Manas Kumar Patra

Neutrophil Functions and Cytokines Expression Profile in Buffaloes with Impending Postpartum Reproductive Disorders

The study was conducted to correlate the periparturient immune status in terms of neutrophil functions and cytokine expression in peripheral blood mononuclear cell culture with impending postpartum reproductive disorders in buffaloes. Forty pregnant buffaloes were observed for occurrence of postpartum reproductive disorders (PRD), i.e., metritis, endometritis and delayed uterine involution etc., during one week prepartum to four weeks postpartum period. A representative number (n = 6) of buffaloes that did not develop any PRD were included in group I (healthy, control), while the animals which experienced PRD were assigned into group II (PRD, n = 8). The blood samples were collected at weekly interval from one week prepartum to four weeks postpartum period considering the day of calving as ‘d 0’. Differential leucocytes counts, superoxide and hydrogen peroxide production activity in isolated neutrophils and the mRNA expression profile of cytokines i.e., IL-2, IL-4 and IFN-γ in PBMC culture were studied in all the samples. A higher total leucocytes, neutrophil and band cells count along with impaired neutrophil functions i.e., lowered level of production of superoxide and hydrogen peroxide before parturition and during early postpartum period were observed in buffaloes developing PRD. Further, a lower expression of IL-2, IFN-γ and IL-4 mRNA in PBMC culture was observed at calving in buffaloes that subsequently developed PRD at later postpartum. Thus, suppression in neutrophil function and cytokine expression at prepartum to early postpartum period predisposes the buffaloes to develop postpartum reproductive disorders. Hence, monitoring of neutrophils function and cytokine expression profile would be effective to predict certain reproductive disorders at late pregnancy or immediately after parturition in buffaloes. In future, this may be a novel approach for determining suitable management and therapeutic decisions for prevention of commonly occurring reproductive disorders in farm animals.

Upregulation of TLR-4 and proinflammatory cytokine transcripts as diagnostic indicator of endometritis in buffaloes

The aim of present study was to examine the relative mRNA transcription of toll-like receptor 4 (TLR-4), CD14, interleukin-1β (IL-1β), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) in endometritic buffaloes. Reproductive tracts were collected from 60 buffaloes at postmortem. The genitalia were diagnosed for endometritis based on positive white side test of uterine fluid, presence of >5% polymorphonuclear cells in uterine cytology, and endometrial histopathology. The endometritis-positive genitalia were categorized as luteal (n = 10) and follicular (n = 10) based on the presence of corpus luteum. Further, 20 normal genitalia (luteal, n = 10 and follicular, n = 10) were also included as control. The endometrial tissue scrapings were collected ex vivo from all the samples, total RNA was extracted, complementary DNA was transcribed for each sample, and relative quantification of mRNA was done by real-time polymerase chain reaction. The threshold cycle values of all the samples were normalized with the housekeeping β-actin gene and relative fold change was calculated using 2−ΔΔCt method. Present study revealed a variable upregulation of TLR-4, CD14, IL-1β, IL-8, and TNF-α transcription in endometritic buffaloes either at luteal or follicular stage. Predominantly at luteal stage, the significant upregulation of TLR-4, IL-8, and TNF-α transcripts was 4.66-, 21.19-, and 42.49-fold, respectively, indicated the possibility of cytokine-based diagnostic method for the early indication of endometritis before the onset of cyclic estrus. Nevertheless, further research in respect to threshold level of each cytokine in uterine biopsies involving more number of animals is required to establish a potential marker gene(s) for the diagnosis of endometritis and for devising novel therapeutic intervention.

Effect of leukotriene B4 and oyster glycogen in resolving subclinical endometritis in repeat breeding crossbred cows

In the present study an innovative attempt was made using Leukotriene B4 (LTB4) and Oyster glycogen (OG), non-specific immunomodulators, for the treatment of subclinical endometritis (SCE) in repeat breeding crossbred cows. Thirty six subclinical endometritis repeat breeding cows were randomly divided into 3 equal groups (G-I, G-II and G-III). The cows in treatment groups G-I and G-II received single intrauterine (iu) infusion with 50 ml of 30 nmol/L LTB4 and 10 mg/ml OG, respectively, whereas the cows in G-III served as control. Blood samples collected just before treatment (0 hr), 24 hr post-treatment and at subsequent estrus were assayed for total leukocyte count (TLC), lipid peroxide (LPO) and nitric oxide (NO). Following treatment the recovery rate was assessed at subsequent estrus by determining the pH of cervico-vaginal mucus (CVM), white side test and percentage of polymorphonuclear (PMN) cells in uterine cytology. The recovery rate in G-I (83.3%) and G-II (75.0%) was greater (P

Endometrial transcripts of proinflammatory cytokine and enzymes in prostaglandin synthesis are upregulated in the bitches with atrophic pyometra

Inflammatory markers of endometrial origin are valuable in order to differentiate the pyometra from cystic endometrial hyperplasia in the bitch. In the present study, we hypothesized that histological categorization would distinguish the differential regulation of the proinflammatory genes in the endometrium of bitches with pyometra. Ovariohysterectomy was done on bitches with confirmatory diagnosis of pyometra (nu2009=u200918). Using endometrium to myometrium ratio of 0.79 as threshold, the uteri (nu2009=u20098/group) were categorized into hyperplastic pyometra (HP) and atrophic pyometra (AP). Two samples were excluded as the diagnosis was inconclusive. In parallel, endometrial tissue was collected for total RNA extraction to study the differential expression of TLR4, IL-6, IL-8, COX-2 and PGFS through real time PCR. Diestrus uterus of non-pyometra bitches (nu2009=u20096) served as control. The mean fold change (2-ΔΔCt) for the target genes was determined using β-actin as endogenous control and non-pyometra uterus as calibrator group. Except TLR4, other inflammatory genes were upregulated significantly by 1.82 to 3.74 times in the AP as compared to HP with maximum upregulation of COX-2 and PGFS. Further, correlation matrix with Spearmans rho revealed that IL-8 had strong positive correlation with COX-2 and PGFS in the AP group (Pu2009<u20090.05). It is concluded that histological grading of pyometra into HP and AP revealed differential regulation of inflammatory cytokines and enzymes in the PG synthetic pathway in the canine endometrium that has diagnostic potential under clinical settings.

Endometritis Increases Pro-inflammatory Cytokines in Follicular Fluid and Cervico-vaginal Mucus in the Buffalo Cow

ABSTRACT Emerging evidence shows that some of the pro-inflammatory cytokines are elevated not only in the endometrium but also in the follicular fluid of cows with endometritis. Developing a cervico-vaginal mucus (CVM) based test has the potential for becoming a pen-side test because of the ease of sample collection. The present study describes the results of two different experiments. The first experiment was conducted to investigate the influence of endometritis on the proinflammatory cytokines of follicular fluid based on the reproductive tracts of buffalo collected at a slaughter house Buffalo genitalia were categorized into purulent endometritis (PE), cytological endometritis (CE), and non-endometritis (NE) based on the white-side test and endometrial cytology, respectively (nu2009=u200914/group). Each group was subdivided into follicular and mid-luteal stage (nu2009=u20097/stage) and the follicular fluid was collected from the largest follicle. Second experiment was done to study the difference in the levels of proinflammatory cytokines in the CVM of repeat breeders with subclinical endometritis presented to the clinic. CVM was collected from the repeaters (nu2009=u200910) and non-repeaters (nu2009=u200910) through aseptic trans-vaginal aspiration. The pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNFα were quantitated through bovine specific ELISA kits. Significantly higher concentrations of pro-inflammatory cytokines (IL-1β, IL-8, IL-6, and TNFα) along with low intra-follicular estradiol in buffaloes of PE and CE groups suggest that endometritis impedes the follicular steroidogenesis. Significantly higher concentration of IL-1β and TNF-α in the CVM of repeaters indicate their potential as a pen-side diagnostic test for CE.