Kisspeptin stimulates oestradiol biosynthesis by upregulating steroidogenic transcripts and proliferation markers in the bubaline granulosa cells in vitro.

Kisspeptin (Kp), an upstream regulator of GnRH release, is essential for the development and function of reproductive axis. Previously, we demonstrated the localization of Kp and its receptor (Kiss1r) in the active follicle in the bubaline ovary. Present study aimed to determine the effect of Kp on granulosa cell (GCs) functions, especially oestradiol (E2 ) and progesterone (P4 ) production, and differential expression of genes regulating the proliferation, apoptosis and steroidogenesis in the buffalo. The ovaries with 6-10 mm size follicles obtained from the cyclic buffaloes after slaughtering were used for isolation of GCs for in vitro study. The primary GCs culture was treated with Kp (0, 10, 50 and 100 nM) and incubated for 48 h. Production of E2 and P4 was estimated in the culture supernatant by ELISA. The expression of gonadotropin receptors (FSHR and LHR), steroidogenic genes (STAR, 3ß-HSD, CYP19A1), proliferation marker (PCNA), apoptotic factors (CASP3 and BCL2) and Kp signalling molecule (extracellular signal-regulated kinase 1/2, ERK1/2 and p-ERK1/2) was studied in the GCs by qPCR. Significant E2 production was found in the Kp 50 and 100 nM groups (p < .05), whereas P4 production was reduced in Kp 100 nM group (p < .05). There was concomitant upregulation of FSHR, ERK1/2, STAR and CYP19A1 in the Kp 100 nM treated GCs. In addition, Kp at 100 nM stimulated the proliferation of GCs by upregulating the expression of BCL2 (5.0 fold) and PCNA (94.9 fold). Further, high immunoreactivity of p-ERK1/2 was observed in the Kp-treated GCs. It was concluded that Kp at 100 nM concentration stimulated E2 production by upregulating the steroidogenic pathway through ERK1/2, STAR and CYP19A1 and modulating PCNA and BCL2 expressions in the GCs. Further experiments are warranted using Kp antagonist in different combinations to establish the signalling pathway in Kp-mediated steroidogenesis in the GCs for developing strategies to control ovarian functions.

Evaluation of sexed semen-based artificial insemination in Tharparkar cattle under organized farm condition.

Sexed semen facilitates additional female calf production for the expansion of a herd at a faster rate and also curtails the surplus production of unwanted male calves. A study was conducted to evaluate the performance of sexed semen in indigenous Tharparkar cows based on 114 artificial inseminations (AI) performed at natural oestrus using two protocols i.e., single AI (n = 48) and double AI (n = 66). Overall, the first service conception rate (CR) was significantly higher in double (53.0%) than single (33.3%) AI protocol. The odds ratio of conception rate in double AI was 2.26 (χ2 = 4.4, df = 1, p = .04) with respect to single AI. The time that elapsed since the detection of oestrus to insemination was also analysed. In a single AI protocol, the CR was higher (p < .05) at 16 h (54.6%) than insemination at 8 h (27.0%) following the onset of oestrus. Yet, the CR using double AI protocol did not differ (p = .73) significantly when AIs were performed either at 8 h and 24 h (51.9%) or 16 h and 24 h (57.1%) post onset of oestrus. Besides, like the single AI protocol, the parity of the animals also influenced the CR, being higher in heifers (n = 22) than those of parous (n = 92) cows (72.73 vs. 40.43%, χ2 = 7.48, df = 1, p = .006) in the present study. The odds ratio of conception in heifers was 3.93 with respect to parous cows. Overall, the birth of female calf was 91.7%. In conclusion, the present study indicates a future promise of the sexed semen for the production of more female offspring from Tharparkar cattle.

Secretory leucocyte protease inhibitor (SLPI) as an adjunct prognostic biomarker for canine pyometra.

Pyometra is a potentially life-threatening condition that affects intact female dogs in their middle to advance age. Timely diagnosis and appropriate treatment are critical for the survival of patients, especially when pyometra advances to sepsis. This study aimed to investigate the prognostic potential of certain haematology, serum biochemical and inflammatory biomarker, secretory leucocyte protease inhibitor (SLPI) for pyometra in bitches (n = 41). Blood samples were collected after clinical diagnosis of pyometra for haematology and serum biochemistry. Based on the prognosis following medical/surgical treatment, animals were retrospectively categorized into survivor (n = 29) and dead (n = 12). Endometrial tissue sections were obtained from the bitches undergoing ovariohysterectomy (n = 21). Serum concentration of SLPI was quantified using sandwich ELISA and its expression in the endometrium was investigated using RT-qPCR. A marked increase in the total leucocyte count (TLC), neutrophils, blood urea nitrogen (BUN) and serum creatinine was observed in the female dogs that did not survive. Significant elevation in the serum SLPI concentration (3.49 ± 0.44 vs. 2.38 ± 0.13 ng/mL) was observed in the bitches those died after the treatment, in comparison to those survived (p < .01). Additionally, there was a notable upregulation of SLPI in the endometrium in the bitches those died due to pyometra. Based on the ROC analysis results, it was observed that a cut-off concentration of 2.93 ng/mL for SLPI, 27.77 mg/dL for BUN and 16.3 × 103 /µL for TLC could effectively distinguish the prognosis of pyometra-affected dogs. From this study, it can be concluded that upregulation of SLPI in the endometrium and its elevated concentration in peripheral circulation along with TLC and BUN concentration could serve as valuable indicators for predicting the prognosis of pyometra in bitches.

Prognostic Potential of Thrombocyte Indices, Acute Phase Proteins, Electrolytes and Acid-Base Markers in Canine Parvovirus Infected Dogs With Systemic Inflammatory Response Syndrome.

Dogs with canine parvovirus enteritis (CPVE) that develop systemic inflammatory response syndrome (SIRS) frequently have a poor prognosis. The aim of the study was to assess the prognostic potential of thrombocyte indices, acute phase proteins, electrolytes, and acid-base markers in CPVE puppies with SIRS (CPVE-SIRS+) at admission. A case-controlled, prospective, and observational study was performed on 36 CPVE puppies. Mean concentrations of C-reactive protein (CRP), albumin, thrombocyte count, mean platelet volume (MPV), platelet distribution width (PDW), sodium (Na+), potassium (K+), chloride (Cl-) and ionized calcium (iCa) were measured and strong ion difference 3 (SID3), ATOT-albumin and ATOT-total protein were determined in CPVE-SIRS+ survivors and nonsurvivors. A prognostic cut-off value for predicting the disease outcome was determined by receiver operating characteristic (ROC) curve analysis. The mean values of MPV, PDW and CRP were significantly higher and the mean values of albumin, Cl- and ATOT-albumin were significantly lower in CPVE-SIRS+ nonsurvivor than CPVE-SIRS+ survivor puppies on the day of admission, but the thrombocyte count, Na+, K+, iCa, SID3 and ATOT- total protein values did not differ significantly. The positive predictive values (PPVs) for survival using cut-off value of MPV (≤15.08 fL), PDW (≤14.85%), CRP (≤180.7 mg/L), albumin (≥1.795 g/dL), Cl- (≥96.00 mmol/L), and ATOT-albumin (≥7.539) were determined as 100%, 100%, 100%, 80%, 100%, and 80%, respectively with better area under ROC curve and sensitivity. Based on sensitivity, specificity, and PPVs from ROC analysis, it is concluded that the determination of Cl- concentration and MPV at admission followed by CRP will serve as the most appropriate biomarkers in predicting the disease outcome of CPVE puppies that develop SIRS.

Serum concentrations of secretory leukocyte protease inhibitor and IL6 can predict the onset of sepsis in pyometra bitches.

As onset of sepsis adversely affects the prognosis of canine pyometra, finding biomarkers that would distinguish sepsis status would be useful in the clinical management. Accordingly, we hypothesized that differential expression of endometrial transcripts and circulating concentration of certain inflammatory mediators would discriminate pyometra-led sepsis (P-sepsis+) from those of pyometra without sepsis (P-sepsis-). Bitches with pyometra (n = 52) were classified into P-sepsis+ (n = 28) and P-sepsis- (n = 24) based on vital clinical score and total leukocyte count. A group of non-pyometra bitches (n = 12) served as control. The relative fold changes in the transcripts of IL6, IL8, TNFα, IL10, PTGS2, mPGES1 and PGFS, SLPI, S100A8, S100A12 and eNOS were determined by quantitative polymerase chain reaction. Furthermore, the serum concentrations of IL6, IL8, IL10, SLPI and prostaglandin F2α metabolite (PGFM) were assayed by ELISA. The relative fold changes in S100A12 and SLPI and mean concentrations of IL6 and SLPI were significantly (p < .05) higher in P-sepsis+ than that of P-sepsis- group. Receiver operating characteristic analysis revealed that serum IL6 had a diagnostic sensitivity of 78.6% and a positive likelihood ratio (LR+) of 2.09, at a cut-off value of 15.7 pg/mL to diagnose P-sepsis+ cases. Similarly, serum SLPI had a sensitivity of 84.6% and an LR+ of 2.23, at a cut-off value of 2.0 pg/mL. It was concluded that SLPI and IL6 would serve as putative biomarkers for pyometra-led sepsis in bitches. Monitoring SLPI and IL6 would be a useful adjunct to the established haemato-biochemical parameters in customizing the treatment strategies and arriving at the decision for management of pyometra bitches with critical illness.

Hematological and serum biochemical parameters and profiling of cytokine genes in lumpy skin disease in Vrindavani cattle.

Lumpy skin disease (LSD) is a notifiable re-emerging transboundary viral disease of bovines that inflicts heavy losses in affected livestock farms. Genetic variations contribute substantially to the inter-individual differences in the immune response against disease agents. The present study aimed to evaluate the genetic basis of differential immune response in Vrindavani cattle by comparing the hematological, biochemical and cytokine genes' expression profiles of LSD-affected and unaffected animals. After 21 days of the outbreak at the farm, the animals were grouped as affected (those who developed symptoms) and unaffected/healthy (those who did not). Standard hematological and biochemical parameters were evaluated in both the groups. Expression profiling of important Th1 (IL2, INFG and GMCSF) and Th2 (IL4, IL6 and IL10) cytokines was also performed via a relative quantification approach using real-time PCR. Erythrogram and leucogram analyses revealed significant differences in total leucocyte count (TLC: 14.18 ± 0.74 versus 11.38 ± 0.68 x103/µL), hemoglobin (Hb: 8.66 ± 0.42 versus 10.84 ± 0.17 g%) and percentage of neutrophils (46.40 ± 1.98 versus 35.40 ± 2.11%), lymphocytes (49.40 ± 1.99 versus 62.40 ± 1.86) and monocytes (4.20 ± 0.37 versus 2.40 ± 0.40) between the affected and healthy animals, respectively. The production of liver enzymes (SGOT and SGPT) was significantly higher in affected animals (74.18 ± 4.76 and 59.51 ± 2.75) when compared to the healthy counterparts (65.95 ± 9.18 and 39.21 ± 3.31). The expression profiling of Th1 and Th2 cytokines revealed significant differences between the two groups, except IL10. The expression of IL2, GMCSF and IL6 were upregulated in healthy animals while that of INFG, IL4 and IL10 were upregulated in LSD-affected animals. The highest abundance was observed for IL2 transcripts in healthy animals among all assessed cytokines with log2fold change of 1.61 as compared to affected counterparts. Overall, the immune response in healthy animals (after exposure to LSD virus) was predominated by the expression of Th1 cell proliferation and there was an increased production of pro-inflammatory cytokines as compared to the affected counterparts. The results revealed that the effective immune response to LSD in cattle consists of changes in hematological and biochemical parameters and altered expression profile of cytokines with enhanced phagocytosis and lymphocyte recruitment. Furthermore, optimal expression of Th1 cytokines is required for maintaining optimal health against infectious insult with LSD virus in cattle.

Early-Life Intervention of Lactoferrin and Probiotic in Suckling Piglets: Effects on Immunoglobulins, Intestinal Integrity, and Neonatal Mortality.

The aim of this study was to determine the effects of early-life bovine lactoferrin and host specific probiotic interventions on growth performance, mortality, and concentrations of immunoglobulin A and immunoglobulin G and transforming growth factor beta 1 (a marker of intestinal integrity) in serum of neonatal piglets. A total of eight piglet litters from parity matched sows were randomly divided into four groups and assigned to one of the four interventions: control (sterile normal saline), bovine lactoferrin (100 mg bovine lactoferrin), probiotic (1 × 109 colony forming unit (cfu) of swine origin Pediococcus acidilactici FT28 probiotic), and bovine lactoferrin + probiotic (100 mg bovine lactoferrin and 1 × 109 CFU of P. acidilactici FT28 probiotic). All the interventions were given once daily through oral route for first 7 days of life. The average daily gain (p = 0.0004) and weaning weight (p < 0.0001) were significantly improved in the probiotic group. The piglet survivability was significantly higher in bovine lactoferrin and probiotic groups than control group in Log-rank (Mantel-Cox) test. The concentrations of immunoglobulin A on day 21 in bovine lactoferrin, probiotic, and bovine lactoferrin + probiotic groups increased significantly (p < 0.05). Immunoglobulin G concentrations on day 7 and 15 in bovine lactoferrin and bovine lactoferrin + probiotic groups and on day 15 in probiotic group were significantly (p < 0.05) elevated, whereas, the concentration of transforming growth factor-ß1 was significantly (p < 0.05) increased from day 7 to 21 in all the supplemented groups. In conclusion, the early-life bovine lactoferrin and P. acidilactici FT28 probiotic interventions reduced the mortality in the suckling piglets by promoting the systemic immunity and enhancing the intestinal integrity.

Efficacy of Microencapsulated Probiotic as Adjunct Therapy on Resolution of Diarrhea, Copper-Zinc Homeostasis, Immunoglobulins, and Inflammatory Markers in Serum of Spontaneous Rotavirus-Infected Diarrhoetic Calves.

The objective of this study was to assess the efficacy of a microencapsulated probiotic as an adjunct therapy in rotavirus-positive diarrhea of neonatal calves that received supportive treatment or supportive along with microencapsulated probiotic treatment, for 5 days. We examined whether microencapsulated Lactobacillus acidophilus NCDC15 probiotic treatment in rotavirus-infected diarrhoetic calves led to faster resolution of diarrhea, amelioration of zinc-copper imbalance, improved the immunoglobulin A and immunoglobulin G, and decreased the inflammatory markers in serum. Calves with rotavirus-positive diarrhea < 4-week age and fecal scores ≥ 2 were randomly assigned into two groups. The supportive along with microencapsulated probiotic treatment significantly (p < 0.05) increased zinc and immunoglobulin A concentrations and decreased copper, tumor necrosis factor-α, and nitric oxide level in serum on days 3 and 5 from pretreatment values; the immunoglobulin G concentration was elevated (p < 0.05) on day 5. The mean resolution time of abnormal fecal score was 5.3 and 3.3 days in supportive treatment and supportive along with microencapsulated probiotic groups, respectively, in log-rank Mantel-Cox test. The calves in the supportive along with microencapsulated probiotic treatment group had faster resolution of diarrhea than supportive treatment group in Dunn's multiple comparisons test. This study demonstrates that supportive treatment along with microencapsulated probiotic administered to naturally rotavirus-infected diarrhoetic calves at onset of diarrhea led to faster resolution of diarrhea, improved zinc and immunoglobulin levels, and decreased the inflammatory parameters in serum of rotavirus-infected diarrhoetic calves.

Differential Expression of Prostaglandin Receptors in Canine Uterus With Pyometra.

The objective of the study was to ascertain the role of prostaglandins Viz., PGE2 and PGF2α, and their respective receptors in the pathophysiology of canine pyometra. Normal (n = 6) and pyometra (n = 8) affected uterus were collected from bitches undergoing ovariohysterectomy. Pyometra was graded according to histopathological alterations. The levels of PGE2 and PGF2α were estimated in the endometrium. The differential expression in the mRNA of PGF2α receptor (FP) and PGE2 receptors (EP1, EP2, EP3, and EP4) were studied in the endometrium and myometrium of the pyometra-affected uterus. Normal uterus served as calibrator. Elevation of both PGE2 and PGF2α levels in the endometrium of pyometra-affected bitches was observed. The FP receptor gene in the endometrium and myometrium of pyometra-affected bitches was downregulated (P < .05). Out of all EP receptors, only EP2 receptor has shown upregulation in both endometrium and myometrium of pyometra affected uterus. EP3 receptor got downregulated in both endometrium and myometrium in pyometra. Thus, downregulation of FP, EP3 receptors in the myometrium reinforces the lack of contractility in pyometra-affected bitches favoring bacterial proliferation and subsequent pus accumulation. Moreover, upregulation of EP2 receptors in the pyometra bitches suggests the scope of selective pharmacological inhibition of EP2 receptors as an adjunct therapy in the treatment of pyometra.

Curcumin inhibits lipopolysaccharide and lipoteichoic acid-induced expression of proinflammatory cytokines and production of PGE2 in the primary bubaline endometrial stromal cells.

Infection of the uterus with Gram-positive Trueperella pyogenes and Gram-negative Escherichia coli is a common cause of postpartum endometritis in the cattle and buffalo and the condition is treated with antimicrobial drugs. The presence of drug residues in the milk and development of resistant bacteria necessitate the evaluation of alternate therapies for endometritis. Accordingly, we tested the immunomodulatory effect of curcumin in the bubaline endometrial stromal cells after treatment with the lipoteichoic acid (LTA) of Gram-positive Staphylococcus aureus and lipopolysaccharide (LPS) of Gram-negative E. coli that activate toll-like receptors (TLR-2 and TLR-4, respectively). Confluent primary culture of endometrial stromal cells was treated with LTA (1 µg/mL) and/or LPS (0.1 µg/mL), in the presence or absence of curcumin (30 µM for 24 h). PGE2 was assayed in the supernatant and the relative expression of proinflammatory cytokines (PICs) (IL1B, IL6, IL8 and TNFA) transcripts were quantified using real-time PCR. LTA was not effective in stimulating PGE2 production or upregulating the PIC expression except IL8. LTA+LPS increased PGE2 production and upregulated IL6 and IL8 genes. Curcumin inhibited the basal and LTA+LPS induced production of PGE2 and upregulation of PIC production. It was apparent that LPS, but not LTA, is a potent stimulator of PGE2 from the bubaline endometrial stromal cells. Curcumin downregulated the expression of LPS and/or LTA induced PICs and PGE2 and may be an alternate to antimicrobial drugs for the therapeutic management of endometritis.

Assessment of certain biomarkers for predicting survival in response to treatment in dogs naturally infected with canine parvovirus.

Canine parvovirus (CPV) enteritis is an important cause of morbidity and mortality in puppies despite aggressive treatment. Identification of reliable biomarkers for CPV enteritis is essential to determine the severity, duration of hospitalization, and predict the clinical outcome. Meanwhile, the biomarkers will assist in decision-making with clients about the further course of treatment or euthanasia. The present study was conducted to evaluate the changes of total leukocyte count (TLC), neutrophil count, and serum concentrations of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), intestinal fatty acid binding protein-2 (IFABP-2), albumin, ceruloplasmin (Cp), cortisol, free triiodothyronine (FT3) and free thyroxine (FT4) in survivors and non-survivors as a predictor of the clinical outcome. Marked leukopenia, neutropenia, hypoalbuminemia, elevated levels of CK-MB, IFABP-2, Cp, and cortisol were noticed in CPV-infected dogs than healthy dogs but, LDH, FT3 and FT4 concentrations did not differ significantly. The CPV-infected non-survivors had persistent leukopenia, neutropenia and elevated CK-MB, IFABP-2, Cp and cortisol concentrations at 72 h of commencement of treatment. In CPV-infected survivors, TLC and neutrophil count were significantly increased, and CK-MB, IFABP-2, Cp and cortisol concentrations were significantly decreased at 72 h of commencement of treatment. The positive predictive values (PPVs) for survival using cut-off value of TLC (>3.2 × 103/µL), neutrophil count (>1.65 × 103/µL), CK-MB (≤234.50 U/L), IFABP-2 (≤7.61 ng/mL), Cp (≤0.605 g/L) and cortisol (≤16.90 ng/mL) were determined as 89.47%, 88.88%, 94.73%, 93.33%, 94.44% and 89.47%, respectively with better area under receiver operating characteristic (ROC) curve as well as sensitivity. The magnitude of decrease in TLC, neutrophil count, and increase in CK-MB, IFABP-2, Cp and cortisol concentrations at 72 h of initiation of treatment in dogs with parvoviral enteritis could be useful indicators for the prognosis of the disease. Based on sensitivity (%) and specificity (%) from ROC curve analysis and PPV (%), it is concluded that serum CK-MB concentration will serve as the most useful biomarker followed by Cp and absolute neutrophil count.

Effect of dietary n-3 polyunsaturated fatty acid supplementation on the expression of genes involved in progesterone biosynthesis in the corpus luteum of goat (Capra hircus).

Emerging evidence indicates that dietary n-3 polyunsaturated fatty acids (PUFA) alter the fatty acid composition of corpus luteum (CL) and directly affect the luteal function in the cow, which is independent of the inhibitory effect on the endometrial PGF2α production. The present study, thus, investigated the effects of n-3 PUFA rich fish oil (FO) supplementation on the transcriptional modulation of genes involved in the biosynthesis of progesterone (P4 ) in the CL collected during the luteolytic phase of oestrous cycle in the goat. On the day of synchronized oestrus, goats (n = 6/group) were fed an isocaloric diet supplemented with either FO or palm oil (PO). The dose of oil supplementation was 0.6 mlkg-1 body weight, and the duration was 55-57 days. The FO provided 156 mgkg-1 body weight of n-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The CL was collected by laparotomy on day 16 post-oestrus, and the relative abundance of P450 side-chain cleaving enzyme, steroid acute regulatory protein (StAR) and 3ß-hydroxy steroid dehydrogenase (3ß-HSD) genes was quantitated by real-time PCR. The results indicated that the dietary FO significantly upregulated the expression of 3ß-HSD by 1.13-fold and downregulated StAR by ~2-fold as compared to PO group (p < .05). It is concluded that dietary FO differently affected the expression of genes involved in P4 synthesis in the CL during the luteolytic window of the oestrous cycle in the goat.

Effect of dietary n-3 polyunsaturated rich fish oil supplementation on ovarian function and interferon stimulated genes in the repeat breeding cow.

Dietary n-3 polyunsaturated fatty acids (n-3 PUFA) improve utero-ovarian functions and embryonic survival in postpartum dairy cows. Because early embryonic mortality is the major cause of repeat breeding (RB) in cows, there was investigation of the effect of dietary supplementation of n-3 PUFA [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] rich fish oil (FO) from -2 to +2 weeks of artificial insemination on the size of preovulatory follicle (POF), serum progesterone (P4) and relative abundance of the mRNA of interferon stimulated genes (ISG) that encode for these proteins in the peripheral blood leukocytes (PBL) in the RB cow (n = 12). The diet of control group was supplemented with palm oil (PO). The results indicated serum concentrations of EPA and DHA were greater by 4.6- and 3.5-fold, respectively at the end of feeding study in the RB cows of the FO group. The diameter of POF was larger by 2.2 mm in FO group; however, serum P4 did not vary from day 14-20 post-artificial insemination. Greater abundance of ISG mRNA transcripts such as ISG15, RTP4, Mx2 and OAS1 in the PBL of pregnant cows of FO group indicates day 20 conceptuses produced more IFN-τ. It is concluded that supplementation of FO during the breeding period increased the size of POF and enhanced the abundance of ISG mRNA transcripts in RB cows that became pregnant.

Effect of n-3 PUFA-rich fish oil supplementation during late gestation on kidding, uterine involution and resumption of follicular activity in goat.

We have shown that dietary supplementation of n-3 polyunsaturated fatty acid (n-3 PUFA)-rich fish oil (FO) around the breeding time improved the utero-ovarian functions in the goat. Here, we investigated the effect of FO supplementation during the periparturient period on serum n-3 PUFA, prostaglandin F2α metabolite (PGFM), placental expulsion, uterine involution, resumption of oestrus and neonatal vigour. Rohilkhandi goat in advanced gestation (n = 16) was divided into two equal groups. One group was supplemented with FO containing 26% n-3 long-chain PUFA at the rate of 156 mg per kg body weight, while the control group was fed isocaloric palm oil (PO) from -3 to +3 week of kidding. Dietary FO increased serum concentration of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by 7.3- and 6.6-fold, respectively, after 6 weeks of supplementation. Goats in FO group expelled the foetal membranes 99.1 min earlier (p < .01) than those of PO group. Further, dietary FO significantly decreased the serum PGFM on day 7 post-partum. However, no difference was found on uterine involution, which was complete by day 20 post-partum in either group. Resumption of follicular activity by day 5 post-partum was 87.5% in the FO as compared to 25% in the PO group (p < .05). Similarly, occurrence of behavioural oestrus by day 90 post-partum was 57.1% in goats of the FO group while none of does was in the PO group (p < .01) expressed oestrus. It was concluded that feeding FO-rich diet during -3 to +3 weeks of kidding decreased the PGFM till day 7 post-partum, hastened the expulsion of foetal membranes and reduced the time from kidding to first post-partum oestrus in Rohilkhandi does.

Immunomodulatory effect of curcumin on lipopolysaccharide- and/or flagellin-induced production of prostaglandin E2 and relative expression of proinflammatory cytokines in the primary bubaline endometrial stromal cells.

Developing alternate therapies for bovine endometritis is important in the context of drug residues in the milk and emergence of antimicrobial resistant bacteria. In this regard, we studied the immunomodulatory effect of curcumin 30 µM, on lipopolysaccharide- (LPS) and/or flagellin (100 ng/ml each)-induced prostaglandin E2 (PGE2 ) and proinflammatory cytokines (PIC) using primary bubaline endometrial stromal cells. After 24 hr treatment, the supernatant was assayed for PGE2 while cells were used for relative quantification of cytokines like IL-1ß, IL-6, IL-8 and TNF α transcripts using a control group as calibrator. LPS was found to possess potent stimulatory effect on PGE2 production, whereas the flagellin was not as potent as LPS in stimulating the PGE2 production either per se or in combination with LPS. LPS markedly up-regulated the transcripts of IL-8 and IL-6 as compared to IL-1ß and TNF α in the bubaline endometrial stromal cells. Except for IL-8, flagellin did not up-regulate other PICs. There was no additive effect between LPS and flagellin on the up-regulation of inflammatory cytokines. Curcumin inhibited the LPS-induced up-regulation of PIC with strong down-regulation of IL-8. The inhibitory effects of curcumin on the inflammatory mediators suggest a potential in the treatment of bovine endometritis.

Kiss1 and its receptor: molecular characterization and immunolocalization in the hypothalamus and corpus luteum of the buffalo.

ABSTARCT The neuropeptide kisspeptin (Kp) through its receptor Kiss1r regulates the HPG axis by controlling GnRH release. Since buffalo is a seasonal breeder with problems of delayed puberty and postpartum anestrus, we characterized the Kiss1 and Kiss1r and investigated the immunolocalization in the hypothalamus and corpus luteum (CL). Kiss1 and Kiss1r genes were amplified from gDNA covering the coding region, cloned and sequenced with accession numbers MF168937 and MG820539, respectively. The Kiss1 DNA sequence had two exonic segment contained coding sequence (cds); 408 bp encoding a predicted protein of 136 aa with conservation of Kp-10 and shared 94.5-98.3% identity with ruminants. Kiss1r DNA sequence consisted of five exons with a cds of 1134 bp encoding a protein of 378 aa. Phylogenetic analysis of Kiss1 and Kiss1r revealed that it formed a monophyletic clade with cattle, which branched from sheep and goat. Immunofluorescence study revealed the presence of Kiss1 and Kiss1r in the neuronal soma and perinuclear area of preoptic and arcuate regions of the hypothalamus and luteal cells of the CL. This is the first report on molecular characterization of bubaline Kiss1 and Kiss1r genes that confirmed the presence of conserved Kp-10 like other ruminants and kisspeptinergic system is present in the hypothalamus and CL.

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 (n = 18). Using endometrium to myometrium ratio of 0.79 as threshold, the uteri (n = 8/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 (n = 6) served as control. The mean fold change (2-ΔΔCt) for the target genes was determined using ß-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 Spearman's rho revealed that IL-8 had strong positive correlation with COX-2 and PGFS in the AP group (P < 0.05). 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.

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 (n = 14/group). Each group was subdivided into follicular and mid-luteal stage (n = 7/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 (n = 10) and non-repeaters (n = 10) through aseptic trans-vaginal aspiration. The pro-inflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNFα were quantitated through bovine specific ELISA kits. Significantly higher concentrations of pro-inflammatory cytokines (IL-1ß, 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-1ß and TNF-α in the CVM of repeaters indicate their potential as a pen-side diagnostic test for CE.

Expression of pathogen recognition receptors and pro-inflammatory cytokine transcripts in clinical and sub-clinical endometritis cows.

The present study was carried out to examine the expression profile of pathogen recognition receptors (CD14 and toll-like receptor 4) and pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNFα) in endometrial tissue of cows with endometritis at different stages of estrous cycle. Genital tracts were collected from 60 cows at slaughter from the killing village. The genitalia were examined for clinical endometritis (CE) and subclinical endometritis (SCE) through physical examination, white side test of cervico-vaginal mucus, endometrial cytology and histopathology. The stage of estrous cycle for each genitalia was determined by visual examination of both the ovaries and classified as either follicular (F) or luteal (L). Depending on the degree of inflammation and stage of estrous cycle, the genitalia were categorized in four groups i.e., FCE, FSCE, LCE, and LSCE with six genitalia in each group. Furthermore, 12 healthy genitalia comprise of six each of follicular (FN) and luteal (LN) were included as control. Endometrial tissue scrapings were collected ex vivo from all the genitalia. Total RNA was extracted and cDNA was transcribed for each sample and relative quantification of mRNA of target genes was done by real-time PCR. The results revealed a significant up-regulation of CD14 (11 fold) and IL-8 (13 fold) in follicular stage and IL-6 (8 fold) and TNFα (29 fold) in luteal stages in SCE cows. However, the majority of pro-inflammatory cytokine and pathogen recognition receptors expressed at significant higher level in both follicular and luteal stages in cows with CE. Thus, it is concluded that the endometrial transcripts of pathogen recognition receptors and pro-inflammatory cytokines expressed differentially in cows with endometritis, whereas the fold change is dependent on the severity of inflammation and the stage of cyclicity. Therefore, endometrial transcript profile with a defined threshold level could be used as a possible diagnostic marker in cows with SCE.

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.