Polymorphism in 5' untranslated region of heat-shock protein 70 gene as marker of post-partum anoestrus in Murrah buffaloes.

The enormous production potential of buffaloes has never been accomplished due to various reproductive insufficiencies. Among them, post-partum anoestrus, a multifactorial disorder, is predominant but any genetic association is yet to be established. This study focused to identify novel polymorphisms in heat-shock protein 70 (HSP70) gene and its possible association with post-partum anoestrus in Murrah buffaloes. A 579-bp fragment from 5' untranslated region of HSP70 gene was amplified by polymerase chain reaction from blood genomic DNA of 614 animals maintained under similar management conditions. In phase-I experiment, custom sequencing and restriction enzyme (RE) digestion of the amplified fragment were performed in 40 buffaloes with similar post-partum oestrous conditions over previous consecutive three or more gestations-20 animals each showing post-partum anoestrus (>120 days after parturition) and normal cyclicity (<65 days after parturition). While in phase-II experiment, herd screening by RE analysis was performed in remaining 574 animals. Four transversions at T-75G, C+31G, T+38G and C+97A and three transition mutations at T-153C, T+33C and A+44G positions were observed. Polymorphism at T+38G site revealed significant (p < .05) variation, where homozygous G was present only in post-partum anoestrous animals while nucleotide T was present randomly in both groups of phase-I animals whereas phase-II experiments revealed homozygous G in 55 animals. Regression analysis in relation to average post-partum interval against genotypic frequencies at T+38G also depicted significant association. HSP70 gene polymorphism at T+38G position can therefore be used as genetic marker for excluding probable post-partum anoestrous buffaloes from herd for breeding programmes.

Serum MX2 Protein as Candidate Biomarker for Early Pregnancy Diagnosis in Buffalo.

Interferon-tau (IFN-τ)-induced molecular markers such as ubiquitin-like modifier (ISG15), 2',5'-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance genes (MX1 and MX2) have generated immense attention towards developing diagnostic tools for early diagnosis of pregnancy in bovine. These molecules are expressed at transcriptional level in peripheral nucleated cells. However, their presence in the serum is still a question mark. This study reports sequential changes in expression of MX2 transcript in whole blood and serum MX2 protein level on days 0, 7, 14, 21, 28 and 35 in pregnant (n = 9) buffalo heifers, and on days 0, 7 and 14 in non-inseminated (n = 8) and inseminated non-pregnant (n = 10) control animals. In non-inseminated and inseminated non-pregnant heifers, the differential expression of MX2 transcript and MX2 protein level remained similar between day 7 and 14 post-oestrus. However, in pregnant heifers, on 14th and 28th day post-insemination MX2 transcript was 16.38 ± 1.57 and 28.16 ± 1.91 times upregulated as compared to day 0. Similarly, serum MX2 protein concentration followed analogous trend as MX2 transcript and increased gradually with the progression of pregnancy. Correlation analysis between expression of MX2 transcript and its serum protein level showed a significant positive correlation in pregnant animals, while it was random in other two groups. Therefore, MX2 surge at transcriptional and serum protein level after day 14-28 of pregnancy in buffalo holds potential for its use in early pregnancy detection.

Urinary metabolomics reveals potential biomarkers for early detection of pregnancy in Mithun (Bos frontalis) cows.

The present study investigated the urinary metabolic profiles of early pregnant and non-pregnant Mithun to identify potential pregnancy detection biomarkers. Urine samples were collected on days 0, 10, 18, 35 and 45 of gestation from pregnant (n = 6) and on days 0, 10 and 18 from non-pregnant (n = 6) Mithun. Urinary metabolites were assessed using proton nuclear magnetic resonance (1H NMR) spectroscopy and identified 270 metabolites. Statistical analyses demonstrated pronounced distinctions in metabolite profiles between pregnant and non-pregnant samples. Twenty-five metabolites that could discriminate between pregnant and non-pregnant Mithun based on Variable Importance in Projection (VIP) scores >1 were identified. Upon further examination of six metabolites (kynurenine, kynurenate, 3-hydroxykynurenine, quinolinate, tyrosine and leucine) identified with high VIP scores, ROC curve analyses demonstrated their significant predictive potential, with AUC values ranging between 0.50 and 0.85. Additionally, a combined panel of top 25 metabolites yielded an AUC value of 0.85. Pathway analysis identified seven potential metabolic pathway modulations during early gestation, with particular emphasis on phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan pathway and pathways involved in the metabolism of various amino acids. In conclusion, kynurenine, kynurenate, 3-hydroxykynurenine, quinolinate, tyrosine, and leucine show promise as non-invasive urinary biomarkers for early pregnancy detection in Mithun. SIGNIFICANCE: This study presents the first report on the metabolic profile of urine from early pregnant and non-pregnant Mithun (Bos frontalis). The metabolites like kynurenine and its derivatives (kynurenate, 3-hydroxykynurenine and quinolinate), tyrosine and leucine were documented signature urinary metabolites associated with early pregnancy in Mithun. The identified combination of metabolites holds promise as predictive biomarkers for non-invasive urinary-based early pregnancy diagnostics in Mithun. In addition, this study identified changes in metabolic pathways that involve phenylalanine, tyrosine, tryptophan and related amino acids and biomarkers identified were either precursors or products within these metabolic pathways.

Exploration of urinary metabolite dynamicity for early detection of pregnancy in water buffaloes.

Early and precise pregnancy diagnosis can reduce the calving interval by minimizing postpartum period. The present study explored the differential urinary metabolites between pregnant and non-pregnant Murrah buffaloes (Bubalus bubalis) during early gestation to identify potential pregnancy detection biomarkers. Urine samples were collected on day 0, 10, 18, 35 and 42 of gestation from the pregnant (n = 6) and on day 0, 10 and 18 post-insemination from the non-pregnant (n = 6) animals. 1H-NMR-based untargeted metabolomics followed by multivariate analysis initially identified twenty-four differentially expressed metabolites, among them 3-Hydroxykynurenine, Anthranilate, Tyrosine and 5-Hydroxytryptophan depicted consistent trends and matched the selection criteria of potential biomarkers. Predictive ability of these individual biomarkers through ROC curve analyses yielded AUC values of 0.6-0.8. Subsequently, a logistic regression model was constructed using the most suitable metabolite combination to improve diagnostic accuracy. The combination of Anthranilate, 3-Hydroxykynurenine, and Tyrosine yielded the best AUC value of 0.804. Aromatic amino acid biosynthesis, Tryptophan metabolism, Phenylalanine and Tyrosine metabolism were identified as potential pathway modulations during early gestation. The identified biomarkers were either precursors or products of these metabolic pathways, thus justifying their relevance. The study facilitates precise non-invassive urinary metabolite-based pen-side early pregnancy diagnostics in buffaloes, eminently before 21 days post-insemination.

New approaches in buffalo artificial insemination programs with special reference to India.

Buffalo farming has made remarkable progress in productivity mainly because of controlled breeding with artificial insemination (AI) that has proved its worth in breed improvement and breeding managements across the livestock species. Artificial insemination is practiced very little in Europe and East Asian countries with coverage of only 5% buffaloes in Italy, 3.7% in Azerbaijan, 0.3% in Egypt, and 0.1% in Romania although in Bulgaria, 80% buffaloes in large cooperative state farms are subjected to AI. In Turkey, it began in 2002 near Hatay with Italian semen provided by the Food and Agriculture Organization (FAO) Network project. In India, where buffaloes are the most valuable livestock species, research on buffalo specific artificial breeding technologies and adoption of AI by buffalo owners are widely acknowledged. Resultantly, average milk yield of buffaloes in India increased from 3.4 kg in 1992 to 93 to 4.57 kg/day/buffalo in 2009 to 10. In the new millennium, mega projects such as the National Project for Cattle and Buffalo Breeding and the National Dairy Plan were initiated with focus on genetic upgradation of bovine and buffalo population through streamlining AI services and support system in the country. Artificial insemination started in India in the year 1939, and the frozen semen was introduced during late 1960s. During the year 2010 to 11, India produced 63 million bovine frozen semen straws including over one million buffalo semen straws through 49 semen stations. Artificial insemination services are provided through 71,341 AI stations clocking 52 million inseminations with overall conception rate of 35% in bovine and buffalo population. Research is being conducted for improved AI conception rates with synchronization programs and improved frozen-thawed semen quality, and success rates are at par with AI in cattle.

Developmental competence and expression profile of genes in buffalo (Bubalus bubalis) oocytes and embryos collected under different environmental stress.

The study examined the effects of different environmental stress on developmental competence and the relative abundance (RA) of various gene transcripts in oocytes and embryos of buffalo. Oocytes collected during cold period (CP) and hot period (HP) were matured, fertilized and cultured in vitro to blastocyst hatching stage. The mRNA expression patterns of genes implicated in developmental competence (OCT-4, IGF-2R and GDF-9), heat shock (HSP-70.1), oxidative stress (MnSOD), metabolism (GLUT-1), pro-apoptosis (BAX) and anti-apoptosis (BCL-2) were evaluated in immature and matured oocytes as well as in pre-implantation stage embryos. Oocytes reaching MII stage, cleavage rates, blastocyst yield and hatching rates increased (P < 0.05) during the CP. In MII oocytes and 2-cell embryos, the RA of OCT-4, IGF-2R, GDF-9, MnSOD and GLUT-1 decreased (P < 0.05) during the HP. In 4-cell embryos, the RA of OCT-4, IGF-2R and BCL-2 decreased (P < 0.05) in the HP, whereas GDF-9 increased (P < 0.05). In 8-to 16-cell embryos, the RA of OCT-4 and BCL-2 decreased (P < 0. 05) in the HP, whereas HSP-70.1 and BAX expression increased (P < 0.05). In morula and blastocyst, the RA of OCT-4, IGF-2R and MnSOD decreased (P < 0.05) during the HP, whereas HSP-70.1 was increased (P < 0.05). In conclusion, deleterious seasonal effects induced at the GV-stage carry-over to subsequent embryonic developmental stages and compromise oocyte developmental competence and quality of developed blastocysts.

Differences in developmental competence and gene expression profiles between buffalo (Bubalus bubalis) preimplantation embryos cultured in three different embryo culture media.

The objective of this study was to compare effects of in vitro culture systems on embryonic development and expression patterns of developmentally important genes in preimplantation buffalo embryos. After IVM/IVF presumptive zygotes were cultured in one of three systems: undefined TCM-199, mCR2aa medium supplemented with 10 % FBS and defined PVA-myo-inositol-phosphate-EGF medium. No (P > 0.05) differences at 2-cell, 4-cell and 8-cell to 16- cell stages were observed among the three cultured media used, however, increased (P < 0.05) blastocyst yield, cell number and hatching rate were found in defined medium compared to undefined media. The expression patterns of genes implicated in embryo metabolism (GLUT-1), anti-apoptosis (BCL-2), imprinting (IGF-2R), DNA methylation (DNMT-3A) and maternal recognition of pregnancy (IFNT) were increased (P < 0.05) in hatched blastocysts derived from defined medium compared to undefined media. In conclusion, serum-free, defined medium improved developmental competence of in vitro cultured buffalo embryos. Whether these differences in morphological development and gene expression have long-term effects on buffalo calves born after embryo transfer remains unknown. However, it is possible that early adaptations of the preimplantation embryo to its environment persist during fetal and post-natal development.

Manipulation of reproductive performance of lactating buffaloes using melatonin and controlled internal drug release device treatment during out-of-breeding season under tropical conditions.

Twelve lactating Murrah buffalo, divided into control and treatment group of six animals each, were used to study the effect of melatonin and controlled internal drug release (CIDR) device treatment on the resumption of ovarian activity during out-of-breeding season (summer solstice). Treated group implanted with melatonin (18-mg melatonin/50-kg body weight) for 45 days and then animals of both groups received CIDR for 9 days. All animals received intramuscular 500 IU eCG, at day before CIDR removal, and 10-µg GnRH at day after CIDR withdrawal. All animals were subjected to estrus detection daily. Blood samples in conjunction with transrectal ultrasonography were performed once a week to determine serum concentrations of melatonin, progesterone, and antioxidant enzyme activities, as well as to monitor the ovarian activity. Melatonin treatment resulted in an increase (P < 0.01) in the overall mean superoxide dismutase activity. Melatonin and CIDR increased the diameter of CL (P < 0.01) and plasma progesterone concentration (P < 0.05). In addition, melatonin and CIDR exhibited superior ability to maintain presence of CL at Day 21 and Day 30 after artificial insemination and achieved higher percentage of conception rate than control. In conclusion, the CIDR treatment preceded by melatonin improved the reproductive performance in lactating buffaloes during out-of-breeding season under tropical conditions.

Relation between antioxidant status and postpartum anestrous condition in Murrah buffalo.

AIM: Objective of the present study was to investigate the relation between antioxidant status and postpartum anestrous (PPA) condition in Murrah buffalo. MATERIALS AND METHODS: Jugular blood samples were collected from two different groups of Murrah buffaloes each group consisting of 20 animals. Group I was of PPA and Group II were of cyclic buffaloes. The animals selected were examined for confirmation for cyclic and acyclic condition (>120 days) after calving by routine transrectal ultrasonography. Heard record was also used for cross confirmation. RESULTS: The analysis of antioxidants in plasma and hemolysates revealed that the levels of vitamin E, ß-carotene and reduced glutathione in plasma and superoxide dismutase (SOD) in hemolysate were significantly higher in cyclic animals than PPA animals. The levels of vitamin C, SOD and glutathione peroxidase in plasma did not show any significant difference among the two groups studied. The low antioxidant level in affected animals may predispose them toward PPA condition. CONCLUSION: Stress imposed by pregnancy and lactation affected the reproductive performance in PPA animals which might be inherently more susceptible to these stressors than those who were normal cyclic as all the animals were maintained under similar feeding and management practices.

Liposome-based semen extender is suitable alternative to egg yolk-based extender for cryopreservation of buffalo (Bubalus bubalis) semen.

Demand for alternative of egg yolk in freezing extenders have increased in recent years due to variability in egg yolk composition, risk of microbial contamination and presence of steroid hormones. The alternative to egg yolk-based extender (EY) can be soya lecithin-based extender (SL) and liposome-based extender (LP). However, the efficacy of SL is still a matter of debate. Few studies have been performed on the effect of LP but to date evaluation of buffalo semen cryopreserved in LP has not been studied. Therefore, this study was designed to compare SL and LP with conventional EY for evaluation of post-thaw quality of buffalo semen. Results showed that total, progressive and rapid sperm motility were found significantly higher (P<0.05) in LP among these extenders. In vitro assessment of post-thaw sperm longevity has also resulted in better maintenance of sperm kinetics and motility in LP in comparison to other extenders. Furthermore, sperm cryopreserved in LP travelled significantly more (P<0.05) distance in cervical mucus as compared to SL and EY. Therefore, it can be concluded that the LP is more efficient than SL and EY for the cryopreservation of buffalo semen.

Hemato-biochemical and hormonal profiles in post-partum water buffaloes (Bubalus bubalis).

AIM: The objective of the present study was to compare serum as well as follicular fluid (FF) biochemical and hormonal profiles along with hematological parameters in postpartum estrus, anestrus, and cystic buffaloes. MATERIALS AND METHODS: Postpartum buffaloes were selected in three different groups (within 40-60 days of parturition at estrus-Group-I, postpartum >90 days at anestrum-Group-II, and postpartum cystic buffaloes in Group III). The animals selected were examined for follicular wave dynamics by routine trans-rectal ultrasonography and FF was collected by transvaginal ultrasound-guided ovum pick up technique. All hematological and biochemical parameters were analyzed by automatic analyzers while hormonal profiles analyzed by commercially available ELISA kits. RESULTS: In the present investigation, estrum and anestrum animal differ significantly in hemoglobin levels. Serum estradiol differs significantly in estrus and anestrus while no significant difference in progesterone concentration was noted among all three stages. The results of our study suggest that significant higher increase in total protein (TP), calcium and glucose values in estrum while urea, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase significantly higher in anestrum animals. CONCLUSION: The conclusion of the present study is that TP and albumin, calcium, urea, glucose affects oocyte development and quality.

Effectiveness of melatonin and controlled internal drug release device treatment on reproductive performance of buffalo heifers during out-of-breeding season under tropical conditions.

Sixteen Murrah buffalo heifers, divided into control and treatment groups of eight animals each, were used to study the effect of melatonin and controlled internal drug release (CIDR) device treatment on the resumption of ovarian activity during out-of-breeding season (summer solstice). Treated group was implanted with melatonin (18 mg of melatonin per 50 kg of body weight) for 45 days and then heifers of both groups received CIDR for 9 days. All heifers received intramuscular 500 IU eCG on the day before CIDR removal and 10 µg GnRH on the day after CIDR withdrawal. All animals were subjected to estrus detection daily. Blood sampling in conjunction with transrectal ultrasonography were performed twice weekly to determine serum concentrations of melatonin, progesterone, LH, and antioxidant enzyme activities, as well as to monitor the ovarian follicular activity. Melatonin treatment resulted in an increase (P < 0.01) in serum melatonin and a decrease (P < 0.01) in serum progesterone and LH. In addition, melatonin had no significant effect on the frequency of LH pulses. Furthermore, melatonin treatment increased (P < 0.01) the diameter of the largest follicle and the number of large follicles between Days 0 and 35 of melatonin treatment. However, melatonin exhibited superior ability to maintain CL at 21 days after artificial insemenation (AI) and increased the percentage of conception to threefold higher than control. In conclusion, melatonin implantation successfully improved the diameter of largest follicles and the ability to maintain CL at 21 days after AI in buffalo heifers during out-of-breeding season under tropical conditions.