Krishnaswamy Narayanan

Sustained delivery of exogenous melatonin influences biomarkers of oxidative stress and total antioxidant capacity in summer-stressed anestrous water buffalo (Bubalus bubalis)

High ambient temperature during summer in tropical and subtropical countries predisposes water buffaloes (Bubalus bubalis) to develop oxidative stress having antigonadotropic and antisteroidogenic actions. Melatonin is a regulator of seasonal reproduction in photoperiodic species and highly effective antioxidant and free radical scavenger. Therefore, a study was designed to evaluate the effect of sustained-release melatonin on biomarkers of oxidative stress i.e., the serum malondialdehyde (MDA) and nitric oxide (NO), and the total antioxidant capacity (TAC). For the study, postpartum buffaloes diagnosed as summer anestrus (absence of overt signs of estrus, concurrent rectal examination, and RIA for serum progesterone) were grouped as treated (single subcutaneous injection of melatonin at 18 mg/50 kg body weight dissolved in sterilized corn oil as vehicle, n = 20) and untreated (subcutaneous sterilized corn oil, n = 8). Blood sampling for estimation of serum TAC and MDA (mmol/L) and NO (μmol/L) was carried out at 4 days of interval from 8 days before treatment till 28 days after treatment or for the ensuing entire cycle length. Results showed serum TAC concentration was higher in the treatment group with a significant (P < 0.05) increasing trend, whereas MDA and NO revealed a significant (P < 0.05) decline. Serum MDA and NO were higher in control compared with those of treatment group. Moreover, buffaloes in the treatment group showed 90% estrus induction with 18.06 ± 1.57 days mean interval from treatment to the onset of estrus. These results report that melatonin has a protective effect by elevating antioxidant status and reducing oxidative stress resulting in the induction of cyclicity in summer-stressed anestrous buffaloes.

Supplementation of Slow‐Release Melatonin Improves Recovery of Ovarian Cyclicity and Conception in Summer Anoestrous Buffaloes (Bubalus bubalis)

The role of melatonin as a protective neurohormone against restoring cyclicity in summer anoestrous animals in photoperiod species has gained wider acceptance. This study was designed to uncover the evidence the slow-release melatonin (MLT) has on initiation of ovarian cyclicity and conception rate (CR) in summer anoestrous buffaloes. Thus, buffaloes diagnosed as summer anoestrous (absence of overt signs of oestrus, concurrent rectal examination and radioimmunoassay for serum progesterone at 10 days interval) were grouped as untreated (Group I, sterilized corn oil, n = 8) and treated (Group II, single subcutaneous injection of MLT @18 mg/50 kg bwt in sterilized corn oil, n = 20). Animals treated and detected in oestrus were artificially inseminated (AI) followed by division into Group III (second dose of MLT on 5th day post-AI, n = 8) and Group IV (no melatonin administration, n = 10). Blood samples were collected at 4 days interval for estimation of serum MLT, progesterone and oestrogen using radioimmunoassay kit. Mean oestrous induction rate (OIR), oestrous induction interval (OII), interoestrous interval (IOI) and CR were estimated. Compared to control, concentration of melatonin was significantly (p < 0.05) higher in treated group ranging from 14.34 ± 1.72 to 412.31 ± 14.47 pg/ml whereas other two hormones did not show any concentration difference. Melatonin-administered buffaloes showed significantly (p < 0.05) higher (90%) OIR with OII of 18.06 ± 1.57 days. Results showed improvement in conception rate in buffaloes administered with post-insemination melatonin. It can be concluded from the study that slow-release melatonin supplementation restored cyclicity in summer anoestrous animals resulting in improvement in conception rate in buffaloes.

Differential expression of ten candidate genes regulating prostaglandin action in reproductive tissues of buffalo during estrous cycle and pregnancy

Prostaglandins (PGs) are the key mediators of several female reproductive functions, including luteolysis, ovulation, fertilization, implantation, pregnancy, and parturition. The present study was conducted in buffalo endometrial and luteal tissues between nonpregnant and two stages of pregnancy (29-38 days of pregnancy, 48-56 days of pregnancy) tissue samples. The genes involved from synthesis upto receptor level effect of PGs (PGF2α and PGE2) were studied for their relative mRNA expression. We have collected the endometrial and luteal tissues from slaughtered animals and confirmed the stages by external examination and crown vertebral rump length measurement of the foetus. The mRNA expression of COX-2 and PGFS genes revealed high significant rise in the transcript at pregnancy stage I as compared to the late luteal phase of nonpregnant. However, EP2 and EP3 genes were highly upregulated in pregnancy stage II. The expression of PLA2G4A and PGT genes showed difference in their transcripts in pregnancy, however, the difference was nonsignificant as compared to the nonpregnant stage. The findings emerged from this study also suggested the strict regulation at COX-2 mRNA level than at synthase enzymes level. Among the four subtypes of EP gene, we have observed highly significant expression difference in EP2 followed by EP3 after implantation.

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.

Dietary fish oil supplementation improved ovarian function, conceptus growth and certain reproductive variables in Marwari mares

We investigated the effect of dietary fish oil supplementation on the development of the ovarian follicles, corpus luteum (CL), conceptus and certain reproductive events in Marwari mares, since it is reported to improve reproduction in cows. Accordingly, non-lactating mares (n = 20) were randomly assigned into two groups (10 per group) and fed either the control diet (CTR) or a diet enriched with fish oil (FOS) to supplement n-3 polyunsaturated fatty acids (PUFA) at the rate of 64 mg/kg body weight/day for 70 days or until 45 days postovulation in the mares that became pregnant. Estrus was detected using a teaser and insemination was performed using frozen thawed semen in the experimental mares. Development of the ovarian follicle, CL and conceptus were recorded using trans-rectal ultrasonography. Plasma concentrations of progesterone and estradiol were estimated by radioimmunoassay. In the FOS group, the diameter of the largest follicle from day 4 of estrus until ovulation, and the diameter of the CL on day 7 post-ovulation (D7PO) were greater (P<0.05). However, on day 15 post-ovulation (D15PO), the CL diameter increased significantly in the pregnant mares. Dietary fish oil significantly improved the development of the embryo as evidenced by an increase in the diameter of the embryonic vesicle on day 15 post-ovulation (D15PO), and the embryo proper on day 28 post ovulation (D28PO). *Corresponding author: Sanjay Kumar Ravi, PhD, Equine Production Campus, ICARNational Research Centre on Equines, P.B. No 80, Bikaner-334001, Rajasthan, India, Phone: +91 151 2232541; Fax: +91 151 2230 114; E-mail: skravivet@gmail.com DOI: 10.24099/vet.arhiv.0056

Transcriptional and translational abundance of Bone morphogenetic protein (BMP) 2, 4, 6, 7 and their receptors BMPR1A, 1B and BMPR2 in buffalo ovarian follicle and the role of BMP4 and BMP7 on estrogen production and survival of cultured granulosa cells

BMPs and their receptors modulate the granulosa cell (GC) function in the follicle of domestic animals. Since little is known on BMPs in the buffalo, the present study was aimed to investigate the expression of BMP2, 4, 6, 7 and their receptors BMPR1A, BMPR1B, BMPR2 in the GC and theca cells (TC) of ovarian follicles and the role of BMP4 and BMP7 on buffalo GC. Follicles were classified into four groups based on size and E2 level in the follicular fluid as follows: (i) Group1(4-6 mm; <0.5 ng/mL) (ii) Group 2 (7-9 mm; 0.5-5 ng/mL) (iii) Group 3 (10-13 mm; 5-40 ng/mL) and (iv) Group 4 (dominant follicle) (>13 mm; >180 ng/mL). The results revealed that except BMP6, BMP2, 4 7 and receptors BMPR1A, BMPR1B and BMPR2 showed a minimum of 1.5-2 fold increase in mRNA expression in the GC of dominant follicle as compared to other follicle classes. In the dominant follicle, a two-fold increase in BMP4 and BMP7 expression was observed in the TC. At 100 ng/mL, the BMP4 and BMP7 either alone or in combination maximally down-regulated CASPASE3 and stimulated the transcripts of PCNA, FSHR and CYP19A1 that was supported by E2 secretion in the granulosa cell culture suggesting their role in cell survival and E2 production. In conclusion, GC and TC of dominant follicles express BMP 2, 4, 6, 7 and their receptors BMPR1A, BMPR1B and BMPR2. BMP4 and BMP7 stimulate E2 production and promote GC survival.

Expression and functional role of Bone Morphogenetic Proteins (BMPs) in cyclical corpus luteum in buffalo (Bubalus bubalis)

The role of growth factors in the modulation of ovarian function is an interesting area of research in reproductive biology. Recently, we have shown the expression and role of IGF, EGF, VEGF and FGF in the follicle and CL. Here, we report the presence of Bone Morphogenetic Proteins (BMPs) and their functional receptors in the corpus luteum (CL) of buffalo. The bubaline CL was classified into four stages according to the morphology and progesterone (P4) concentration. The qPCR, immunoblot and immunohistochemistry studies revealed that BMP2 and BMP Receptors (BMPR1A, BMPR1B and BMPR2) were significantly upregulated during the mid stage whereas BMP4 and BMP7 were upregulated during the early stage of CL (P<0.05). Studies on primary luteal cell culture (LCC) using mid CL showed a significant time and concentration dependent effect of BMP4 and BMP7 (P<0.05). At 100ngml-1, the BMPs maximally stimulated the transcripts of StAR, CYP11A1 and 3βHSD that paralleled with P4 accretion in the media (P<0.05). Further, the BMP4 as well as BMP7 upregulated the transcripts of PCNA and downregulated CASPASE3 in the LCC at the same concentration (P<0.05). Though the combined effect of BMP4 and 7 was significantly higher (P<0.05) than that of individual one, it was not additive. In conclusion, the expression of BMPs and their receptors were dependent on the stages of CL in the buffalo. Treatment of LCC with BMPs in vitro confirmed the presence of functional receptors that stimulated the P4 production and luteal cell survival. Moreover, the results support the concept that the upregulation of P4 and its biosynthetic pathway enzymes such as CYP11A1, StAR and 3βHSD in the CL is likely due to the autocrine and /or paracrine effects of BMP4 and BMP7 under physiological milieu.

Effect of CIDR on Conception Rate in Repeat Breeding Cattle Inseminated with Liquid Semen

A trial was conducted to study the effect of CIDR on conception rate (CR) in repeat breeding (RB) Vrindavani cows following insemination with liquid semen. After insemination with liquid semen, the experimental RB cows were divided into two groups: group-I (n=13) was treated with CIDR between day 5 and 18 post-insemination, while group-II (n=12) was kept as negative control. Pregnancy diagnosis was done on day 60 post-insemination by per rectal palpation. Conception rate (%) in CIDR treated group was 5/13 (38.46%), whereas, it was 2/12 (16.67%) in the control group. Though the increase of CR by 21.79% in the CIDR treated group was statistically non-significant as compared to the control.