Improved quality and fertilizability of cryopreserved buffalo spermatozoa with the supplementation of methionine sulfoxide reductase A.

BACKGROUND: The excessive reactive oxygen species produced during semen-freezing and -thawing damage the macromolecules resulting in impairment of cellular functions. Proteins are the primary targets of oxidative damage, wherein methionine residues are more prone to oxidation and get converted into methionine sulfoxide, thus affecting the protein function. The methionine sulfoxide reductase A (MsrA) catalyzes the conversion of methionine sulfoxide to methionine and restores the functionality of defective proteins. OBJECTIVES: To establish the expression of MsrA in male reproductive organs, including semen and its effect on quality of cryopreserved semen upon exogenous supplementation, taking buffalo semen as a model. MATERIALS AND METHODS: The expression of MsrA was established by immunohistochemistry, PCR, and Western blots. Further, the effect of recombinant MsrA (rMsrA) supplementation on the quality of cryopreserved spermatozoa was assessed in three treatment groups containing 1.0, 1.5, and 2.0 µg of rMsrA/50 million spermatozoa in egg yolk glycerol extender along with a control group; wherein the post-thaw progressive motility, viability, membrane integrity, and zona binding ability of cryopreserved spermatozoa were studied. RESULTS: The MsrA was expressed in buffalo testis, epididymis, accessory sex glands, and spermatozoa except in seminal plasma. In group 2, the supplementation has resulted in a significant (p < 0.05) improvement as compared to the control group in mean progressive motility (47.50 ± 2.50 vs. 36.25 ± 2.63), viability (56.47 ± 1.85 vs. 48.05 ± 2.42), HOST (50.76 ± 1.73 vs. 44.29 ± 1.29), and zona binding ability of spermatozoa (149.50 ± 8.39 vs. 29.50 ± 2.85). DISCUSSION AND CONCLUSION: In the absence of native MsrA of seminal plasma, the supplementations of rMsrA may repair the oxidatively damaged seminal plasma proteins and exposed sperm plasma membrane proteins resulting in better quality with a fivefold increase in fertilizability of frozen-thawed spermatozoa. The findings can be extended to other species to improve the semen quality with the variation in the amounts of rMsrA supplementation.

Effect of different levels of n-3 polyunsaturated fatty acids rich fish oil supplementation on the ovarian and endometrial functions in the goat (Capra hircus).

Decreasing the ratio of n-6 to n-3 polyunsaturated fatty acid (PUFA) through dietary manipulation improves the reproductive functions in the dairy cow. This study was conducted to determine the effect of dietary supplementation of n-3 PUFA rich fish oil that provided different levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to determine the optimum dose with respect to endometrial and ovarian functions in the doe. Normal cyclic goats (n = 20) were divided into four groups (n = 5/group) and the fish oil was supplemented for 55 days such that EPA and DHA content were 0, 72, 156 and 312 mg/Kg body weight for CON, LOW, MED and HIGH groups, respectively. The diet was made isonitrogenous and isocaloric. Estrus was synchronized by two PG regimen at 11 days apart with first PG on day 25 of supplementation. Experimental does were observed from second PG till day 17 post-estrus. Ovarian ultrasound revealed that the follicular attributes were comparable among the groups; however, the diameter of CL on day 5 post-estrus was significantly larger in the MED group. Fish oil supplementation even at the lowest dose significantly decreased the serum estradiol (E2) and basal as well as oxytocin (OXT) induced PGFM during the late luteal phase of the estrous cycle (P < 0.05). Further, the MED group showed a significant decrease in the serum E2 on day 17 post-estrus as compared to LOW group. A significant increase in the progesterone (P4) on day 14 post-estrus was recorded in the MED group. Neither the effect of treatment nor time was significant either on day 16 or 17 post-estrus with respect to PGEM following OXT challenge (P > 0.05). It is concluded that the dietary fish oil inhibited OXT induced PGF2α release and decreased E2 during the late luteal phase of the estrous cycle in the doe, which will have a favourable effect on the maternal recognition of pregnancy. In addition, MED level (156 mg/Kg body weight) increased the CL size and circulating P4 during the luteal phase.

Effect of dietary n-3 polyunsaturated fatty acid rich fish oil on the endometrial prostaglandin production in the doe (Capra hircus).

Recently, we showed that dietary supplementation of n-3 PUFA rich fish oil (FO) decreased the metabolites of serum prostaglandin (PG) F2α and E2 during the window of pregnancy recognition in the doe. In this study, we investigated its effect on the changes on endometrial PG production in vitro. Cycling does (n = 12) of Rohilkhand region were divided into two equal groups and fed a concentrate diet supplemented with either FO containing 26% n-3 PUFA (TRT; n = 6) or palm oil (CON; n = 6) @ 0.6 mL/kg body weight for 57 days. Estrus was synchronized by two injections of PGF2α analogue viz, on day 25 and 36 of supplementation and laparo-hysterotomy was performed to obtain endometrial tissue on day 16 of the synchronized estrus. Endometrial explant culture was done using a defined medium.The basal PG production was assayed at 6 and 12 h. Endometrial explant was stimulated with oxytocin (OXT) and/or recombinant ovine interferon tau (roIFN-τ) and PGs were assayed at 3 and 12 h post-treatment. The relative expression of genes related to PG metabolism in the endometrium was done by Quantitative Real Time PCR technique (qRT-PCR). There was a significant (P < 0.05) decline in the basal production of PGF2α and PGE2 in the TRT as compared to the CON group. The cultured endometrial tissue produced PGF2α in a time- dependent fashion in both the groups (P < 0.05). Neither OXT nor roIFN-τ had a significant (P > 0.05) effect on the PGF2α and PGE2 production in the TRT group. Similarly, the PG production in the OXT and roIFN-τ was comparable with the control in TRT. Expression of mRNA for cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2) and PGF synthase (PGFS) was lower (P < 0.05) whereas, PGE synthase and peroxisome proliferator-activated receptors such as PPAR-γ and δ was increased (P < 0.05) in n-3 PUFA fed doe. In conclusion, dietary supplementation of FO decreased the endometrial production of PGF2α and PGE2 by downregulating the COX-2, cPLA2 and PGFS transcripts in the doe. The findings suggest that n-3 PUFA influence embryo survival by modulating the endometrial PG.

Supplementation of vitamin E, selenium and increased energy allowance mitigates the transition stress and improves postpartum reproductive performance in the crossbred cow.

Dairy cow undergoes tremendous physiological challenges during the transition period leading to negative energy balance (NEBAL), impaired immunity and oxidative stress that ultimately compromises the postpartum fertility. Accordingly, we investigated the effects of antioxidant supplementation and increased energy allowance on transition stress and fertility of crossbred cow. Advanced pregnant crossbred cows (n = 26) of 2-4 parity and lactation potential of >10 L/day were divided into two equal groups (n = 13 cows/group). Cows were fed diets either (i) supplemented with vitamin E (80 IU/kg DM), Selenium (Se; 0.3 mg/kg DM) and increased energy allowance in the form of 20% additional concentrate (TRT) or (ii) basal diet without any supplementation as control (CON). Vitamin E and Se were supplemented with wheat flour bolus from -4 to 8 week of calving whereas energy allowance was increased from 2 to 8 week of calving on daily basis to individual animal (where 0 is day of calving). Blood samples were collected on weekly interval from -4 to 8 week of calving. Oxidative stress was assessed by estimation of malondialdehyde (MDA), total antioxidant capacity (TAC), superoxide dismutase (SOD) and catalase (CAT). Immunity level was assessed via estimation of phagocytic activity (PA) of granulocytes and lymphocyte proliferation assay (LPA). Postpartum fertility was assessed by interval to first postpartum estrus (day) and pregnancy rate. Cows in TRT had a significantly lower MDA, higher TAC and decreased activity of SOD and CAT than that of CON (P < 0.05). Phagocytic activity increased at -1, 0 and 3-8 weeks postpartum (P < 0.05) while LPA showed difference (P < 0.05) at parturition, week 4 and 8 postpartum. Marked improvement in the fertility was recorded in terms of early resumption of postpartum estrus (P < 0.001) and higher pregnancy rate (P < 0.05).

Effect of dietary supplementation of omega-3 polyunsaturated fatty acid (PUFA) rich fish oil on reproductive performance of the goat (Capra hircus).

Dietary supplementation of n-3 PUFA decreases the luteolytic PGF2α and improves the pregnancy rate in the dairy cow. However, its effect in the goat is not known. Accordingly, we studied the effect of supplementation of n-3 PUFA rich Fish oil (FO) on different reproductive events in the goat. Cycling goats (n = 30) were divided into two equal groups and fed an isocaloric and isonitrogenous diet supplemented with either FO (TRT; n = 15) or palm oil (PO) (CON; n = 15) @ 0.6 mL/kg body weight for 72 days during the breeding season. Estrus synchronization was done on day 25 and 36 of supplementation using two PG regimen and the goats in estrus were bred. Mean interval from PGF2α administration to the onset of estrus was 12 h longer (P < 0.05) in the TRT group than that of CON. The number of preovulatory follicles (POF) and ovulation rate was significantly higher in FO supplemented goats (P < 0.05) by 39.64 and 41.35%, respectively. Though the corpus luteum diameter was significantly higher (P < 0.05) on day 5, 8 and 11 post-breeding in the TRT group, mean serum progesterone (P4) did not differ significantly (P > 0.05). Mean concentration of serum estradiol (E2) was significantly (P < 0.01) lower in the FO supplemented group during day 0-60 post-breeding which could be due to significantly low serum cholesterol (P < 0.01). Though the serum concentration of PGF2α metabolite (PGFM) and PGE2 metabolite (PGEM) in the pregnant goats was significantly (P < 0.05) lower in the TRT group on day 16 and 17 post-breeding, the ratio of PGEM to PGFM remained unaffected suggesting a favourable effect of FO supplementation on the early pregnancy. The number of embryos, twinning rate and kidding rate were high in FO supplemented group though it was non-significant. However, gestation length, birth weight of kids and neonatal behaviour were comparable between the groups (P > 0.05). In conclusion, supplementation of n-3 PUFA rich FO significantly increased the number of POF and ovulation rate with numerical increase in the kidding rate. Further, it decreased the serum E2 and PGFM during the critical window of pregnancy recognition in the doe.

Potential antibacterial activity of berberine against multi drug resistant enterovirulent Escherichia coli isolated from yaks (Poephagus grunniens) with haemorrhagic diarrhoea.

OBJECTIVE: To evaluate the antimicrobial efficacy of berberine, a plant alkaloid. METHODS: Five multi-drug resistant (MDR) STEC/EPEC and five MDR ETEC isolates from yaks with haemorrhagic diarrhoea were selected for the study. Antibacterial activity of berberine was evaluated by broth dilution and disc diffusion methods. The binding kinetics of berberine to DNA and protein was also enumerated. RESULTS: For both categories of enterovirulent Escherichia coli (E. coli) isolates, berberine displayed the antibacterial effect in a dose dependent manner. The MIC(50) of berberine chloride for STEC/EPEC isolates varied from 2.07 µM to 3.6 µM with a mean of (2.95 ± 0.33) µM where as for ETEC strains it varied from 1.75 to 1.96 µM with a mean of (1.87 ± 0.03) µM. Berberine bind more tightly with double helix DNA with Bmax and Kd of (24.68±2.62) and (357.8±57.8), respectively. Berberine reacted with protein in comparatively loose manner with Bmax and Kd of (18.9±3.83) and (286.2±113.6), respectively. CONCLUSIONS: The results indicate clearly that berberine may serve as a good antibacterial against multi drug resistant E. coli.

Food supplementation as an incentive to improve pre-antiretroviral therapy clinic adherence in HIV-positive children--experience from eastern India.

OBJECTIVE: To evaluate the importance of food supplementation as incentive in improving preantiretroviral therapy (pre-ART) adherence, and second its impact on health of HIV-infected children by a clinic-based observational study. METHODS: HIV-seropositive children aged between 2 and 12 years were followed-up sequentially for 2 years without and with food supplementation, respectively, with monitoring of disease parameters. The outcome morbidity parameters were compared and correlated. RESULT: Study showed significant improvement in clinic adherence (r = 0.165, p = 0.027) along with increased mean clinic visit (6.65 ± 1.43 vs. 8.01 ± 1.52, p = 0.000) and mean CD4 count (p = 0.028) with incentive. Provision of incentive correlated well (Pearson's r = 0.345) with number of visits which in turn had strong correlation with weight gain (r = 0.548), episodes of AIDS-defining illnesses (r = -0.412), hospitalization (r = -0.279). CONCLUSION: Food incentive could enhance pre-ART phase clinic adherence that decreases disease-related morbidities, setting the stage for improved treatment and care of seropositive children in future.

Pre-exchange 5% albumin infusion in low birth weight neonates with intensive phototherapy failure--a randomized controlled trial.

OBJECTIVE: To evaluate the role of 5% albumin infusion before exchange transfusion in reducing post-exchange unconjugated serum bilirubin (UCB) levels in low birth weight (LBW) neonates with intensive phototherapy failure. METHODS: In a placebo-controlled Randomized Controlled Trial, 42 healthy LBW (birth weight between 1000 and 2499 g and gestational age ≥ 32 weeks) neonates were randomly allocated into intervention and control groups. Post-exchange UCB at 6 and 12 h were compared in the two groups along with the duration of post-exchange phototherapy, repeat-exchange requirement, adverse effects of albumin and hospital stay. RESULTS: The intervention group (n = 21) with mean birth weight 1619 ± 324 g, gestational age 34.5 ± 1.65 weeks, peak UCB 19 ± 3.85 mg dl(-1), was demographically comparable with the control group (n = 21) (1660 ± 320 g, 34 ± 1.6 weeks, 19.4 ± 3.59 mg dl(-1), respectively). Significant reduction in the post-exchange UCB (10.55 ± 1.53 mg dl(-1) at 6 h; 5.86 ± 1.21 mg dl(-1) at 12 h in albumin group; 15.26 ± 1.78 mg dl(-1) at 6 h; 11.69 ± 1.52 mg dl(-1) at 12 h in control group) and phototherapy duration (23.8 ± 3.2 h vs. 40.3 ± 7.2 h) was observed in the intervention group (p < 0.0001). Repeat exchange requirement was reduced by 86% (RR = 0.14; 95%CI: 0.19-1.06). Mean duration of hospital stay was significantly lower (10.1 ± 5.8 days vs. 12.4 ± 6.6 days) (p = 0.021). No albumin transfusion-related complications were observed.

Generalized epimerase deficiency galactosemia.

Galactosemia is caused by inherited deficiencies in one of three enzymes involved in the metabolism of galactose: galactose-1-phosphate uridyltransferase (GALT), galactokinase (GALK), and uridine diphosphate galactose-4-epimerase (GALE). The rarest and most poorly understood form of galactosemia is due to epimerase deficiency. We are reporting such a rarest form of galactosemia presenting with progressively increasing cholestatic jaundice and failure to thrive at one month of age. After confirmation of decreased epimerase level in RBC hemolysate, the patient was put on galactose restricted diet and vitamins supplementation, which reversed the clinical signs as well as altered liver function. Patient is on regular follow-up and now at 15 months of age he has no marked developmental delay.