BackgroundEndometritis is a major cause of infertility in the mare. Therefore, the diagnosis of this disease is very important in veterinary practice. The objective of this study was to compare bacteriological and cytological results obtained from the mare uterus using biopsy (EB) and cytobrush (CB) techniques and relating these findings to the presence of polymorphonuclear cells (PMNs) in endometrial tissue as the gold standard for detection of endometritis. In particular, we tested the hypothesis that endometrial cytology and microbiology data obtained from material collected using the EB and CB techniques are similar, so that the CB technique could preferentially be used to detect subclinical endometritis in clinical practice.MethodsA total of 69 mares suspected of subclinical endometritis because of previous reproductive history and 15 maiden mares were enrolled in this study. Material collected from both EB and CB was smeared on sterile glass slides for cytological examinations and on culture media for microbiological examinations. Bacteriological cultures and cytological samples were classified as negative (no growth or mixed cultures of more than three microorganisms; <2% PMNs) or positive (pure growth of microorganisms; >2% PMNs) for endometritis.ResultsPositive growth was observed in 43% of CB samples and in 54% of EB samples (difference not significant). The growth of β-hemolytic streptococci was always connected with positive cytology. This relationship was not observed for growth of E. coli or for non-pathogenic flora. The sensitivity of bacterial growth and cytology from EB was 0.63 and 0.73 respectively. The sensitivities of bacterial growth and cytology from CB were 0.50 and 0.71 respectively.ConclusionMicrobiological and cytological results obtained from CB are similar to those obtained from EB and based on these findings the CB technique may be recommended for collection of materials from the mare’s uterus in clinical practice.

Programmed necrosis (necroptosis) is an alternative form of programmed cell death that is regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent, but is a caspase (CASP)-independent pathway. In the present study, to determine if necroptosis participates in bovine structural luteolysis, we investigated RIPK1 and RIPK3 expression throughout the estrous cycle, during prostaglandin F2α (PGF)-induced luteolysis in the bovine corpus luteum (CL), and in cultured luteal steroidogenic cells (LSCs) after treatment with selected luteolytic factors. In addition, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 μM) on cell viability, progesterone secretion, apoptosis related factors and RIPKs expression, were evaluated. Expression of RIPK1 and RIPK3 increased in the CL tissue during both spontaneous and PGF-induced luteolysis (P < 0.05). In cultured LSCs, tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM) up-regulated RIPK1 mRNA and protein expression (P < 0.05). TNF + IFNG also up-regulated RIPK3 mRNA expression (P < 0.05), but not RIPK3 protein. Although Nec-1 prevented TNF + IFNG-induced cell death (P < 0.05), it did not affect CASP3 and CASP8 expression. Nec-1 decreased both RIPK1 and RIPK3 protein expression (P < 0.05). These findings suggest that RIPKs-dependent necroptosis is a potent mechanism responsible for bovine structural luteolysis induced by pro-inflammatory cytokines.

The mechanisms governing corpus luteum (CL) function in domestic dogs remain not fully elucidated. The upregulated expression of cyclooxygenase 2 and prostaglandin (PG) E2 synthase (PGES) at the beginning of the canine luteal phase indicated their luteotrophic roles, and the steroidogenic activity of PGE2 in the early canine CL has been confirmed in vitro. Recently, by applying a cyclooxygenase 2 (COX2)-specific inhibitor (firocoxib [Previcox]; Merial) from the day of ovulation until the midluteal phase, the luteotrophic effects of PGs have been shown in vivo. This is a follow-up study investigating the underlying endocrine mechanisms associated with the firocoxib-mediated effects on the canine CL. Experimental groups were formed with ovariohysterectomies performed on Days 5, 10, 20, or 30 of firocoxib treatments (10 mg/kg bw/24h; TGs = treated groups). Untreated dogs served as controls. A decrease of steroidogenic acute regulatory (STAR) protein expression was observed in TGs. The expression of PGE2 synthase was significantly suppressed in TGs 5 and 10, and both PGE2 and PGF2 levels were decreased in luteal homogenates, particularly from CL in TG 5. Similarly, expression of the prolactin receptor (PRLR) was diminished in TGs 5 and 20. The expression of PGE2 receptors PTGER2 (EP2) and PTGER4 (EP4), the PG-transporter (PGT) , and 15-hydroxy PG dehydrogenase (HPGD) was not affected in TGs. Our results substantiate a direct luteotrophic role of PGs in the early canine CL, i.e., by upregulating the steroidogenic machinery. Additionally, the possibility of an indirect effect on PRL function arises from the increased prolactin receptor expression in response to PGE2 treatment in canine lutein cells observed in vitro.

Abstract.We studied the secretory function of the corpus luteum (CL) in cows following different estrus synchronization protocols. Estrus was synchronized using one (n=4) or two injections (n=5) of prostaglandin F2α (PGF2α; dinoprost), two injections of different analogues of PGF2α (aPGF2α), luprostiol (n=5) and cloprostenol (n=5), at eleven-day intervals, a gestagen implant (norgestomet, n=5, for 10 days) or norgestomet together with a subsequent dinoprost injection on the day of implant removal (n=5). CL samples were collected by ovariectomy on Day 7-8 of the estrous cycle. Luteal strips were stimulated with LH (100 ng/ml) or prostaglandin E2 (PGE2, 10 -6 M) for 24 h in culture media. The progesterone (P4) and PGE2 concentrations in the media were measured by enzyme immunoassay. In the control CL (spontaneous estrus; n=5), LH and PGE2 stimulated P4 and PGE2 (P<0.001). The effects of both factors on P4 were reduced in the CL following dinoprost-and cloprostenol-synchronized estrus (P<0.05) and were absent in the luprostiol-synchronized CL (P>0.05). In the norgestomet-synchronized CL, the stimulatory effects of LH and PGE2 were higher compared with the CL synchronized by aPGF2α (P<0.05). Pharmacological manipulation of the estrous cycle using aPGF2α may cause lower P4 secretion. Estrus synchronization inhibited CL sensitivity to luteotropic factors. Therefore, attention should be focused on the estrous synchronization method in both in vivo and in vitro studies of CL functions in cattle. Key words: Cattle, Corpus luteum (CL), Estrus synchronization, Progesterone, Prostaglandin E2 (PGE2), Prostaglandin F2α (PGF2α) analogue (J. Reprod. Dev. 55: [170][171][172][173][174][175][176] 2009) he physiological processes of corpus luteum (CL) formation, growth and maintenance might be regulated by many different factors including luteinizing hormone (LH) and prostaglandin (PG)s [1,2]. After ovulation, as the CL forms from the wall of the ruptured follicle, it grows and rapidly vascularizes [3]. The CL is a complex tissue composed of parenchymal (steroidogenic) and nonparenchymal (fibroblast, vascular smooth muscle, pericyte and endothelial) cells [3,4]. Disturbances in physiological conditions and pharmacological manipulations during follicle growth and maturation, ovulation and as well during early CL formation influence the future secretory function of this new formed endocrine gland.The main function of the CL is secretion of progesterone (P4), an important hormone for the establishment and maintenance of a successful pregnancy [1,2,5]. In domestic animals, LH, which is released in a pulsatile fashion from the anterior pituitary, is one of the most potent regulators of P4 production [6]. LH stimulates basal P4 production in small luteal cells via the LH receptor. In addition to pituitary LH, intraluteal substances produced by the component cells of the CL play important roles in regulating P4 production during the luteal stages and pregnancy [1,2,7]. Arachidonic acid metabolites, such as PGE2 and PGI2, have been prop...

BackgroundUntil recently, the corpus luteum (CL) was considered to be the main source of progesterone (P4) during pregnancy in the domestic cat (Felis catus). However, other possible sources of P4 have not been ruled out. Although feline placental homogenates were found to be capable of synthesizing P4, expression of the respective steroidogenic enzymes has not been investigated at the molecular level. Therefore, in the present study, expression of the two major factors involved in the synthesis of P4 - 3beta-hydroxysteroid dehydrogenase (3betaHSD) and steroidogenic acute regulatory protein (StAR) - was investigated in the feline CL and placenta during the course of pseudopregnancy and pregnancy.MethodsThe mRNA levels of StAR and 3betaHSD were determined using Real Time PCR and their localizations were determined by immunohistochemistry. Placental P4 concentrations, after ethyl extraction, were measured by EIA.ResultsLuteal 3betaHSD and StAR mRNA levels were strongly time-dependent, peaking during mid-pregnancy. The placental 3betaHSD mRNA level was significantly upregulated towards the end of pregnancy. In the CL, 3betaHSD and StAR protein were localized in the luteal cells whereas in the placenta they were localized to the maternal decidual cells. Placental P4 concentrations were low in early pregnant queens, but increased along with gestational age.ConclusionsThese results confirm that the placenta is an additional source of P4 in pregnant queens and can thereby be considered as an important endocrine organ supporting feline pregnancy.

Abstract. The present study compared the changes in isoflavones (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cyclic and pregnant heifers after feeding with soy bean. Twelve healthy heifers were divided into three groups: cyclic heifers (days 8-12 of the estrous cycle; control group; n= 4), an early pregnancy group (2 months pregnant; n=4) and a late pregnancy group (8 months pregnant; n=4). All heifers were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on an HPLC system. In the blood plasma of the early-and late-pregnant heifers, we found lower concentrations and time-dependent decreases in daidzein and genistein in comparison to cyclic heifers (P<0.05). Moreover, we noticed significant increases of equol and para-ethyl-phenol in the blood plasma of the earlypregnant heifers (P<0.05). In contrast, in the blood plasma of the late-pregnant heifers, we did not find an increase in the isoflavone metabolite concentrations compared with the early-pregnant heifers (P>0.05). In conclusion, physiological status (cyclicity or pregnancy) of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the heifers. The stage of pregnancy affects isoflavone absorption, biotransformation and metabolism differently and results in higher concentrations of active metabolites of isoflavones during early pregnancy in comparison to their lower concentrations during late pregnancy. Therefore, we surmise that cows are more sensitive to active isoflavone metabolite actions during early pregnancy than cyclic heifers and heifers in late pregnancy. Key words: Cow, Estrous cycle, Isoflavone, Phytoestrogen, Pregnancy (J. Reprod. Dev. 54: [358][359][360][361][362][363] 2008) ost of the fodder commonly used for feeding ruminants has been reported to contain phytoestrogens. In particular, alfalfa, red clover and soy bean contain 5 to even 25% phytoestrogens, such as coumestrol, genistein, daidzein, formonentin and biochanin A [1][2][3]. Red clover silage containing isoflavones [1], alfalfa containing coumestrans [4] as well as soy bean containing up to 25% of daidzein and genistein have been reported to cause infertility in cattle. The role of phytoestrogens in causing infertility is mainly recognized by accompanying signs of estrogenism including mammary development, swelling of the vulva and enlargement of the uterus [4]. Many cows suffer behavioral abnormalities such as irregular estrus, nymphomania and even anestrus [5]. Cows fail to conceive from servicing. These data are in agreement with our previous studies showing that long term feeding of a soy diet significantly increases the mean insemination rate and causes infertility in cattle [6], modulates prostaglandin secretion and action [7,8] and inhibits LH-stimulated progesterone (P4) secret...

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