S.H. Hayes

Relationship between endogenous progesterone and follicular dynamics in lactating dairy cows with ovarian follicular cysts.

Abstract Two experiments were conducted to examine circulating concentrations of progesterone (P4) in cows with ovarian follicular cysts (OFCs) and to relate differing levels of P4 to subsequent follicular events. In experiment 1, peripheral concentrations of P4 were determined in cows diagnosed with OFCs. Nonpregnant, lactating Holstein and Jersey cows (n = 32) were diagnosed as having OFCs by rectal palpation. Ovarian follicular cysts were then examined by transrectal ultrasonography to confirm the presence of OFCs (follicle diameter, ≥17 mm; absence of luteal tissue). At confirmation, a blood sample was collected for quantification of P4. The concentration of P4 at confirmation was classified as low (<0.1 ng/ml), intermediate (0.1–1.0 ng/ml), or high (1.0–2.0 ng/ml). More OFCs were associated with intermediate (66%) than with either low (28%) or high (6%) concentrations of P4. In experiment 2, the fate of follicles (diameter, ≥10 mm) that formed in the presence of an OFC was determined and related to circulating concentrations of P4 during follicular development. Follicles (n = 59) that formed in the presence of an OFC ovulated (n = 19), formed a cyst (n = 30), or underwent normal growth and regression (NGR; n = 10). Endogenous P4 in the 7-day period during follicular development was classified as low (if P4 dropped to <0.1 ng/ml for 1 day or longer), intermediate (if P4 averaged between 0.1 and 1.0 ng/ml and never dropped to <0.1 ng/ml), or high (if P4 averaged >1.0 ng/ml and never dropped to <0.1 ng/ml). In the presence of intermediate P4, 75% of observed follicles formed cysts, compared with 10% that ovulated and 15% that experienced NGR. In the presence of low P4, 53%, 41%, and 6% of follicles ovulated, formed a follicular cyst, or experienced NGR, respectively. Thus, an association between intermediate P4 and the formation of OFCs was established.

Cellular Mechanisms by Which Oxytocin Mediates Ovine Endometrial Prostaglandin F2α Synthesis: Role of Gi Proteins and Mitogen-Activated Protein Kinases

Abstract Oxytocin stimulates a rapid increase in ovine endometrial prostaglandin (PG) F2α synthesis. The overall objective of these experiments was to investigate the cellular mechanisms by which oxytocin induces endometrial PGF2α synthesis. The objective of experiment 1 was to determine whether Gi proteins mediate oxytocin-induced PGF2α synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. Pertussis toxin, an inhibitor of Gi proteins, had no effect on the ability of oxytocin to induce PGF2α synthesis (P > 0.10). The objective of experiment 2 was to determine whether any of the three mitogen-activated protein kinases (MAPKs), extracellular signal regulated protein kinase (ERK1/2), c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK), or p38 MAPK, mediate oxytocin-induced PGF2α synthesis. Eleven ovary-intact ewes were given an injection of oxytocin (10 IU; i.v.; n = 5) or physiological saline (i.v.; n = 6) on Day 15 postestrus. Uteri were collected 15 min after injection and caruncular endometrium was dissected. Endometrial homogenates were prepared and subjected to Western blotting. Membranes were probed for both total and phosphorylated forms of all three classes of MAPK. All classes of MAPK were detected in ovine endometrium, but oxytocin treatment had no effect on the expression of these proteins (P > 0.10). ERK1/2 was the only phosphorylated MAPK detected and its concentrations were higher in oxytocin-treated ewes (P < 0.01). The objective of experiment 3 was to further investigate the role of ERK1/2 during oxytocin-induced PGF2α synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. PD98059, a specific inhibitor of ERK1/2 activity, blocked the ability of oxytocin to stimulate PGF2α synthesis in a dose-dependent manner (P < 0.05). These results indicate that the ovine oxytocin receptor is not coupled to Gi proteins. These results indicate that oxytocin induces phosphorylation of ERK1/2 and that this MAPK appears to mediate oxytocin-induced PGF2α synthesis in ovine endometrium.

Cellular mechanisms by which oxytocin stimulates uterine PGF2α synthesis in bovine endometrium: Roles of phospholipases C and A2

The objective of these experiments was to identify the cellular mechanisms by which oxytocin stimulates prostaglandin (PG) F2 alpha synthesis in bovine endometrial tissue. Uteri were collected on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to assess PGF2 alpha release or phospholipase (PL) C activity. Oxytocin (10(-6) M) stimulated PGF2 alpha release and PLC activity within 30 min of incubation (P < 0.01). The highest stimulation was observed at 100 min (P < 0.01). Oxytocin stimulated PLC activity at 10(-9) M and higher doses, whereas an increase in PGF2 alpha release was not detected until 10(-8) M (P < 0.09). Melittin, a stimulator of PLA2 activity, stimulated PGF2 alpha release at 10(-6) M and higher doses (P < 0.01). Aristolochic acid, an inhibitor of PLA2 activity, blocked the ability of oxytocin to stimulate PGF2 alpha release at 10(-5) M and higher doses (P < 0.01). Aristolochic acid (10(-4) M) reduced the stimulation of PGF2 alpha release induced by A1F4-, a nonspecific stimulator of G protein (10(-5) M) and melittin (10(-4) M; P < 0.05). Aristolochic acid had no effect on the ability of oxytocin or A1F4- to stimulate PLC activity (P > 0.10). By comparing the time course of stimulation and dose-response relationships between PGF2 alpha and PLC activity, it appears that oxytocin may stimulate PGF2 alpha secretion by activating PLC. The effects of melittin and aristolochic acid indicate that PLA2 may play a role in mediating the stimulatory effect of oxytocin on PGF2 alpha secretion, as well.

Cellular mechanisms by which oxytocin mediates uterine prostaglandin F2α synthesis in bovine endometrium: role of calcium

The objective of these experiments was to determine the role of Ca2+ during oxytocin-stimulated prostaglandin (PG) F2 alpha release from bovine endometrial tissue in vitro. Uteri were collected from dairy cows on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to determine phospholipase C activity or PGF2 alpha release. A23,187 (a calcium ionophore) and maitotoxin (an activator of voltage-gated L-type calcium channels) stimulated release of PGF 2 alpha in a concentration-dependent manner (P < 0.05). Thapsigargin (induces accumulation of Ca2+ in the cytoplasm by inhibiting endoplasmic reticulum Ca2+/ATPase pumps) stimulated release of PGF2 alpha in a concentration-dependent manner as well (P < 0.13). Oxytocin (10(-6) M), AIF4- (a nonspecific activator of G-proteins; 10(-5) M), A23,187 (10(-5) M), and melittin (a stimulator of phospholipase A2; 10(-4) M) stimulated PGF2 alpha release when explants were incubated in Ca(2+)-free medium (P < 0.10); however, oxytocin, A23,187, or melittin were unable to stimulate PGF2 alpha release when explants were incubated in Ca(2+)-free medium containing the calcium chelator EGTA (P < 0.10). This treatment did not prevent oxytocin or AIF4- from stimulating phospholipase C activity (P < 0.08). CoCl2 (a nonspecific Ca2+ channel blocker) and methoxyverapamil (a specific voltage-gated L-type Ca2+ channel blocker) prevented oxytocin from stimulating PGF2 alpha release (P < 0.05). Our results suggest that both extracellular and intracellular Ca2+ may be required for oxytocin to stimulate PGF2 alpha secretion in bovine endometrial tissue.

Comparison of in vitro digestibility estimates using the DaisyII incubator with in vivo digestibility estimates in horses.

The objective of this study was to determine if in vitro methodologies developed for the Ankom Daisy(II) incubator could produce accurate estimates of in vivo equine DM digestibility (DMD) and NDF digestibility (NDFD) when equine feces were used as the inoculum source. Four mature geldings were utilized in a 4 × 4 Latin square design experiment with a 2 × 2 factorial arrangement of dietary treatments (timothy hay, alfalfa hay, timothy hay plus oats, and alfalfa hay plus oats), in which the geldings were individually housed and fed. During each 5-d total fecal collection period, feces were collected and composited daily and used to calculate in vivo digestibility. Digestion of the 4 treatment diets was evaluated in vitro using the Daisy(II) incubator. Each incubation vessel of the Daisy(II) was assigned to 1 of the horses and contained 18 filter bags (6 containing the assigned treatment hay, 6 containing hay-oat mix, and 6 containing oats). Three incubation periods were evaluated: 30, 48, and 72 h. Although the 30- and 48-h in vitro estimates were consistently less than the in vivo estimates, they ranked diets in the same order as the in vivo method. For the alfalfa oat diet, timothy diet, and the timothy oat diet, the mean 72-h in vitro DMD and in vivo DMD were not different (P = 0.1444). However, for the alfalfa diet, the DMD estimate from 72-h in vitro incubation was less than the in vivo estimate (P < 0.010). For NDFD, the timothy diet was the only diet, in which the mean 72-h in vitro NDFD estimate was not different than the in vivo estimate. However, the in vitro method correctly ranked the alfalfa-based diets as having greater NDFD estimates than the timothy-based diets. Of the 3 incubation periods, the 72-h period provided digestibility estimates most similar to the in vivo data. Using the methodologies described in this research, the Daisy(II) incubator and equine feces can be used to estimate in vivo DMD of horse feeds.

Differential regulation of estrous behavior and luteinizing hormone secretion by estradiol-17β in ovariectomized dairy cows

Holstein cows (n = 9) were used in an experiment to characterize the behavioral and endocrine responses to estradiol-17β when administered at rates designed to maintain peripheral concentrations within a physiological range. Cows were pretreated with progesterone for 3 d. Three days after progesterone treatment was completed, each cow was assigned to one of five estradiol-17β treatment groups (Doses 0 to 4), calculated to produce and maintain 0, 3, 6, 9, or 12 pg/mL in peripheral blood for 8 h. The experiment was conducted in eight replicates (with 3 to 7 cows each), with no dose repeated in any replicate. In each replicate, at least one additional cow was given an injection of estradiol-17β (500 μg im, in a corn oil vehicle) to facilitate estrus detection. Estrus was detected by visual observation for 30 min at 4 h intervals. Estrus was defined as a cow that stood to be mounted at least twice during the 50 h interval over which estrus was observed. Jugular venous blood samples were collected at 2 h intervals throughout the infusion and observation periods for quantification of luteinizing hormone (LH). Cows that received the highest dose (Dose 4, n = 7) all showed estrus, whereas those that received the two lowest doses (Dose 0, n = 5; Dose 1, n = 6) did not. Over the course of the experiment, five cows received each dose at least once. Of these, three showed estrus at Doses 2, 3, and 4, whereas the other two showed estrus only at Dose 4. Therefore, individual cows differed in the amount of estradiol-17β needed to induce estrus. There was a linear effect of dose on duration of estrus (P < 0.01). Estrus was shorter for Dose 2 (8.0 h) than for Dose 4 (18.4 h). The onset of estrus (after start of infusion) tended to be later for Dose 2 (20.0 h) than for Doses 3 and 4 (14.0 and 13.4 h, respectively; P = 0.15). Preovulatory-like surges of LH were induced in all cows at Doses 2, 3, and 4. Surges also were detected in 3 of 5 cows receiving Dose 1. The magnitude of the LH surge was less for Doses 1, 2, and 3 than for Dose 4 (P = 0.06). In contrast to the timing of estrus, the timing of the LH surge (after start of infusion) was not different among doses (P = 0.88). Thus, the hypothalamic centers responsible for regulating expression of estrus and secretion of LH responded differently to estradiol-17β.

Effect of oxytocin on expression of cytosolic phospholipase A2 mRNA and protein in ovine endometrial tissue in vivo

The induction of endometrial prostaglandin (PG) F2alpha synthesis by oxytocin is dependent upon activation of phospholipase (PL) A2 and mobilization of arachidonic acid. The objective of this study was to determine if oxytocin stimulates PGF2alpha synthesis by inducing synthesis of cytosolic PLA2 (cPLA2). In Experiment 1, 15 ovariectomized ewes were given progesterone and estradiol to simulate an estrous cycle. Ewes were then given an injection of oxytocin on Day 14 of the simulated estrous cycle. Jugular blood samples were collected and assayed for 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM). Uteri were collected at 0, 7.5, 25, 90, or 240 min postinjection (n = 3 ewes/time point). Total RNA was isolated from caruncular endometrium and subjected to dot-blot analysis. Oxytocin induced a rapid and transient increase in serum PGFM (P < 0.01). However, endometrial concentrations of cPLA2 mRNA did not change following oxytocin administration (P > 0.10). In Experiment 2, 11 ovary-intact ewes were given oxytocin (n = 5) or saline (n = 6) on Day 15 after estrus. Jugular blood samples were collected and assayed for serum concentrations of PGFM. Uteri were collected at 15 min postinjection. Homogenates were prepared from caruncular endometrium and subjected to Western blot analysis. Concentrations of PGFM were higher in oxytocin treated ewes compared to saline treated ewes at 15 min postinjection (P < 0.01). Endometrial concentrations of cPLA2 protein were greater in the cytosolic than in the microsomal fraction (P < 0.01). Oxytocin did not affect the amount of cPLA2 protein in either fraction (P > 0.10). In conclusion, oxytocin did not effect expression of either cPLA2 mRNA or protein in ovine endometrium. Oxytocin may stimulate PGF2alpha synthesis by activating cPLA2 protein that is already present in an inactive form.

Effect of Dietary Starch Source and Concentration on Equine Fecal Microbiota

Starch from corn is less susceptible to equine small intestinal digestion than starch from oats, and starch that reaches the hindgut can be utilized by the microbiota. The objective of the current study was to examine the effects of starch source on equine fecal microbiota. Thirty horses were assigned to treatments: control (hay only), HC (high corn), HO (high oats), LC (low corn), LO (low oats), and LW (low pelleted wheat middlings). Horses received an all-forage diet (2 wk; d -14 to d -1) before the treatment diets (2 wk; d 1 to 14). Starch was introduced gradually so that horses received 50% of the assigned starch amount (high = 2 g starch/kg BW; low = 1 g starch/kg BW) by d 4 and 100% by d 11. Fecal samples were obtained at the end of the forage-only period (S0; d -2), and on d 6 (S1) and d 13 (S2) of the treatment period. Cellulolytics, lactobacilli, Group D Gram-positive cocci (GPC), lactate-utilizers and amylolytics were enumerated. Enumeration data were log transformed and analyzed by repeated measures ANOVA. There were sample day × treatment interactions (P < 0.0001) for all bacteria enumerated. Enumerations from control horses did not change during the sampling period (P > 0.05). All treatments except LO resulted in increased amylolytics and decreased cellulolytics, but the changes were larger in horses fed corn and wheat middlings (P < 0.05). Feeding oats resulted in increased lactobacilli and decreased GPC (P < 0.05), while corn had the opposite effects. LW had increased lactobacilli and GPC (P < 0.05). The predominant amylolytic isolates from HC, LC and LW on S2 were identified by 16S RNA gene sequencing as Enterococcus faecalis, but other species were found in oat fed horses. These results demonstrate that starch source can have a differential effect on the equine fecal microbiota.

Measures of antioxidant status of the horse in response to selenium depletion and repletion.

Selenium plays a role in the antioxidant mechanism via the selenoenzyme glutathione peroxidase (GSH-Px). Change in Se status because of Se depletion or supplementation is associated with a change in GSH-Px activity and could potentially affect antioxidant status. This study evaluated the impact of change in Se status on measures of antioxidant status and oxidative stress in adult horses. Twenty-eight horses were blocked by age and gender and were randomly allocated to 1 of 4 dietary treatment groups: low Se (LS), adequate Se (AS), high organic Se (SP), and high inorganic Se (SS). For 196 d, LS, SP, and SS received a low-Se diet (0.06 mg Se/kg DM) to allow for depletion of Se stores, whereas AS received an adequate Se diet (0.12 mg Se/kg DM). Then, for the next 189 d, LS and AS were maintained on the same diets, whereas SP was supplemented with Se-yeast and SS with sodium selenite to allow for a total dietary Se intake of 0.3 mg Se/kg DM. Blood samples were collected throughout the study. Variables of interest included whole blood Se and GSH-Px activity, serum vitamin E concentration, total antioxidant capacity (TAC), serum malondialdehyde (MDA), and triiodothyronine and thyroxine concentrations. Data were analyzed using ANOVA with repeated measures. Whole blood Se and GSH-Px activity decreased in LS, SP, and SS during the depletion phase and increased in SP and SS with supplementation (treatment × time, P < 0.001). At the conclusion of the supplementation period, GSH-Px activity was greater in SP and SS compared with AS and LS (P < 0.05). Vitamin E status remained adequate throughout the study, and no differences existed between treatments. Serum TAC did not change in response to Se depletion or repletion. Serum MDA was greater for AS than LS during depletion (P < 0.05) but similar across treatments after supplementation. Overall, change in Se status did not have a large impact on TAC or MDA, possibly because the horses maintained an adequate vitamin E status. However, Se supplementation at 0.3 mg/kg DM increased GSH-Px activity above that of the horses fed an adequate diet based on the 2007 NRC recommendations, indicating a potential benefit to feeding greater Se diets to horses kept in low-Se areas.

Digestive capacity in weanling and mature horses.

The ability of young and mature horses to digest DM, OM, and NDF was compared using 6 weanling colts and 6 mature (13.2 ± 3.0 yr) geldings. Each colt was paired with a gelding, and the pair was adapted to a diet containing 67% alfalfa cubes and 33% concentrate for 21 d. During the adaptation period, horses were accustomed to housing and all handling procedures. The adaptation period was also used to adjust the amount of feed offered to minimize orts and to maintain similar rates of intake within a pair. After the adaptation period, a 5-d fecal collection period using fecal collection harnesses ensued. The average age of the weanling colts at the start of the 5-d collection period was 181.8 ± 2.9 d. On the morning of the first collection day, Co-EDTA (9 mg Co/kg BW(0.75)) and ytterbium-labeled hay fiber (9 mg Yb/kg BW(0.75)) were added to the concentrate portion of the diet, and horses were closely observed for complete consumption of the markers before additional feed was offered. The fecal collection bags were emptied every 1 to 2 h, and each collection was weighed and subsampled for later measurement of Co and Yb concentrations, which were used to determine the mean retention time (MRT) of the fluid and particulate phases of digesta, respectively. The remaining feces for each horse were composited each day and then subsampled for measurement of DM digestibility (DMD), NDF digestibility (NDFD), and OM digestibility (OMD). During the fecal collection period, DMI was similar between colts and geldings (91.4 and 91.2 g/kg BW(0.75), respectively). There were no differences between colts and mature geldings for DMD, OMD, or NDFD. Across both ages, the MRT of the particulate phase was 24.9 h compared with 21.8 h for the fluid phase (P = 0.002). However, MRT for the particulate phase was not different between colts and mature geldings (24.7 and 25.2 h, respectively). There was no difference in the MRT for the fluid phase between colts and mature geldings (21.5 and 22.0 h, respectively). The results indicated that the digestibility of DM, OM, and NDF in a diet consisting of good-quality cubed forage and concentrate is similar for weanling colts and mature geldings.