Anti-inflammatory and Intestinal Barrier-protective Activities of Commensal Lactobacilli and Bifidobacteria in Thoroughbreds: Role of Probiotics in Diarrhea Prevention in Neonatal Thoroughbreds.

We previously isolated the commensal bacteria lactobacilli and bifidobacteria from the Thoroughbred intestine and prepared the horse probiotics LacFi(TM), consisting of Lactobacillus ruminis KK14, L. equi KK 15, L. reuteri KK18, L. johnsonii KK21, and Bifidobacterium boum HU. Here, we found that the five LacFi(TM) constituent strains remarkably suppressed pro-inflammatory interleukin-17 production in mouse splenocytes stimulated with interleukin-6 and transforming growth factor-ß. The protective effects of the probiotic on impaired intestinal barrier function were evaluated in Caco-2 cells treated with tumor necrosis factor-α. Evaluation of transepithelial resistance showed that all the strains exhibited intestinal barrier protective activity, with significant suppression of barrier impairment by L. reuteri KK18. The LacFi(TM) constituent strains were detected in neonatal LacFi(TM)-administered Thoroughbred feces using polymerase chain reaction denaturing gradient gel electrophoresis and culture methods. These five strains were found to be the predominant lactobacilli and bifidobacteria in the intestinal microbiota of LacFi(TM)-administered Thoroughbreds. Administration of LacFi(TM) to neonatal Thoroughbreds decreased diarrhea incidence from 75.9% in the control group (n=29 neonatal Thoroughbreds) to 30.7% in the LacFi(TM)-administered group (n=101 neonatal Thoroughbreds) immediately after birth to 20 weeks after birth. LacFi(TM) treatment also prevented diarrhea especially at and around 4 weeks and from 10 to 16 weeks. The duration of diarrhea was also shorter in the probiotics-administered group (7.4 ± 0.8 days) than in the control group (14.0 ± 3.2 days). These results indicate that the LacFi(TM) probiotics regulates intestinal function and contributes to diarrhea prevention.

Papillary renal adenoma of distal nephron differentiation in a horse.

A 20-year-old thoroughbred mare had a mass in the right kidney. The mass was encapsulated with fibrous capsule and composed of variably-sized papillary projections lined by a single layer of flattened to cuboidal neoplastic epithelial cells with no cytological and nuclear atypia. Immunohistochemically, the neoplastic cells were broadly positive for cytokeratin AE1/AE3 and granular staining for alpha-1-antitrypsin was focally detected; this immunohistochemical property was similar to that of the normal distal nephron. From these results, this case was diagnosed as papillary renal adenoma of distal nephron differentiation.

Expression of inhibins, activins, insulin-like growth factor-I and steroidogenic enzymes in the equine placenta.

In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the placenta using either immunohistochemistry or in situ hybridization. Inhibin/activin betaA-subunit and its mRNA were confined to maternal endometrial glands, whereas immunopositive betaB-subunit was not detected in either endometrial glands or microcotyledons. Cytochrome P450 side chain cleavage enzyme was detected by immunohistochemistry in both endometrial glands and microcotyledons, whereas cytochrome P450 17alpha-hydroxylase/lyase was absent in these tissues. Immunopositive signals for 3beta-hydroxysteroid dehydrogenase and cytochrome P450 aromatase were localized in microcotyledons but not in endometrial glands. Immunohistochemistry revealed that IGF-I was highly expressed in microcotyledons around Day 130, and decreased as pregnancy progressed. Changes in the expression of IGF-I were correlated with the number of PCNA positive cells in the placenta. The present study demonstrated the presence and localized the site of expression of activin, IGF-I and steroidogenic enzymes in equine placental tissues during the latter two-thirds of gestation; the results suggest that activin and IGF-I may be involved in the regulation of placental development.

Plasma concentrations of ir-inhibin, inhibin A, inhibin pro-alphaC, FSH, and estradiol-17beta during estrous cycle in mares and their relationship with follicular growth.

The relationship among plasma levels of immunoreactive (ir)-inhibin, inhibin A, inhibin pro-alphaC, FSH, estradiol-17beta and follicular growth were investigated during the normal estrous cycle in mares. Seven mares were used for two successive normal estrous cycles. Follicular diameters and ovulation were obtained by transrectal ultrasonography, and blood samples were collected daily by jugular venipuncture for hormonal assay. The results showed that inhibin A was inversely correlated (r = -0.59, p < 0.0001) with FSH indicating its contribution to negative feedback control of FSH secretions from the pituitary gland. Estradiol-17beta increased during the follicular phase reaching a peak (37.9 +/- 3.8 pg/mL) 2 d before ovulation. Estradiol-17beta was positively correlated (r = 0.78, p < 0.0001) with inhibin A. The high levels of inhibin A and estradiol-17beta were associated with the growth of the preovulatory dominant follicle and inversely correlated with FSH suggesting that both hormones are products of the large dominant follicles and were responsible for the decline in FSH secretion during the follicular phase of estrous cycle. In conclusion, an inverse relationship between inhibin A and FSH was clearly demonstrated indicating that inhibin A has a key role in the negative feedback control of FSH from the pituitary gland. In addition, inhibin A and estra-diol-17beta secretions were associated with the growth of the preovulatory dominant follicle and were positively correlated.

Localization and secretion of inhibins in the equine fetal ovaries.

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.

The testis as a major source of circulating inhibins in the male equine fetus during the second half of gestation.

Immunolocalization of the inhibin (a) and inhibin/activin (beta3A and betaB) subunit proteins in equine fetal testes was investigated to determine the ability of the fetal testis to produce inhibins at 120, 150, 200, and 250 days of gestation. In addition, concentrations of immunoreactive (ir)-inhibin, inhibin pro-aC, and inhibin A in both the maternal and fetal circulation were measured. It was found that plasma concentrations of ir-inhibin, inhibin pro-alphaC, and inhibin A were much higher (P < .05) in the fetal than in the maternal circulation at any stage of gestation examined. Similarly, while fetal testicular homogenate contained increased amounts of inhibins, the inhibins were undetectable in homogenates of maternal ovaries and placentae. At 120 days of gestation, all 3 subunit proteins were localized to the interstitial cells, while the immunoreactivity for the inhibin/activin 3B subunit protein was also observed in Sertoli cells. The intensity of immunoreactivity for the 3 subunit proteins in interstitial cells increased as pregnancy advanced to day 200, and, at this stage, immunoreactivity for the inhibin alpha subunit protein was observed in the fetal testes in a pattern consistent with localization in Sertoli cells. Thus, the inhibin/activin betaA subunit protein was confined to interstitial cells during the gestational periods examined. We conclude that equine fetal testes secrete large amounts of inhibins, including dimeric inhibin A and possibly other dimeric forms, such as inhibin B and activins, into the fetal circulation. These results suggest that these proteins may play some important roles in the development of fetal testes during gestation.