Effects of equine chorionic gonadotropin administered via the Baihui acupoint on follicular ovarian dynamics and the luteal function of cattle during an ovulation synchronization treatment regimen for fixed-time artificial insemination.

Objectives were to evaluate effects of a smaller than typically used dose of equine chorionic gonadotropin (eCG) during a fixed-time artificial insemination (FTAI) treatment regimen. Transrectal ultrasonic (US) examinations were conducted on dairy cows on Day 0 (D0) and the treatment regimen was initiated with administrations of an intravaginal progesterone (P4) implant, estradiol benzoate (im), and prostaglandin F2α (PGF2α; im). On D8, the P4 implant was removed and PGF2α and estradiol cypionate were administered to all animals. Subsequently, cows were randomly assigned to three groups and eCG was administered to Groups 1, 2, and 3 in doses of 300 (im); 100 (im); and 100 (Baihui acupoint) IUs, respectively. The B-mode and power-flow US cineloops were performed to assess follicular dynamics and evaluate various morphological and vascular characteristics of the corpus luteum. Blood samples were collected to quantify serum P4 concentrations. There were no differences between the ovulation synchronization treatment regimens for all follicular dynamic variables tested; however, cows in Group 3 differed from Group 2 having a larger follicle diameter (FD) on D10 (P = 0.06) and larger preovulatory FD (P = 0.09), as well as a blood perfusion area of the dominant follicle wall on D8 (P = 0.07). There were no differences in responses to the ovulation synchronization treatment regimens for the luteal variables evaluated subsequent to ovulation. In conclusion, the Baihui acupoint was effective as an alternative route for eCG dose reduction when FTAI treatment regimens were imposed without detrimentally affecting values for reproductive variables.

Rumen-protected B vitamin complex supplementation during the transition period and early lactation alters endometrium mRNA expression on day 14 of gestation in lactating dairy cows.

Greater metabolic demands in high-producing dairy cows are believed to be a cause of sub-fertility in these animals. Previously, supplementation with vitamin B complex molecules has shown benefits in improving milk production, health, and reproductive efficiency of dairy cows. The primary aim of this project was to determine the effects of rumen-protected vitamin B complex supplementation of 100 g of Transition VB (Jefo, St. Hyacinthe, QC, Canada) and 4 g of Lactation VB (VB; Jefo), during the transition and early lactation periods, respectively, compared with a control diet containing no supplementation on d 14 endometrial outcomes of pregnancy. In the vitamin B supplemented cows, we expect to see a change in the mark-up of endometrial genes important for embryo survival before implantation. Multiparous Holstein cows were enrolled into the study 3 wk before parturition and were randomly assigned to either the VB or control treatment. Twice-a-week blood samples, weekly milk samples, and daily feed intake were collected. Cows were enrolled onto a double-ovsynch protocol at 33 ± 3 d postpartum and inseminated by timed artificial insemination. Milk production and components, concentrations of BHB, haptoglobin, and progesterone in serum, and ovarian dynamics were also measured, but no treatment effect was observed. The uterus was flushed on d 14 after artificial insemination (around 72 DIM) for conceptus collection, and endometrial samples were collected at the same time. Overall, 42 cows were flushed and 13 embryos were collected. Analysis of mRNA expression of genes related to embryo development, immune system, adhesion, and regulation of vitamin B molecules showed that OXTR, MUC5B, MUC1, IL1B, SPP, TRD, FZD8, and FOLR1 genes were significantly upregulated in the VB group. Vitamin B supplementation had no effect on the size of the embryo and ovulatory follicle or corpus luteum diameter at embryo collection. In conclusion, the benefits of strategic dietary VB supplementation during the transition and early lactation might be directly linked to endometrial functions required for embryo survival during the peri-implantation period.

Mechanisms of intracellular calcium homeostasis in developing and mature bovine corpora lutea.

Although calcium (Ca(2+)) is accepted as an intracellular mediator of prostaglandin F2 alpha (PGF2alpha) actions on luteal cells, studies defining mechanisms of Ca(2+) homeostasis in bovine corpora lutea (CL) are lacking. The increase in intracellular Ca(2+) concentration ([Ca(2+)]i) induced by PGF2alpha in steroidogenic cells from mature CL is greater than in those isolated from developing CL. Our hypothesis is that differences in signal transduction associated with developing and mature CL contribute to the increased efficacy of PGF2alpha to induce a Ca(2+) signal capable of inducing regression in mature CL. To test this hypothesis, major genes participating in Ca(2+) homeostasis in the bovine CL were identified, and expression of mRNA, protein, or activity, in the case of phospholipase Cbeta (PLCbeta), in developing and mature bovine CL was compared. In addition, we examined the contribution of external and internal Ca(2+) to the PGF2alpha stimulated rise in [Ca(2+)]i in LLCs isolated from developing and mature bovine CL. Three differences were identified in mechanisms of calcium homeostasis between developing and mature CL, which could account for the lesser increase in [Ca(2+)]i in response to PGF2alpha in developing than in mature CL. First, there were lower concentrations of inositol 1,4,5-trisphosphate (IP3) after similar PGF2alpha challenge, indicating reduced phospholipase C beta (PLCbeta) activity, in developing than mature CL. Second, there was an increased expression of sorcin (SRI) in developing than in mature CL. This cytoplasmic Ca(2+) binding protein modulates the endoplasmic reticulum (ER) Ca(2+) release channel, ryanodine receptor (RyR), to be in the closed configuration. Third, there was greater expression of ATP2A2 or SERCA, which causes calcium reuptake into the ER, in developing than in mature CL. Developmental differences in expression detected in whole CL were confirmed by Western blots using protein samples from steroidogenic cells isolated from developing and mature CL. Localization of these genes in steroidogenic luteal cells was confirmed by immunohistochemistry. Therefore, it is concluded that the cellular mechanisms that allow PGF2alpha to induce a calcium signal of greater magnitude in mature than in developing CL involve 1) greater PLCbeta activity with enhanced generation of IP3, 2) an enhanced Ca(2+) release from the ER via unrestrained RYR2 due to a decrease in SRI expression, and 3) a reduction in calcium reuptake to the ER due to lower expression of ATP2A2. Accordingly, the increase in [Ca(2+)]i induced by PGF2alpha in mature large steroidogenic cells had less dependency from extracellular calcium than in those isolated from immature CL.

Effects of selenium supplementation on plasma progesterone concentrations in pregnant heifers.

It is known that selenium (Se) has various functions in animals. Many investigations on the biochemical and physiological effects of Se have been previously reported; however, the detailed function of Se in reproduction is not yet clear. We proposed the possibility that Se plays a notable role in progesterone production. The aim of this study was to clarify the effects of Se supplementation on progesterone levels of pregnant Holstein heifers. Eight Holstein heifers (-Se) were fed basal diet (containing 0.022 ppm of Se) throughout the experiment. While a 0.3 ppm diet of Se (sodium selenite) was fed to another seven animals (+Se) with basal diet. Blood sampling was carried out every week. Plasma Se concentrations were higher in Se-supplemented cows compared with controls (-Se) (P < 0.01) throughout the experiment. Se supplementation increased plasma progesterone in the 29-39 weeks of pregnancy from 4.98 ± 0.64 to 6.86 ± 0.49 ng/mL on average (P < 0.05). The present findings suggest that Se contributes to maintaining the function of the corpus luteum and/or placenta in the latter period of pregnancy.

Localization of ghrelin and its receptor in the reproductive tract of Holstein heifers.

The aim of this experiment was to localize the mRNA and protein of ghrelin and its active receptor, growth hormone secretagogue 1A (GHS-R1A), within the reproductive tract of dairy cattle. Ghrelin is an orexigenic hormone that has been identified as a potent regulator of energy homeostasis. Recent evidence suggests that ghrelin may also serve as a metabolic signal to the reproductive tract. Ghrelin and GHS-R1A have been identified in the reproductive tract of several species, including humans, mice, and rats. However, ghrelin and GHS-R1A expression have not been described within bovine reproductive tissues. Therefore, the ampulla, isthmus, uterine body, corpus luteum, and follicles were harvested from 3 Holstein heifers (15.91±0.07 mo of age) immediately following exsanguination. Duodenum and hypothalamus were collected as positive controls for ghrelin and GHS-R1A, respectively. Tissues were fixed in 10% formalin and embedded in paraffin for microscopy. Additional samples were stored at -80°C for detection of mRNA. Ghrelin and GHS-R1A mRNA and protein were observed in all tissue types within the reproductive tract of dairy heifers; however, expression appeared to be cell specific. Furthermore, ghrelin protein appeared to be localized to the cytoplasm, whereas GHS-R1A protein was found on the plasma membrane. Within the reproductive tissues, ghrelin mRNA and protein were most abundantly expressed in the ampulla of the oviduct. Concentrations of GHS-R1A were lower than those of ghrelin but differed between tissues. This is one of the first studies to provide molecular evidence for the presence of ghrelin and GHS-R1A within the entire reproductive tract. However, implications for fertility remain to be determined.

Immunization of beef heifers against gonadotropin-releasing hormone prevents luteal activity and pregnancy: Effect of conjugation to different proteins and effectiveness of adjuvants.

Three experiments were conducted to evaluate methods of immunization against GnRH on antibody titer, luteal activity, and pregnancy in beef heifers. Experiment 1 evaluated the efficacy of adjuvants with 30 heifers. Control heifers were immunized against human serum albumin (HSA) emulsified in Freund's complete adjuvant (FCA). The other 4 treatments contained GnRH conjugated to HSA (HSA-GnRH) emulsified in FCA, Freund's incomplete adjuvant (FIA), DEAE dextran (DD) + mineral oil (MO), or DD+FIA. Treatment was in the mammary gland for all experiments. Titers against GnRH for heifers immunized against HSA-GnRH with FCA, DD+MO, or DD+FIA were greater than titers for HSA-GnRH with FIA or control heifers (P < 0.01). Body weight was reduced (P < 0.05) in control and FCA heifers compared with FIA, DD+MO, and DD+FIA heifers. Heifers immunized with DD+MO and DD+FIA had fewer granulomas in mammary glands than heifers treated with FCA (P < 0.01). In Exp. 2, 36 heifers were used to determine the effect of the protein conjugated to GnRH on titers against GnRH. Heifers (6/treatment) received a primary immunization against GnRH conjugated to HSA (HSA-GnRH), ovalbumin (OA-GnRH), or keyhole limpet hemocyanin (KL-GnRH), or heifers were immunized against each carrier protein. Antigens were emulsified in DD+FIA. Immunization of heifers against OA-GnRH, KL-GnRH, or HSA-GnRH suppressed luteal activity (P < 0.01) for 23, 16, and 12 wk, respectively, and antibody titers against GnRH were greater (P < 0.01) for 19, 5, and 7 wk, respectively, compared with heifers immunized against the carrier proteins. In Exp. 3, 90 heifers were used to determine the effect of immunization against GnRH on ovarian activity and pregnancy rate. Heifers (30/treatment) received a primary and 2 or 3 booster immunizations against GnRH conjugated to OA, and controls received a primary and 2 booster immunizations against OA. All antigens were emulsified in DD+FIA. At 8 wk after primary immunization, heifers were exposed to fertile bulls for 24 wk. Pregnancy rate was less (P < 0.01) for 3-booster heifers (13%) compared with control (83%) and 2-booster (62%) heifers. We conclude that immunization against GnRH, conjugated to OA and emulsified in DD+FIA, does not influence ADG and produces sufficient titers against GnRH to prevent estrous cycles with few mammary granulomas. Immunization against GnRH with 3 booster immunizations prevented luteal activity and pregnancy in most beef heifers for more than 4 mo.

Effects of prepartum supplementation of linoleic and mid-oleic sunflower seed on cow performance, cow reproduction, and calf performance from birth through slaughter, and effects on intake and digestion in steers.

Two experiments were conducted to determine the effects of sunflower seed supplements with varying fatty acid profiles on performance, reproduction, intake, and digestion in beef cattle. In Exp. 1, 127 multiparous spring-calving beef cows with free-choice access to bermudagrass hay were individually fed 1 of 3 supplements for an average of 83 d during mid to late gestation. Supplements (DM basis) included 1) 1.23 kg/d of a soybean hull-based supplement (control treatment); 2) 0.68 kg/d of linoleic sunflower seed plus 0.23 kg/d of the control supplement (linoleic treatment); and 3) 0.64 kg/d of mid-oleic sunflower seed plus 0.23 kg/d of the control supplement (oleic treatment). During the first 62 d of supplementation, the BW change was 11, 3, and -3 kg for cows fed the control, linoleic, and oleic supplements, respectively (P < 0.001). No difference in BW change was observed during the subsequent period (-65 kg, P = 0.83) or during the entire 303-d experiment (-31 kg, P = 0.49). During the first 62 d of supplementation, cows fed sunflower supplements tended (P = 0.08) to lose more body condition than cows fed the control diet, but BCS was not different (P > 0.22) for any subsequent measurement. At the beginning of the breeding season, the percentage of cows exhibiting luteal activity was greater for cows fed the control diet (43%; P = 0.02) than for cows fed either linoleic (20%) or oleic (16%) supplementation; however, first-service conception rate (67%; P = 0.22) and pregnancy rate at weaning (92%; P = 0.18) were not different among supplements. No differences were detected in calf birth (P = 0.46) or weaning BW (P = 0.74). In Exp. 2, 8 ruminally cannulated steers were used to determine the effects of sunflower seed supplementation on forage intake and digestion. Treatments (DM basis) included 1) no supplement; 2) a soybean hull-based supplement fed at 0.29% of BW/d; 3) whole linoleic sunflower seed fed at 0.16% of BW/d; and 4) whole high-oleic sunflower seed fed at 0.16% of BW/d. Hay intake was not influenced (P = 0.25) by supplement (1.51% of BW/d); however, DMI was greatest (P < 0.01) for steers fed the soybean hull-based supplement (1.93% of BW/d). Sunflower seed supplementation reduced (P < 0.01) NDF and ADF digestibility while increasing (P < 0.01) apparent CP and apparent lipid digestibility. In conclusion, whole sunflower seed supplementation resulted in reduced cow BW gain during mid to late gestation, but this reduction did not influence subsequent cow BW change, pregnancy rate, or calf performance.

Attenuation of luteolytic response following fish meal supplementation in dairy buffaloes (Bubalus bubalis).

Luteolysis of corpus luteum, due to un-inhibited PGF(2α) secretion, has been reported to be a cause of early embryonic mortality in dairy animals. The objective of this study was to determine the effects of fish meal (FM) supplementation on the uterine secretion of PGF(2α) and hence establish its supplementation as an antiluteolytic strategy in dairy buffaloes. Five cycling Murrah buffaloes were supplemented with 250g FM daily for 55 days in addition to their routine feed and seven buffaloes were kept as non-supplemented control. After 30 days of FM supplementation, the oestrus was synchronized in all the buffaloes using Ovsynch protocol. On day 15 of synchronized cycle, animals were challenged with oxytocin (OT; 100IU) intravenously and blood samples were collected at 15min interval, 1h before to 4h after OT challenge. The PGF(2α) response was measured as the venous concentration of 13,14-dihydro-15-keto PGF(2α) (PGFM). The mean hourly concentration of PGFM in FM supplemented buffaloes was lower than in the control buffaloes at all the occasions. During peak response (1h post-OT challenge), PGFM concentration was significantly lower (P<0.05) in FM supplemented buffaloes than in the control (197.4±41.7pg/ml versus 326.3±33.5pg/ml, respectively). Also the percent rise in PGFM after OT-challenge in FM supplemented buffaloes was less than the control (11.73% versus 22.47%). The dietary supplementation did not affect the size of corpus luteum (CL) and plasma progesterone concentration. Plasma glucose and total protein concentrations remained within the normal physiological limits during FM supplementation. The present study indicated that supplementing FM decreased the concentrations of PGF(2α) without alterations in the size of CL and plasma progesterone concentrations in dairy buffaloes.

[Effects of acupuncture on the luteal function of rats with dysfunctional embryo implantation].

OBJECTIVE: To observe the influence of acupuncture on the luteal function of rats with dysfunctional embryo implantation, and investigate its mechanism. METHODS: The early pregnant female rats were randomly divided into a normal control group, a model group, an acupoint group and a non-acupoint group. The model rats with dysfunctional embryo implantation were established with Mifepristone. For acupuncture treatment, bilateral "Housanli" (ST 36) and "Sanyinjiao" (SP 6) were selected in the acupoint group, while the points around the acupoint were used in the non-acupoint group. In this experiment, the levels of luteinizing hormone (LH), estrodiol (Es) and progesterone (P) in serum were tested with radioimmunity method, the expression of vascular endothelial growth factor (VEGF) in ovary was examined with immunohistochemical assay and Western-blot, and also, the mRNA expression of VEGF and luteinizing hormone receptor (LHR) in ovary were detected with RT-PCR. RESULTS: The levels of LH and P in serum, as well as the expression of VEGF, VEGF mRNA and LHR mRNA in ovary in the acupoint group were significantly higher than those in the model group and the non-acupoint group (P < 0.05), but there was no obvious difference from that of the normal control group. CONCLUSION: Acupuncturing at "Housanli" (ST 36) and "Sanyinjiao" (SP 6) can increase the levels of LH and P in the serum, and up-regulate the expression of VEGF, VEGF mRNA and LHR mRNA in ovary, which may enhance the luteal function of rats with dysfunctional embryo implantation and improve its embryo implantation.

Characterization of cAMP/PKA/CREB signaling cascade in the bonnet monkey corpus luteum: expressions of inhibin-alpha and StAR during different functional status.

Luteinizing hormone mediates its nuclear action primarily by activating cAMP/Protein kinase A (PKA) pathway leading to phosphorylation of cAMP response element binding (CREB) family of transcription factors. Earlier studies have documented altered cAMP responsiveness of luteal cells during maturation, and in the rhesus monkey, extinction of CREB expression following luteinization and ovulation. In the course of studies aimed at characterizing LH-cAMP signaling pathway, we serendipitously discovered that CREB is after all present in the monkey corpus luteum (CL). The present experiments were carried out to examine the PKA activity, CREB expression and RT-PCR expression of inhibin-alpha (Inh-alpha) subunit and steroidogenic acute regulatory protein (StAR) in CL obtained from a variety of model systems. PKA activity in the CL was maintained throughout the luteal phase. Messenger RNA expression by RT-PCR and Northern analyses and protein levels employing antibodies specific to total- and phospho-forms demonstrated presence of CREB in the CL. Additionally, immuno-histo/cytochemical analyses, Electrophoretic mobility shift assays and chromatin immunoprecipitation assays for Inh-alpha and StAR genes further confirmed the presence of CREB in the CL. The present study, contrary to an earlier report, demonstrates the presence of CREB (both transcript and protein) in the monkey CL. Also, analysis of expression of Inh-alpha and StAR genes (considered to be cAMP responsive), during different functional status of CL suggests that LH regulates their expression perhaps by cAMP/PKA/CREB pathway.

Expression of phospholipase A2 group IVA (PLA2G4A) is upregulated by human chorionic gonadotropin in bovine granulosa cells of ovulatory follicles.

Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2-4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 microM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.

The regulated expression of the pregnancy-associated plasma protein-A in the rodent ovary: a proposed role in the development of dominant follicles and of corpora lutea.

Compelling evidence exists displaying that the intrafollicular IGF-I system constitutes an obligatory mediator of FSH action in the murine ovary. Within this system, the ovarian IGF binding protein-4-directed protease (IGFBP-4ase) may have a critical role. Human IGFBP-4ase has been proved identical to the previously well-characterized pregnancy-associated plasma protein-A (PAPP-A). This communication reports the cloning and sequencing of the mouse PAPP-A cDNA as well as its expression and cellular localization in the mouse ovary. PAPP-A mRNA was undetectable in ovaries of untreated immature 25-d-old mice. Treatment with PMSG led to a marked time-dependent increase in PAPP-A expression in well-defined subsets of granulosa cells and follicles. Specifically, PAPP-A expression was detectable exclusively in centrifugally residing membrana granulosa cells of antral follicles during a 3- to 36-h period post PMSG. PAPP-A expression then fell to nondetectable levels in dominant preovulatory follicles at 48 h post PMSG. Treatment of PMSG-primed mice with human CG caused a rapid reinduction of PAPP-A expression in granulosa cells of dominant follicles and was sustained at relatively high levels throughout the ovulation and luteinization. These results suggest a role for gonadotropin-stimulated PAPP-A gene expression in the physiologic processes of dominant follicle development, ovulation, and luteogenesis in the mammalian ovary. The early onset and extended duration of gonadotropin-dependent PAPP-A expression in granulosa cells may serve to degrade the antigonadotropin IGFBP-4. Accordingly, successful antral follicle development, ovulation, and corpus luteum formation may be contingent on an IGFBP-4-deplete/PAPP-A-replete circumstance, hence resulting in an IGF-I-replete intrafollicular microenvironment.

Physiological classification of anovulatory conditions in cattle.

Evaluation of follicular growth patterns by ultrasound combined with measurement of circulating reproductive hormones has allowed designation of three functionally critical follicular sizes during the final stages of follicular growth: emergence (-4 mm), deviation (-9 mm), and ovulation (variable from 10 to 20 mm). Classification of anovulatory conditions on the basis of these three critical points is logical and provides for rational diagnosis and treatment of the underlying physiological condition. In extreme undernutrition, there is growth of follicles to emergence but not to deviation; however, the underlying pathophysiology is not defined because of relatively few scientific investigations of this condition. Anovulatory conditions with growth of follicles to deviation but not to ovulatory size have been extensively studied. Undernutrition and/or suckling can cause this anovulatory condition. It is characterized by a greater negative feedback effect of estradiol on GnRH/LH pulses than found in normally cycling cows. Another anovulatory condition that is common in high producing lactaing dairy cows is characterized by growth of follicles to larger than ovulatory size, such as is observed in cows with follicular cysts. This condition is characterized by an insensitivity of the hypothalamus to the positive feedback effects of estradiol. Thus, these last two common anovulatory conditions appear to be primarily due to changes in the responsiveness of the hypothalamus to estradiol. Treatments that increase circulating progesterone concentrations can help in the treatment of these two conditions by potentially altering GnRH/LH pulses and allowing the final stages of follicular growth or resetting the hypothalamic responsiveness to the positive feedback effects of estradiol.

Luteal and clinical response following administration of dinoprost tromethamine or cloprostenol at standard intramuscular sites or at the lumbosacral acupuncture point in mares.

OBJECTIVE: To determine whether administration of a microdose of prostaglandin at the BAI HUI acupuncture point offers any advantage over IM injections for luteolysis, ovulatory interval, or systemic response in mares. ANIMALS: 17 mature cycling mares, 3 to 20 years of age and weighing 400 to 500 kg. PROCEDURE: Conventional and microdoses of the prostaglandin dinoprost tromethamine (PGF2alpha), the analogue cloprostenol, or sterile water (control) were administered to mares in 7 treatment groups. Treatments were assigned by dose, administration site (semimembranosus, semitendinosus, or lumbosacral region), and treatment type (PGF2alpha, analogue, or sterile water). Mares were observed for ovulatory interval and systemic response to treatment, including heart, and respiratory rates, rectal temperature, and sweat score. Plasma progesterone concentrations were also determined at the time of treatment and at 24-hour intervals for 96 hours following treatment. RESULTS: Ovulatory interval was shortened and progesterone concentrations decreased in prostaglandin-treated mares, compared with control mares, regardless of dose or treatment site. However, no differences in ovulatory interval were observed among prostaglandin-treated mares. Mares treated with conventional doses of PGF2alpha had greater systemic responses than mares treated with microdoses of PGF2alpha or sterile water. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of prostaglandins at the BAI HUI acupuncture point does not appear to offer any advantage over administration at standard IM injection sites for induction of luteolysis or to shorten the ovulatory interval. However, administration of a microdose of the analogue cloprostenol was effective at inducing luteolysis and shortening ovulatory interval regardless of administration site.

Opposite effect of prolactin and prostaglandin F(2 alpha) on the expression of luteal genes as revealed by rat cDNA expression array.

It is well established that prolactin (PRL) sustains, while prostaglandin F(2 alpha) (PGF(2 alpha)) curtails, progesterone production by the rat corpus luteum (CL). We have previously shown that the actions of both molecules converge on the 20 alpha-HSD gene and control its expression in a dramatically opposed manner. In this investigation, we have found twelve more genes that are inversely regulated by PRL and PGF(2 alpha). In addition to 20 alpha-HSD, PGF(2 alpha) stimulated and PRL inhibited PGF(2 alpha)-receptor, phospholipase C delta(1) and TGF beta(1) expression. In contrast PRL stimulated and PGF(2 alpha) inhibited the LH receptor, 11 beta-HSD2, sterol carrier protein 2, mitochondrial glutathione S-transferase (GST), GST mu(2), inhibitory DNA-binding proteins 1, 2, and 3, and calcium binding protein 2. We have also identified new target genes for PRL and PGF(2 alpha). PGF(2 alpha) stimulated the expression of genes involved in cell signaling such as cell adhesion kinase-beta, ERK3, FRA2, IL-2 receptor, and 14-3-3 proteins. PGF(2 alpha) also up-regulated the expression of the sodium channel beta(1), Na/K ATPase, annexin IV, GST7pi, and P450 reductase. In contrast PGF(2 alpha) inhibited the expression of two genes involved in cell cycle: cyclin D2 and retinoblastoma related protein (Rb2/p130). It also inhibited genes involved in estradiol (P-450(AROM)) and cholesterol biosynthesis (HMG-CoA synthase), as well as genes involved in tissue remodeling: VEGF and TIMP3. PRL had a profound inhibitory effect on the expression of genes encoding the ADP-ribosylation factor 3, annexin V and c-jun, yet increased the expression of P450scc, 3beta-HSD, and SR-B1 (HDL-receptor), all genes involved in steroidogenesis. PRL also stimulated the expression of beta(2)-microglobulin, TIMP2, cytochrome c oxidase IV, cathepsin H and L, and copper-zinc superoxide dismutase as well as elongation factor SIII, heat shock protein-60 and mitochondrial ATP synthase-D. In conclusion, this investigation has revealed a "yin-yang" relationship between PRL and PGF(2 alpha) in regulating certain critical genes in the rodent CL, and has demonstrated novel regulation by these factors of other important genes involved in luteal function.

LH pulses and the corpus luteum: the luteal phase deficiency LPD).

The proper function of the GnRH pulse generator in the hypothalamus is essential for normal ovarian function, hence also for proper function of the corpus luteum. During the luteal phase LH pulses stimulate progesterone release, which is essential for normal endometrial transformation. Approximately one-half of all luteal phase deficiencies (LPD) are due to improper function of the GnRH pulse generator. Obviously, following ovulation the increased serum progesterone levels oversuppress the GnRH pulse generator, resulting in too few LH pulses and therefore improper luteal function. Also, latent hyperprolactinemia may lead to an LPD which can be effectively treated with plant extracts containing dopaminergic (prolactin-suppressing) compounds. Our increasing knowledge of auto- and paracrine mechanisms between nonsteroidogenic and steroidogenic cells now allow subclassification of LPDs of ovarian origin. The so-called small luteal cells are LH-responsive. If they develop improperly the regularly occurring LH pulses are unable to stimulate progesterone secretion from the small luteal cells, which results in what we call the small luteal cell defect. In addition, there is also evidence that the large luteal cells may function improperly. Hence, basal progesterone release is too low while LH-stimulated progesterone release from the small luteal cells appears to be intact. This subclassification of luteal phase deficiency results in the suggestion of different treatments. In cases where the corpus luteum is LH-responsive, such as the hypothalamic corpus luteum insufficiency and the large luteal cell defect, HCG treatment or pulsatile treatment with GnRH is advisable. In the case of LH/hCG-unresponsive small luteal cell defect a progesterone substitution is suggested.

Effect of diet quantity and urea supplementation on oocyte and embryo quality in sheep.

The objective was to investigate the effects of dietary energy and urea supplementation on oocyte and embryo quality in sheep using in vivo and in vitro experimental models. Sixty-three ewes were fed grass meal at 0.5 or 2.0 times maintenance energy requirements (MER). The diet was supplemented with feed grade urea (U) for half of the ewes on each energy treatment. Ewes were stimulated with 1000 IU eCG and either slaughtered on the day of pessary withdrawal, for in vitro embryo production, or mated and slaughtered on Day 5 for embryo recovery. Urea decreased cleavage rate (48.3 vs 39.7%) and consequently blastocyst rate (41.6 vs 36.8%) but the differences were not significant. Oocytes from animals on 2.0 MER had a lower cleavage rate (54.9 vs 36.0%) and blastocyst yield (49.3 vs 31.4%) than those on 0.5 MER. However, there was an interaction between urea and energy for cleavage (P = 0.04) and blastocyst yield (P = 0.03) indicating a variable response to urea in the presence of high energy. This was manifested by a decrease in cleavage rate in the presence of urea and high energy (22%, 8 of 36), and a reduction in blastocyst development (19%, 7 of 36). When blastocyst development rate was expressed as a proportion of cleaved oocytes there was no difference between groups; in addition, there was no difference between groups in terms of blastocyst hatching rate (overall mean 66.1%) or blastocyst cell number on Day 8 (overall mean +/- SEM, 138.4 +/- 9.0, n=61). The effect of urea on cleavage rate in vivo was more severe. Urea supplementation reduced (P<0.001) the cleavage rate (93 vs 62%). Despite this, the yield of blastocysts was unaffected. Oocytes from ewes on 0.5 MER exhibited a lower (P<0.05) cleavage rate than those on 2.0 MER (66 vs 87%). This effect was also apparent at the blastocyst stage (40.0 vs 50.9%), although the difference was no longer significant. There were no differences in hatching rate (overall mean 70.7%) or blastocyst cell numbers (overall mean +/- SEM, 166.3 +/- 15.6, n=40). Collectively, these results suggest that both high dietary energy and urea content influence subsequent embryo development in vitro, and the deleterious effects of urea are likely influenced by concomitant energy intake.

Control of ovarian follicular growth and maturation by the corpus luteum and the placenta during pregnancy in sheep.

Ovarian follicular growth and maturation and its control throughout pregnancy have not been described fully in sheep. Experiment 1 characterized the size and maturation (steroid production in vitro and aromatase activity) of ovarian follicles obtained at days 20, 50, 80 and 110 of pregnancy compared with those obtained at day 12 of the oestrous cycle. There was no difference in the number of small follicles (< 3 mm in diameter) between cyclic and pregnant ewes, regardless of the stage of pregnancy. There was a marked reduction (P < 0.01) in the number of medium follicles (3-5 mm) starting at day 80 of pregnancy. Large follicles (> 5 mm) were not detected at day 110 of pregnancy. In vitro testosterone output by follicles was constant throughout pregnancy. Oestradiol output remained steady until day 80, but decreased markedly at day 110 of pregnancy. This decrease was associated with a reduction in aromatase activity in follicles obtained at this stage. Experiment 2 examined the effect of administration of high concentrations of progesterone between day 100 and day 120 after mating on resumption of follicular growth in ewes that underwent Caesarean section at day 99 of pregnancy. In ewes that underwent Caesarean section, progesterone supplementation was successful in mimicking the profile found in pregnant ewes, but did not prevent re-initiation of follicular growth, as demonstrated by the presence of large follicles (> 5 mm) at day 120 after mating. Experiment 3 examined the effects of PGF(2alpha)-induced regression of the corpus luteum of day 100 of pregnancy on resumption of follicular growth. High concentrations of PGF(2alpha) (0.28 mg kg(-1) body weight) administrated at day 100 of pregnancy were required to initiate regression of the corpus luteum. At day 120 after mating, the mean (+/- SEM) diameter of the largest follicle in PGF(2alpha)-treated ewes (3.40 +/- 0.47 mm) was significantly greater (P < 0.05) than that in control pregnant ewes (2.52 +/- 0.34 mm). Experiment 4 examined the effect of removal of the fetus and of the corpus luteum at day 100 of pregnancy on resumption of ovulation. Removal of the corpus luteum by PGF(2alpha) treatment at the time of removal of the fetus resulted in earlier occurrence of short luteal phases (27.8 versus 40.6 days, PGF(2alpha)-treated versus non-treated) but did not alter the timing of the first normal luteal phases (41 days). In conclusion, the results from these experiments indicate that placental compounds play a major role in inhibiting follicular growth and maturation during late pregnancy in sheep.

Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis.

Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2-4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon gamma (IFN) and tumor necrosis factor alpha (TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2. 0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.