0.03) and did not differ between treatment groups (P>0.01). However, average plasma PGFM concentrations were greater (P<0.0001) in infected than in control cows. These data indicate that plasma PGFM concentrations are greater in postpartum cows with spontaneous uterine infections then in herdmates free of infection.

Stimulation of development of in vitro-matured and in vitro-fertilized bovine embryos by platelets.

In vitro-fertilized bovine embryos were incubated in Menezo's B2 medium (MB2) supplemented with 2 mg/mL of BSA. In Exp. 1, eight-cell stage embryos were allotted to one of the following groups: control medium (MB2), MB2 with 20 ng/mL of platelet-activating factor (PAF), 1 x 10(7) bovine blood platelets (Platelets), oviductal cells (BOEC), BOEC and 20 ng/mL of PAF (BOEC+PAF), or BOEC and 1 x 10(7) platelets (BOEC+Platelets). In Exp. 2, eight-cell embryos were allotted to one of the following groups: control medium (MB2), MB2 with 1 x 10(7) platelets (Platelets), 1 x 10(7) platelets and 10 micrograms/mL of platelet-derived growth factor antibody (Platelets+anti-PDGF), 1 x 10(7) platelets and 1 microgram/mL of indomethacin (Platelets+Indomethacin), or 1 x 10(7) platelets and 3 micrograms/mL of mianserin (Platelets+Mianserin). Embryos were incubated at 39 degrees C in 5% CO2 in groups of five until 8 d after in vitro fertilization (IVF). In Exp. 1, Platelets stimulated embryo development to the morula, blastocyst, and expanded blastocyst stages. Embryo development was greatest in the BOEC+Platelets group on d 7 and 8 after IVF. Only embryos incubated in the BOEC+Platelets treatment group reached the hatched blastocyst stage on d 8. In Exp. 2, embryos incubated in the Platelets treatment group had the greatest (P < .05) proportion develop beyond the eight-cell stage. Embryos incubated in the Platelets + anti-PDGF group had less (P < .05) development beyond the eight-cell stage and to the morula stage. These results indicate that the stimulatory effects of PDGF on bovine embryo development may be derived from both the oviductal epithelium and platelets.

Role of platelet-derived growth factor in development of in vitro matured and in vitro fertilized bovine embryos.

This experiment was designed to determine whether the stimulatory effects of bovine oviductal epithelial cells (BOEC) on development of early bovine embryos are due to platelet-derived growth factor (PDGF). Four hundred and twenty five 8-cell bovine embryos derived from in vitro maturation and in vitro fertilization procedures were equally and randomly allotted to one of the following culture treatment groups: control medium alone (Menezo's B2 medium; MB2), MB2 with 1 ng PDGF ml-1 (PDGF), 1 ng PDGF ml-1 and 10 micrograms anti-PDGF antibody ml-1 (PDGF + Ab), BOEC or BOEC and 10 micrograms anti-PDGF antibody ml-1 (BOEC + Ab). All embryos were cultured in 100 microliters of serum-free MB2 medium supplemented with 2 mg fatty-acid-free bovine serum albumin ml-1. Embryos for all treatment groups were incubated at 39 degrees C and 5% CO2 in humidified air in groups of five embryos per well in 96-well culture plates until 7 days after in vitro insemination. A higher proportion of embryos developed to > 8-cell and to the morula stage following culture with PDGF, BOEC or BOEC+Ab than with MB2 alone. Incubation of PDGF and BOEC-treated embryos with anti-PDGF reduced development to the morula and blastocyst stages. However, anti-PDGF did not completely inhibit blastocyst development when added to BOEC. In addition, embryos incubated with BOEC and anti-PDGF contained a reduced number of inner cell mass cells compared with embryos incubated with BOEC alone. These results indicate that PDGF provides a developmental stimulus similar to BOEC for bovine embryos at the fourth cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Jugular plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha in prepubertal beef heifers treated with progestogen then challenged with oxytocin.

Prepubertal Angus crossbred heifers (n = 24) between 8 and 10 mo of age were used to determine if progestogen treatment would enhance jugular concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) after oxytocin (OT) injections. Heifers were stratified by age and weight and allotted to randomized treatments in a 2 x 2 factorial arrangement. Heifers were treated with either a norgestomet (NOR) implant (6 mg) for 9 d or no implant (0 mg; BLK). On d 8 of NOR treatment, jugular veins were catheterized and, on d 9, blood samples were collected every 15 min for 165 min. The first four samples were used to determine basal PGFM concentrations (an indirect measure of uterine PGF2 alpha release). After collection of the fourth sample, either OT (100 IU) or saline (0 IU; SAL) was injected via the jugular catheter. After the 165-min sample was collected, NOR implants were removed. Beginning 48 h after implant removal, a second 165- min blood sampling period was initiated. Average progesterone concentrations were less than 1 ng/ml during both bleeding periods. Within treatment, PGFM concentrations were similar between the first and second sampling periods; therefore, data within treatment were combined. Basal PGFM concentrations were higher (P < .01) in NOR-treated than in BLK heifers. Oxytocin did not increase PGFM concentrations in BLK-OT heifers; however, a marked increase in PGFM was detected in the NOR-OT heifers in response to oxytocin. Average PGFM concentration was greatest (P < .0001) in NOR-OT heifers, and PGFM profiles differed (P < .0001) between NOR-OT and each of the other treatment groups. Results from this study indicate that NOR increases basal PGFM and may "condition" the uterus to respond to OT in prepubertal heifers.

Effect of intrauterine bacterial infusions and subsequent endometritis on prostaglandin F2 alpha metabolite concentrations in postpartum beef cows.

Multiparous Angus and crossbred Angus cows were used to determine the effect of induced endometritis on plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) and progesterone (P4) and on duration of the estrous cycle of treatment. Beginning on the day of calving (d 0), blood samples were collected on alternate days. On three consecutive days, ranging from d 8 to 14 of the first postpartum estrous cycle, uterine horns were inoculated transcervically with either 3 x 10(9) colony forming units (cfu) of Actinomyces pyogenes and 1.5 x 10(9) cfu of beta-hemolytic Escherichia coli (treated; n = 9) in sterile PBS or with sterile PBS alone (control; n = 9). Samples of uterine fluid were collected by transcervical aspiration twice weekly from just before the start of each series of inoculations until the end of the experiment. Endometrial biopsies were collected transcervically between d 4 to 6 and 11 to 13 after inoculation. Based on clinical observations and results of bacterial cultures, all treated cows developed acute uterine infections. Controls did not develop uterine infections. Endometrial biopsies indicated that there were no significant diffuse or focal cellular reactions in response to the infection. The interestrous interval was greater (P less than .0003) for treated (27.7 +/- 1.0 d) than for control (20.6 +/- 1.0 d) cows, but P4 concentrations were similar between the two groups. Mean PGFM concentration and PGFM profiles were similar (P greater than .10) between treated and control cows before bacterial infusions. Bacterial infusions increased mean PGFM concentration (P less than .0001) and changed the shape of the PGFM profile (P less than .02).(ABSTRACT TRUNCATED AT 250 WORDS)

Concentrations of 13, 14-dihydro-15-keto-prostaglandin F(2)alpha, estradiol-17beta and progesterone during the peripubertal period in heifers.

Twenty prepubertal Holstein heifers were utilized to assess plasma 13, 14-dihydro-15-keto-prostaglandin F(2)alpha (PGFM), serum progesterone (P(4)) and estradiol-17beta (E(2)) concentrations as well as the E(2):P(4) ratio during the onset of puberty in cattle. All animals were maintained as a group along with a sterile marker bull to assist in the detection of estrus. Upon detection of the first estrus (Day=O), daily blood samples were collected from a jugular vein until the heifers had completed 3 estrous cycles. The average body weight and age at first estrus were 247.6+/-4.8 kg and 304.0+/-7.5 days, respectively. Frequency of abnormal length estrous cycles was greater (P<0.02) during the first (40%) and second (35%) cycles than during the third estrous cycle (0%). All heifers had normal cycle lengths (18 to 24 days) by the third estrous cycle. Serum P(4) was greater during the third cycle (P<0.05) from Day 10 to Day 4 before the next estrus compared with the same period of the first estrous cycle. Serum E(2) did not peak until the day of estrus in the first cycle, whereas E(2) reached a maximal level 2 days before estrus in the third estrous cycle. Serum E(2) was higher (P<0.0001) 2 days before estrus in the third cycle than in the first estrous cycle. Plasma PGFM reached maximum concentrations 3 days before estrus in the third cycle compared with 1 day before estrus at the end of first estrous cycle. As estrus approached during the third cycle, PGFM rose 1 day before E(2) rose and P(4) declined, while the rise in PGFM and E(2) occurred simultaneously, with P(4) declining at the end of the first estrous cycle. During diestrus, the E(2):P(4) ratio was lower (P<0.07) in the third cycle than in the first, but it was higher (P<0.04) at estrus and 1 day before in the third estrous cycle. These data reveal a high incidence of abnormal length estrous cycles during the first two estrous cycles of the peripubertal period, and demonstrate anomalies in uterine and ovarian endocrine activity during the peripubertal period in cattle.

In vitro metabolism of glucose by bovine reproductive tissues obtained during the estrous cycle and after calving.

Two experiments were conducted to determine the effect of physiological state (stage of the estrous cycle or time after calving) on the in vitro metabolism of glucose by reproductive tissues. In Exp. 1, the corpus luteum and ipsilateral uterine horn were collected at surgery from 15 cows at early (d 6 to 7), middle (d 10 to 13), or late (d 17 to 19) stages of the estrous cycle. Luteal or endometrial tissues (45 mg) were incubated (4 h, 37 degrees C) in metabolic flasks containing Nutrient Mixture F-10 (3 mL), increasing concentrations of glucose (1, 2, 5, 10, or 15 mM), and 1 microCi of [U-14C]glucose. Luteal tissue collected at the middle stage of the estrous cycle had greater (P less than .04) rates of glucose uptake (17.8 vs 12.1 ng/mg of wet tissue per min) and oxidation (138.6 vs 67.7 pg/mg of wet tissue per min) and a lower (P less than .02) rate of metabolism of glucose to lactate (10.5 vs 13.3 ng/mg of wet tissue per min) than tissue collected at the late stage. Compared to tissue collected at early and late stages, endometrial tissue collected at the middle stage of the estrous cycle had a similar (P greater than .5) rate of glucose uptake (29.6 vs 27.8 and 26.1 ng/mg of wet tissue per min), a lower (P less than .02) rate of metabolism of glucose to lactate (13.8 vs 16.6 and 16.4 ng/mg of wet tissue per min), and a greater (P less than .1) rate of oxidation of glucose (84.1 vs 65.8 and 72.3 pg/mg of wet tissue per min). In Exp. 2, the previously gravid uterine horn was collected at surgery on d 20 (n = 8) or 30 (n = 7) after calving, or the uterine horn ipsilateral to the preovulatory follicle was collected at first observed estrus after calving (n = 8). Uterine tissue obtained at first estrus after calving had a greater (P less than .003) rate of glucose uptake and oxidation (88.8 vs 77.3 and 75.5 pg/mg of tissue per min) than d-20 or d-30 endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative solid-phase enzymeimmunoassay for 13,14-dihydro-15-keto- prostaglandin F2 alpha in plasma.

Enzymeimmunoassays (EIA) can be viable alternatives to radioimmunoassays (RIA). Indeed, from an environmental perspective, EIA are preferable to RIA. Therefore, the purpose of this project was to develop a quantitative EIA for 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) in bovine plasma. Acetylcholine esterase bound covalently to PGFM, rabbit anti-PGFM, mouse monoclonal anti-rabbit IgG, and PGFM were the principle reagents used for the EIA. Validation experiments indicated that: 1) PGFM standard curves, with doses ranging from 391 to 200,000 fg per microtiter well, were linear; 2) assay sensitivity averaged 391 fg per well; 3) for satisfactory results, PGFM had to be extracted from plasma; 4) content of PGFM in ethyl ether extracts of aliquots from serial dilutions of whole plasma with unknown amounts of PGFM and charcoal-stripped plasma supplemented with known amounts of PGFM did not deviate from parallelism with PGFM standard curves in buffer; 5) correlation between EIA and RIA measurements of PGFM in the same plasma samples was .95; 6) the regression of EIA data on RIA data was linear (Y = .93 x + 83.9; r2 = .91); 7) intra- and interassay coefficients of variation were 3.3 and 10.6%, respectively. The EIA developed in this project is a valid and reliable method for quantitating PGFM in extracts of bovine plasma.

Role of prostaglandin F2 alpha in follicular development and subsequent luteal life span in early postpartum beef cows.

In postpartum cows expected to have corpora lutea (CL) of normal (norgestomet-treated) compared to short (control) life spans, function of the largest follicle increases after an increase in concentrations of prostaglandin F2 alpha (PGF). To determine whether PGF alters follicular growth and subsequent life span of the CL, 43 crossbred beef cows (19 to 22 d postpartum) were assigned to one of four treatments: 1) control (C; n = 10), 2) control+PGF (CPGF; n = 10), 3) norgestomet (N; n = 13), 4) norgestomet+flunixin meglumine (NFM; n = 10). Flunixin meglumine inhibits prostaglandin endoperoxide synthase. On day 0, N and NFM cows received a 6 mg implant of norgestomet. From days 3 through 8, CPGF and NFM cows were injected every 8 hr with 10 mg PGF im or 1 g FM iv, respectively. Implants were removed on day 9. On day 11, each cow received 1000 IU of hCG im to induce formation of CL. Follicular growth was monitored by daily ultrasonography from days 6 through 11. In a majority of the cases (25/32), the largest follicle present on day 6 was still the largest on day 11; frequency of persistence did not differ with treatment. Rate of growth of the largest follicle was greater in CPGF than in N cows (.6 +/- .1 vs .3 +/- .1 mm/d, respectively; P less than .05) but did not differ between C and NFM cows (.4 +/- .1 and .5 +/- .1 mm/d, respectively). Concentrations of estradiol in NFM cows were higher (P less than .05) on day 3 and declined to concentrations similar to those of the other treatments on day 9.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma 13,14-dihydro-15-keto-prostaglandin F2 alpha concentrations in prepubertal dairy heifers challenged with oxytocin.

Twenty-nine prepubertal Holstein heifers were assigned by age to one of three age groups to determine if the prepubertal bovine uterus could respond to an oxytocin stimulus. Group 1 heifers were 6 to 7 months of age (AGE1; n = 11), group 2 heifers were 8 to 9 months of age (AGE2; n = 11) and group 3 heifers were 10 to 11 months of age (AGE3; n = 7). Blood samples were collected via an indwelling jugular catheter. Four samples were collected at 15-min intervals prior to oxytocin administration to determine basal 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) concentrations. Each heifer received 100 IU of oxytocin i.v., blood sampling continued at 5 min intervals for the next 30 min and for an additional 90 min at 15-min intervals. Heifers were considered responders to oxytocin if mean PGFM concentrations increased at least 1.5 times the SD of their basal PGFM concentration. Age of the heifer (P less than .0001) and responder status (P less than .05) affected plasma PGFM. Plasma PGFM was higher in AGE1 and AGE3 heifers than AGE2 (P less than .0001). The number of responders was greatest at AGE3 (P less than .03) with AGE1 and AGE2 being similar. Mean basal PGFM was lower (P less than .04) at AGE2 than AGE1 with AGE3 being intermediate. In addition, basal PGFM at AGE1 tended to be lower (P less than .08) in the responders than in the non-responders, while AGE2 basal PGFM did not differ between responders and non-responders (P greater than .10).(ABSTRACT TRUNCATED AT 250 WORDS)