Local and systemic inflammatory response to the intrauterine infusion of enzymes during estrus in water buffaloes with subclinical endometritis.

Our objective was to determine the effects of intrauterine infusion of proteolytic enzymes in buffaloes with subclinical endometritis (SCE) at estrus on the resolution of endometrial inflammation and reproductive performance. Buffaloes at spontaneous estrus (E1) were screened for SCE by endometrial cytology to identify SCE (≥5% PMN, n = 22) and non-SCE (<5% PMNs, n = 14) animals. All buffaloes underwent uterine ultrasonographic examination, low volume uterine lavage (cytokines and acute phase proteins) and blood sampling (cytokines and acute-phase proteins) at E1. On the same day (E1), SCE buffaloes were randomly selected either for intrauterine infusion of proteolytic enzymes (ENY, n = 11) or saline (PC, n = 11). Buffaloes without SCE were kept as untreated control (NC; n = 14). All buffaloes were re-examined and re-sampled during subsequent estrus (E2), inseminated during the following estrus (E3), and assessed for fertility related outcomes. Proteolytic infusion resulted a reduction in uterine PMN (P < 0.01) in SCE buffaloes. The concentrations of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in uterus, and TNF-α and IL-10 in serum were higher (P < 0.01) at E1 in buffaloes with SCE (PC and ENY) compared to NC. After treatment, uterine IL-1ß and TNF-α (P = 0.02), and serum TNF-α and IL-10 were lower within the animals of ENY group (P < 0.01). Before treatment, buffaloes with SCE had higher concentrations (P < 0.01) of serum and uterine amyloid-A and haptoglobin, which decreased (P < 0.01) after treatment in the ENY group. None of the fertility outcomes differ between the treatment groups. In conclusion, intrauterine infusion of proteolytic enzymes reduced endometrial inflammation; however, did not improve reproductive outcomes.

Escherichia coli membrane-derived oxygen-reducing enzyme system (Oxyrase) protects bubaline spermatozoa during cryopreservation.

The objective of this study was to determine the effectiveness of deoxygenation of semen extender using Escherichia coli membrane-derived oxygen scavenger (Oxyrase) on post-thaw quality of buffalo (Bubalus bubalis) spermatozoa. Sixteen semen ejaculates, four each from four bulls, were each divided into five equal fractions, diluted using Tris-egg yolk extender supplemented with different concentrations of Oxyrase (0, 0.3, 0.6, 0.9, and 1.2 U/ml), designated as treatments T1, T2, T3, T4, and T5, respectively, and cryopreserved. Immediately after thawing, Oxyrase did not improve sperm kinetics and motility; however, it improved the keeping quality (significantly lower deterioration of post-thaw sperm motility after incubation for 120 min) in T3. Further, T3 reduced (p < .05) cholesterol efflux and protected the intactness of the sperm plasma membrane. Flow cytometry with Fluo-3 AM/propidium iodide (PI) dual staining revealed the highest (p < .05) proportion of live spermatozoa with low intracellular calcium in T3. Oxyrase supplementation protected spermatozoa from premature capacitation which was confirmed by low expression of tyrosine-phosphorylated proteins (32, 75, and 80 kDa) and a relatively lower percentage of F-pattern (uncapacitated spermatozoa) in chlortetracycline assay. Importantly, the Oxyrase fortification decreased superoxide anion in a dose-dependent manner indicating reduced availability of oxygen at sperm mitochondrial level. Similarly, in Oxyrase-fortified sperm, malondialdehyde concentration, an index of lipid peroxidation, is also reduced in a dose-dependent manner. In conclusion, we demonstrate that deoxygenation of buffalo semen by Oxyrase has the potential of improving post-thaw sperm quality by overcoming the problem of cryocapacitation and oxidative damage during cryopreservation process.

Effects of intra-uterine infusion of proteolytic enzymes on selected cytokine concentrations, uterine inflammation, and fertility in postpartum water buffalo cows with subclinical endometritis.

The study was conducted to evaluate effects of intrauterine administration of proteolytic enzymes on endometrial inflammation and reproductive performance in postpartum water buffalo cows with subclinical endometritis (SCE). Cows (n = 38) with SCE (≥ 18 % PMN i.e.; polymorphonuclear cells) on day 21 postpartum (21 dpp), were allocated into treatment (TR; n = 19; intrauterine infusion of trypsin, chymotrypsin and papain in 20 ml normal saline on 21 dpp) and control (PC; n = 19; intrauterine administration 20 ml saline) groups. Cows without SCE (< 18 % PMN) were not treated and served as the negative control (NC; n = 30). Ultrasonography and sampling (endometrial cytology, uterine flushing, blood) were conducted on day 21 (before treatment) and 28 postpartum (28 dpp). The PMN % and uterine horn diameter were less on 28 dpp (compared with 21 dpp) in NC and TR group only. Cows with SCE had greater uterine concentrations of interleukin (IL)-1ß, IL-8 and tumor necrosis factor (TNF)-α; but lesser IL-10 than NC group on 21 and 28 dpp. There were greater serum IL-1ß and TNF-α concentrations on 28 dpp in cows with SCE than NC group. Uterine concentrations of IL-1ß were less, whereas IL-6 was greater following enzymatic treatment. Proteolytic enzyme treatment did not result in improvement in pregnancy rate compared with the PC group; however, days to conception were less in TR compared with the other two groups. In conclusion, results indicated a reduction in endometrial inflammation and days nonpregnant after proteolytic enzyme treatment in buffalo cows with SCE.