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Estrus or sexual receptivity determination is utmost important for efficient breeding programs for female buffaloes. Prominent estrus behavioral symptoms are the result of several molecular and neuroendocrine events involving the ovary and the brain. Expression of estrus behavior is poor in buffaloes during the summer season. Hence, the discovery of biomarkers specific to the estrus stage or its related ovarian events, like the presence of dominant ovarian follicle, is helpful for developing an easy estrus determination method. MicroRNA are small non-coding RNA with a potential to be biomarkers. Therefore, the present study targeted to investigate the potential of estrogen responsive miRNAs (miR-24, miR-200c, miR-16, miR-191, miR-223 and miR-203) as estrus biomarkers in buffalo saliva, a non-invasive fluid representing animals' pathophysiology. There was a significant (P < 0.05) increase in the salivary presence of the miR-16, miR-191 and miR-223 at 6th and 18th-19th days than the 0 day (estrus), 10th day and the following consecutive estrus day. These observations may indicate an association between the representative lower presence of these miRNA in saliva and the presence of dominant ovarian follicles. To test this association, pathway analysis, target gene identification, functional annotation and protein-protein interaction networks (PPI) were performed for miR-16, miR-191 and miR-223 by different bioinformatics tools. Interestingly, the top pathways (fatty acid biosynthesis and oocyte meiosis), target genes (FGF, BDNF and IGF1) and PPI hub genes (KRAS, BCL2 and IGF1) of these miRNAs were found essential for ovarian follicular dominance. In conclusion, the miR-16, miR-191 and miR-223 may not be the perfect estrus stage-specific biomarkers. However, their lower presence in saliva at estrus and 9th-10th day of estrous cycles, when the ovary usually has a dominant follicle in buffaloes, may intuitively indicate the follicular dominance. Further studies are needed to prove this association in a large population.

This work was conducted to describe the morphological characters of the tongue of Egyptian water buffalo (Bubalus bubalis). The lingual root and the dorsal middle region of apex and body in addition to the dorsal and ventral surface of lingual tip were devoided from any fungiform papillae. The lingual tip contains conical papillae only. The ventral surface of lingual apex was divided into two portions by the U-shaped fungiform line into papillary and non-papillary region. Histological investigation on the lingual surface epithelium and lamina propria submucosa reflects differences in these layers in different parts of the tongue. By SEM, there are two subtypes of filiform papillae: caudally directed papillae on dorsal surface and rostrally directed papillae on the lateral region of ventral surface of lingual apex. There are two subtypes of conical papillae: small slightly rostrally directed papillae on dorsal and ventral surface of lingual tip and large posteromedially directed papillae on dorsal surface of lingual root. The rounded circumvallate papillae consisted of round bulb surrounded by deep circular groove, which surrounded by circular pad. Higher magnification of filiform papillae indicates the presence of microcrests separated by microgrooves, and these microgrooves consisted of microrodes. The fungiform surface having micropores on the tip of elevated tubercle for taste buds pores. All these observed structures (microcrests, microgrooves, microrodes, tubercles, microridges) in a higher magnification allow animals to transport food particles through the oral cavity and help in the defensive behaviour. There are strong correlations between the tongue anatomical characteristics and its functions., (© 2017 Blackwell Verlag GmbH.)

This study was conducted to test the efficacy of gonadotropic hormone (GnRH)-based synchronization protocols (Ovsynch, Heatsynch, and Ovsynch Plus) in buffaloes under field condition. Based on anamnesis and transrectal palpation twice at 10-day interval and serum progesterone (P 4 ) concentration, 150 anoestrous buffaloes and delayed pubertal heifers were selected to induce oestrus using GnRH-based protocols. These selected animals were randomly divided into three groups: group I: Ovsynch (n = 50), group II: Heatsynch (n = 50), and group III: Ovsynch Plus (n = 50) regimen. Before treatment initiation, blood samples were collected for P 4 , beta-hydroxy butyric acid (β-OHB), and mineral estimation, in addition to the monitoring of oestrus signs. In this investigation, no significant difference (P > 0.05) in oestrus signs was deduced among three groups. Oestrus induction rate (OIR) was comparable (P > 0.05) among the groups (Ovsynch 82%, Heatsynch 86%, and Ovsynch Plus 88%). Conception rate (CR) following fixed time artificial insemination (FTAI) was slightly higher with Ovsynch Plus group (28%) as compared to Ovsynch (24%) and Heatsynch (18%) groups, though non-significant. Furthermore, serum glucose, β-OHB, macrominerals (calcium, potassium, and magnesium), and trace minerals (copper, zinc, and iron) remained comparable (P > 0.05) among the groups. In conclusion, all the protocols (Ovsynch, Heatsynch, and Ovsynch Plus) are efficient in oestrus induction in anoestrous buffaloes under field condition with Ovsynch Plus protocol resulting in higher CR as compared to other protocols.

Buffalo, the most important livestock species in tropical India, remains to be a poor breeder mainly due to embryonic mortality (65%) occurring mostly between 16 and 18 days of pregnancy. Early and accurate diagnosis of pregnancy can thus become a boon for successful herd management in buffalo. However, most of the currently available methods allow diagnosis only after 30 days post AI. Interferon tau (IFNT), the first pregnancy recognition signal in ruminants is one such molecule, which stimulates expression of various Interferon stimulated genes (ISGs) in the peripheral blood mononuclear cells (PBMC's) concomitant with IFNT signaling which occurs around maternal recognition of pregnancy (MRP). Hence, the study was planned to demonstrate the expression dynamics of ISGs (OAS1, MX1, MX2 and ISG15) in PBMCs during peri-implantation period in buffalo and also molecular cloning and expression of suitable ISG coded protein (s) in suitable host. Blood was collected from two groups of multiparous buffaloes: Group1: (n = 10) inseminated/pregnant (Experimental) and Group2: (n = 10) anestrous/non pregnant (Control). The expression profile of ISGs was then analyzed using real time qPCR. Expression profile of most ISGs was observed to increase through day 14 to day 20 post AI and declined thereafter. On the basis of differential gene expression at day 18 post AI, OAS1 and MX2 were identified as suitable ISG candidate biomarkers for accurate pregnancy diagnosis within 18 days post AI. Molecular cloning and expression of selected ISGs in a suitable prokaryotic expression vector was done thereafter. Bulk expression of the recombinant proteins was done and purified by affinity chromatography and confirmed by Western blot using Mouse Monoclonal His-probe antibodies. To conclude, as OAS1 and MX2, showed distinct differential expression at day 18 post AI, they may serve as ideal biomarkers for detection of early pregnancy in buffalo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lysophosphatidic acid (LPA) is an important factor involved in embryo implantation and pregnancy establishment in humans and domestic livestock. LPA exerts its action through six G-protein-coupled receptors (LPA1-LPA6). We investigated the types of LPA receptors expressed in buffalo uterus and also their differential expression in the nonpregnant, and early-pregnant endometrium. The nonpregnant, and early-pregnant (<42 days) uteri were collected from the local slaughterhouse. RT-PCR experiments detected mRNAs of all the six LPA receptors (LPAR1-LPAR6) in both nonpregnant, and early-pregnant endometrial tissues. Their comparative profiling by real-time PCR revealed that the early pregnant endometrium expressed more mRNAs of LPAR1 and LPAR6. All the mRNA fragments were sequenced and submitted to Genbank, NCBI. Western blot studies also showed a similar expression pattern of these two receptor proteins, including higher expression of both LPA1 and LPA6 proteins during early pregnancy. And between these two receptors, LPA6 upregulation was more pronounced than LPA1. In immunohistochemistry, these receptors were found to be localized in the endometrial glandular epithelial cells of both types of uterus. Level of LPA was also higher in early pregnant endometrial tissues. In summary, our study demonstrated expression of all the six LPAR mRNAs in buffalo uterus, wherein the early-pregnant uterus did express comparatively higher mRNA as well as protein of LPA1 and LPA6, indicating their role in pregnancy. The more pronounced expression of LPA6 possibly indicates its greater contribution to mediating LPA signaling in early pregnancy (29-42 days) of buffalo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The current study was aimed to investigate the impact of THI on productive and reproductive indices of PE and F 1 crosses (50% PE and 50% Italian buffaloes) and back crosses (BC) (75% PE and 25% Italian buffaloes) under hot conditions. In this study, 8385 records used PE (1914, 1518, and 1737), F 1 (387, 447, and 657), and BC (495, 585, and 645) for low, medium, and high THI, respectively. The high THI reduced the conception rate after first insemination in PE, F 1 , and BC ((odds ratio, OR) = 1.187, P = 0.007; 2.361, <0.0001 and 1.603, <0.0001, respectively) when compared with low THI. But, stillbirth and calving condition were not significantly influenced by different THI levels in BC and F 1 . BC was more bearable to the harsh environment; they possessed the highest incidence of conception after first insemination (72.70, 72.60, and 62.40%), producing live calves (98.50, 100, and 99.40%) with easy calving condition (98.50, 100, and 99.40%) when compared to PE and F 1 at different levels of THI, respectively. The average daily milk yield and peak of milk production were decreased in PE (4.02 and 5.12%), BC (13.33 and 10.95%), and F 1 (25.29 and 12.20%) from low to high THI. However, BC revealed no significant changes in days open, dry period, calving interval, gestation length, service per conception, and the first service post-partum at different levels of THI when compared with PE and F 1 . Therefore, rearing BC is recommended for improving buffalo productive and reproductive performance under hot conditions.

This article reports genetic analysis of the weight at different ages of Murrah water buffaloes, using random regression models (RRM). Models ranging from third to sixth order polynomial were used to describe direct genetic and animal permanent environmental effects. Contemporary group was included as a fixed effect, and a cubic polynomial was used to model the mean curve of the population. The residual was modeled considering a log-linear function. Two models were selected for study of genetic parameters. The first model included third and sixth order polynomials for direct genetic and animal permanent environmental effects (M36). The second model included sixth order polynomials for all random effects (M66). The estimates of heritability varied from 0.16 + 0.04 (44 days) to 0.38 + 0.04 (568 days) for model M36 and from 0.16 + 0.05 (33 days) to 0.42 + 0.05 (600 days) for model M66. Regarding estimates of the correlation for all effects, the magnitude tended to decline with the increase of the time span between measurements. These results indicate that the species has potential for genetic selection based on weight at different ages, since we found favorable genetic variability within the herd, with selection likely to be more efficient at ages near 600 days., (© 2016 Japanese Society of Animal Science.)

The excellent adaptation of water buffalo (Bubalis bubalis) to swampy environments means that animals are frequently managed in areas with restricted access for reproductive procedures. The objective of this study was to evaluate the effect of the ovum pick-up (OPU) interval on follicular population, oocyte recovery, oocyte quality and in vitro embryo production. Twelve Murrah buffaloes were subjected to two consecutive dominant follicle reductions, and randomly assigned to either 7-day (n=6) or 14-day (n=6) OPU interval groups. Although there was no significant difference in the average number of small (<3mm) and large (>8mm) diameter follicles available per OPU, a higher proportion of medium-sized follicles (3-8mm) were observed in the 14-day interval group (5.129 vs 3.267; p<0.05). The number of recovered oocytes per donor was also significantly higher (4.51 vs. 2.8; p<0.05) in the 14-day interval group, although this was attributed to an increase in the proportion of lower quality oocytes (grades III and IV). After in vitro fertilization, embryo developmental competence from grade I and II oocytes was superior to that from grade III and IV oocytes, irrespective of OPU interval group. There was no significant difference in the proportion of grade I and II oocytes cleaved after sperm co-incubation; however, there was a higher proportion of blastocysts produced in 14-day interval group (28 vs. 6%, p<0.05). No blastocysts were produced from grade III and IV oocytes. This study indicates it is possible to use a 14-day interval for oocyte collection in water buffalo; this approach could be considered as an alternative when access to animals is restricted., (Copyright © 2017 Elsevier B.V. All rights reserved.)

For the first time in literature this study describes the pregnancy-associated glycoprotein (PAG) profile of buffalo cows during gestation and the post-partum period using antiserum raised against PAG-molecules purified from buffalo placenta (AS#860). Ninety-eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and then biweekly until the end of pregnancy. Pregnancy was confirmed by ultrasonography on days 28 and 45, and by rectal palpation from day 60 onwards. Blood samples were suspended for the non-pregnant cows on day 45, while the blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 post-calving. A cut-off value of 1.0 ng/mL was used in order to discriminate between pregnant and non-pregnant buffaloes. We used Linear Mixed models after Log(x+1) transformation to analyse the PAG concentrations. Fifty-two buffalo cows had become pregnant out of 98 synchronized (53%) and 46 remained non-pregnant (47%) as shown by ultrasonography and the PAG analysis. Significant differences (P < 0.001) in PAG concentrations were observed between the pregnant and non-pregnant buffaloes from day 23 as the PAG of the non-pregnant cows was always close to zero. Conversely, the PAG of the pregnant cows increased progressively from day AI until day 105 post-insemination and then stabilized until the end of pregnancy. Regarding pregnancy diagnosis, the sensitivity of PAG-RIA 860 system (ability of the test to correctly identify pregnant buffalo) ranged from 23% on day 23-98% on day 28 post AI; the specificity (ability to correctly identify non-pregnant buffaloes) was 100% throughout the sampling period. PAG progressively decreased from parturition to day 25 post-partum; from day 30 post-partum, the concentrations fell below 1 ng/mL and were close to 0 on the last day of observation (50 d post-partum). In conclusion, our results showed that RIA-860 is highly accurate for diagnosing pregnancy in buffaloes starting from day 28 of gestation. Furthermore, the rapid disappearance of PAG concentration after calving means that a cut-off limit in post-partum for detecting a new pregnancy is not required., (Copyright © 2017. Published by Elsevier Inc.)

Sperm sexing through flow-sorting technology is relatively expensive, requires considerable technical support and is actually not practicable in many developing countries. The aim of this study was to investigate the feasibility of producing enriched pools of X or Y chromosome-bearing sperm by a modified swim-up method. For this purpose semen was collected from five mature Nili-Ravi buffalo bulls for a period of six weeks. The qualifying ejaculates were divided into two aliquots for further processing through modified swim-up or control (untreated). After processing, semen was cryopreserved in tris citric acid extender using standard techniques. Semen quality was assessed at pre dilution, post dilution and post thawing. Validation of technique was done by using SYBR ® green real time PCR using two sets of primers, PLP and SRY for X and Y chromosome of buffalo genes, respectively. Sperm recovery rates, pre freeze and post thaw sperm quality were found significantly higher in X chromosome bearing sperm fraction than Y chromosome bearing fraction and control. Mean fold relative expression of X bearing sperm was significantly higher (4-5 fold) in X chromosome bearing fraction of supernatant than Y chromosome bearing fraction (0.06 fold), similarly mean fold relative expression of Y chromosome bearing sperm was significantly higher in Y chromosome bearing fraction (4 fold) of supernatant than X chromosome bearing fraction (0.15 fold) compared to control (1.00). In conclusion, a modified swim up method proved to be an effective method for Nili-Ravi buffalo sperm sexing as validated by real time PCR., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present study aimed to evaluate the effect of organic and inorganic selenium (Se) supplementation on semen quality and blood serum profiles of buffalo bulls. Nine mature buffalo bulls were divided into three groups: control (non-supplemented); organic Se (10 mg Sel-Plex®/head twice weekly) and inorganic Se (10 mg sodium selenite/head twice weekly). Semen was collected twice a week for 3 months during Se supplementation. Semen properties were evaluated from fresh ejaculate. Moreover, fructose concentration, aspartate and alanine transaminase (AST and ALT) activities, total protein and total cholesterol were assayed in seminal plasma. Additionally AST, ALT, testosterone and Se levels were determined in the blood serum. Results showed that Se supplementation significantly (P < 0.05) influences the semen parameters during 3 months of treatment. Organic Se significantly (P < 0.05) increased the percentage of viable sperms compared to inorganic Se and the control group. Fructose concentration was significantly higher (P < 0.05) in the seminal plasma of organic Se-treated bulls. Serum testosterone and Se concentrations were significantly (P < 0.05) increased in the Se supplemented groups than the control group. In conclusion, Se supplementation improved the parameters of buffalo bull semen and more precisely, organic Se was more effective for the improvement of semen quality and some blood components than inorganic Se., (© 2016 Japanese Society of Animal Science.)

The objectives of this study was to investigate the outcome of mastitis, in its clinical or subclinical forms, on the mean diameter of pre-ovulatory follicle (POF), plasma estradiol concentration on the day of estrus, subsequent luteal profile and subsequent conception rate in buffaloes. Sixty dairy buffalo (Bubalus bubalus) conducted in this study were divided into three groups {healthy (H), n=20; subclinical mastitis (SCM), n=18; and clinical mastitis (CM), n=22}. Ultrasonography of ovaries revealed that mean diameter of POF was larger (P<0.05) in H buffalo (14.35mm) compared to SCM (12.40mm) and CM (10.25mm). Also, plasma estradiol concentration on the day of estrus was higher (P<0.05) in H buffalo compared to SCM and CM counterparts; 34.95 vs. 32.87 and 27.50pg/ml, respectively. Besides, positive correlation was observed between the POF diameter with plasma estradiol concentration in H, SCM and CM buffaloes (r=0.64, 0.74, 0.72 respectively, P<0.05). Moreover, positive correlations (P<0.01) were found on days 9, 12, 16, and 21 post-ovulation between POF diameter and luteal profile. Thus, the conception rate in H buffalo was higher (P<0.05) compared with SCM and CM counterparts; 55% vs. 38.89 and 18.18%, respectively. In conclusion, mastitis in its clinical or subclinical forms disrupts the functioning of the pre-ovulatory follicle on the day of estrus, associated with low follicular estradiol production, resulting in suppression to subsequent luteal profile leading to substantial decrease in pregnancy consequence of buffaloes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We studied the effect of modified Heatsynch and Ovsynch protocols on the ovulatory response (OR), estrus induction rate (EIR) and conception rate (CR) in the anovular postpartum Murrah buffalo (n = 35). In the modified Heatsynch protocol (Group I; n = 12), buffaloes were given two GnRH at 2 h interval on treatment day 0, PGF (PGF2α) on day 7 and estradiol (E 2 ) 1 mg on day 8. Two FTAI were done at 20 h intervals after E 2 administration. In the modified Ovsynch protocol (Group II; n = 15), GnRH was given on day 0, 7 and 16 with a PGF on day 14. Two FTAI were done; one at last GnRH and the other 20 h later. Group III served as untreated negative control (n = 8). During the treatment, ovarian changes were monitored by transrectal ultrasonography and plasma progesterone (P 4 ) and E 2 . Administration of two GnRH at 2 h interval neither increased the OR nor strengthened the subsequent P 4 priming. Interestingly, in group I, none of the buffalo ovulated to E 2 though the EIR was 100% indicating the occurrence of behavioral, but not ovulatory estrus. Administration of GnRH 7 day prior to the commencement of Ovsynch protocol (Group II) did not improve the CR (21.4%), though the OR was 71.4%. No significant difference was found in the diameter of largest follicle between the ovulated and non-ovulated buffalo in response to GnRH suggesting that follicle of ≥9.5 mm is necessary but not sufficient to induce ovulation in the anovular buffalo. In both the protocols, the plasma P 4 was higher on day 7 in those buffaloes that ovulated to GnRH. Buffaloes treated with modified Ovsynch regimens were 5.27 times more likely to become pregnant than modified Heatsynch protocol. It is concluded that modified Ovsynch protocol is superior to modified Heatsynch protocol in terms of OR and CR., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Four rumen-fistulated male swamp buffaloes, 5-year-old with initiated live weight at 360 ± 12 kg, were randomly assigned according to a 4 × 4 Latin square design to investigate the effect of feeding high level of dried Leucaena leaf (DLL) on feed intake, fermentation efficiency and microbial protein synthesis. The dietary treatments were the feeding levels of DLL at 0, 2, 4 and 6 kg/head/day. All buffaloes were supplemented with concentrate mixtures at 0.1% of body weight, and rice straw was fed ad libitum with the availability of water and mineral block at all time. The results revealed that the total feed intake and nutrient digestibility were significantly improved with the increasing levels of DLL feeding, and the highest was in the buffaloes consuming DLL at 6 kg/head/day. Feeding high levels of DLL did not affect on ruminal pH and temperature, while ammonia nitrogen, blood urea nitrogen and volatile fatty acid concentration were significantly enhanced. Moreover, methane production was dramatically reduced by increasing levels of DLL feeding. Total direct counts of the micro-organism population were increased with the increasing levels of DLL feeding. According to the application of quantitative PCR to quantity cellulolytic bacteria (16S rRNA) targets, it was found that the population of total bacteria and Fibrobactor succinogenes was affected by treatments, while Ruminococcus flavefaciens and methanogen population were significantly decreased as buffaloes were fed with DLL. The nitrogen balance and microbial nitrogen supply were remarkably improved with the increasing levels of DLL feeding. Based on this study, it could be concluded that high levels of DLL feeding at 6 kg/head/day could enhance feed intake, nutrient digestibility, rumen fermentation efficiency and microbial protein synthesis in swamp buffaloes fed on rice straw without any adverse effect., (Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.)

Assessment of urine levels of luteinizing hormone (LH) for predicting the reproductive status of animals is in practice. The aim of this study was to predict the period of ovulation based on the urine levels of LH for timed-artificial insemination to increase the conception rate in buffaloes, which are naturally silent-oestrous animals. Level of LH in urine was assessed using ELISA, and a cut-off LH concentration for prediction of ovulation period was obtained using receiver operating characteristic analysis. Artificial insemination was performed before- and after -positive prediction of ovulation period adopting this method, and the rates of conception were assessed. Urine LH level of 105 mIU/ml (n = 14) was derived as a cut-off concentration which predicts the ovulation period. The buffaloes in the positively predicted group (day 1 or 2) inseminated via intracervical route had an increase in the conception rate (83.33%); however, the insemination in the before-positive-prediction group resulted in poor conception rates (day 0; 16.66%) compared to that of the naturally inseminated group (day 0; 75.0%). In conclusion, the urinary LH would possibly be a fairly reliable predictor of the ovulation period. The day when cut-off LH concentration is obtained may be taken as the most favourable time for artificial insemination, so as to attain a much better rate of conception in the buffalo., (© 2017 Blackwell Verlag GmbH.)

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., (© 2017 Blackwell Verlag GmbH.)

We studied the effect of sexual excitation on serum testosterone and nitric oxide (NO) levels in water buffalo bulls with different categories of sexual behavior and their correlation with each other. Buffalo bulls were classified according to their sexual behavior (including reaction time, sexual aggressiveness and mating ability): acceptable (good to excellent) (n=5), fair (n=5), and unacceptable (poor) (n=5) sexual behavior. Blood samples were collected from all animals immediately before and after sexual teasing and/or mounting to estimate the testosterone and NO levels using a commercial radioimmunoassay kit and Griess reaction test, respectively. Comparisons among groups were evaluated using a mixed-design analysis of variance. Pearson's correlation coefficients were calculated to determine the relationship between testosterone and NO levels before and after sexual excitation besides sexual behavior. The level of testosterone before sexual excitation was higher (p≤0.05) in bulls with acceptable and fair sexual behavior than in bulls with unacceptable sexual behavior (0.86±0.01, 0.69±0.02, and 0.29±0.02ng/mL, respectively). The level of NO was higher (p≤0.05) in bulls with acceptable and fair sexual behavior than in bulls with unacceptable sexual behavior (8.00±0.03, 7.66±0.19, and 6.29±0.33μM, respectively). Sexual excitation significantly (p<0.05) increase testosterone and NO levels in bulls with acceptable (1.45±0.01ng/mL and 19.04±0.32μM, respectively) or fair (0.92±0.02ng/mL and 14.95±0.34μM, respectively) sexual behavior, but not in bulls with unacceptable sexual behavior. The unacceptable sexual behavior bulls had significantly lower testosterone and NO levels than the other bulls. There was a strong correlation and association between serum testosterone and NO levels besides sexual behavior of buffalo bulls. In conclusion, the alteration in the testosterone and NO levels after sexual excitation depends on the sexual behavior category of buffalo-bull. Testosterone and NO can be used to create a sexual behavior score. The testosterone and NO levels of can be predicted via evaluation of sexual behavior of buffalo bull., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Buffalo production is spreading globally because of its economic advantage. Then, it has become necessary to improve the reproductive and productive efficiency of these animals, as well as to look for genetic factors that increase this efficiency. The objectives of this study were to characterize the promoter region of the melatonin 1A receptor gene (MTRN1A), to detect possible SNPs and associate them with fertility characteristics, and identify binding sites of transcription factors involved in the regulation of genetic expression in buffaloes in the Amazon. The conventional PCR method was carried out using the two primers designed from the reference sequence deposited in the GenBank AY52466.1. The products of the PCRs were purified, sequenced, and subsequently edited and aligned. Twenty-six SNPs were found, where 73% presented allele frequencies of wild nucleotides above 0.5, and 73% presented deviations from the Hardy-Weinberg equilibrium (P < 0.05) and F IS varying between 0.06 and 1.00, characterizing high degrees of inbreeding within the population. A block of ACAA deletion (position -1483) was observed in 25% of samples. The associations between these SNPs and reproductive characteristics were observed for calving interval and 5 SNPs: -1289, -1139, -911, -724, and -656 (P < 0.05), and three other SNPs: -1395, -724, and -94 (P < 0.05) were associated significantly with age at first calving, and were not associated with calving concentration. The promoter region was characterized by the different types of binding factors, where only 11 sites are significantly strong enough for transcription factor bindings. The ACAA deletion also exhibited a strong association with transcription factors. As a result, it would be necessary to test the SNPs above with other reproductive characteristics of economic relevance to approve the gene as a strong candidate for the selection of buffaloes in the Amazon.

Although it is understood that spermatozoa are subjected to selection processes to form a functional sperm reservoir in the oviduct, the mechanism remains obscure. With the aim to understand the sperm selection process in the oviduct, in the present in vitro study, we analyzed mitochondrial membrane potential and tyrosine phosphorylation status in oviduct-explants bound and unbound spermatozoa. Frozen semen from Murrah buffalo bulls (n=10) used under progeny testing programme were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 4h. Mitochondrial membrane potential (MMP) and tyrosine phosphorylation status of bound and unbound spermatozoa were assessed at 1h and 4h of incubation. The proportion of spermatozoa with high MMP was significantly higher (P<0.001) among the bound spermatozoa (range 84.67-96.56%) compared to unbound (range 8.70-21.03%) spermatozoa. The proportion of tyrosine phosphorylated spermatozoa was significantly higher (P<0.001) among unbound population as compared to bound population. The proportion of spermatozoa displaying tyrosine phosphorylation at acrosomal area was significantly (P<0.05) lower in bound sperm population compared to unbound population. It was inferred that spermatozoa with high MMP and low tyrosine phosphorylation were preferred for oviduct-explants binding in the buffalo., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Spermatogenesis is a complex and highly regulated process. The ability of spermatozoa to perform its function depends on multiple physiological and genetic factors that are not fully understood. Notably, due to lack of transcriptional and translational activity in spermatozoa, posttranslational modifications (PTMs) play key roles in determining their viability. PTMs not only confer structural changes in the proteome of the spermatozoa cells, but also increase the diversity of the proteome and introduce specific modifications that could be translated into functional changes in the affected spermatozoa. Multiple PTMs of active proteins have been identified in the developing spermatogonia. This review summarizes a diverse range of PTMs taking place in the developing spermatozoa, and analyzes their effects on male fertility and sperm viability. In particular, we discuss how SUMOylation, ubiquitination, phosphorylation, acetylation, glycosylation, and disulphide bond formation in proteins play a role in spermatogenesis, sperm maturation, movement of maturing spermatozoa to epididymis, capacitation, hyperactivation, spermatozoa motility, subversion of immune detection by spermatozoa, sperm to egg recognition and fusion, and the fertilization process. When possible, the specific proteins involved in these processes are highlighted. We point to existing knowledge gaps in the field of proteomics, and provide suggestions for future research on sperm viability and male fertility. We discuss briefly, as an example, the observations in water buffalo, Bubalus bubalis, which provides both meat and milk, and therefore is a reliable source for energy and protein needs of human populations. In conclusions, understanding the ways in which PTMs impact mammalian fertility and reproduction is important to make significant strides for diagnostic and therapeutic strategies in the near future.

In our previous study, we demonstrated that the repair efficiency of DNA double-strand breaks declines with increasing age in rat primordial follicles. In the present study, we extended our studies to buffalo (Bubalus bubalis) wherein we studied the expression of BRCA-1 related DNA repair genes in primordial follicles of young (12 months-22 months) and adult (72-96 months) buffaloes. The relative expression of selected genes, as determined by RT-PCR, revealed a significant (p < 0.05) decrease in mRNA levels of BRCA1, MRE11, RAD51, ATM, and H2AX in adult primordial follicles as compared to the young. Western blot analysis revealed a significant (p < 0.05) decrease in the expression of phosphorylated protein levels of BRCA1 and H2AX in adult buffalo primordial follicles. The protein expression profile of young and adult buffalo primordial follicles revealed differential expression of proteins involved in mitochondrial function, cell survival and cell metabolism. Similar to reports from aging rodent and human primordial follicles, our findings support the fact that impairment of DNA repair may be an universal mechanism involved in oocyte aging.

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high-fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high-fertile bulls was found to be higher (p < .05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p < .05) in high-fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r = .678, p = 0.013) and TAC (r = .648, p = .042) were found to be positively correlated with bull fertility and the level of MDA (r = -.718, p = .019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds., (© 2016 Blackwell Verlag GmbH.)

Present study unravels the involvement of different calcium signaling pathways in oxytocin-induced contractions in myometrium of non-pregnant and pregnant buffaloes during early and mid-pregnancy stages. Uteri of pregnant animals were more sensitive than of non-pregnant buffaloes. Phasic contractions and frequency of contraction significantly increased with advancement of pregnancy, while tonic contractions non-significantly and amplitude significantly decreased from six months pregnancy onward. Oxytocin produced concentration-dependent-contraction on isolated myometrial strips of pregnant and non-pregnant buffaloes and the dose response curves (DRCs) of oxytocin were significantly (P < 0.05) shifted to right in the presence of nifedipine (1 μM), in Ca 2+ -free Ringer Locke solution (RLS), ruthenium red (30 μM), ruthenium red + nifedipine, cyclopiazonic acid (CPA; Ca 2+ free RLS as well as RLS), CPA (10 μM)+nifedipine, U-73122 (1 μM) + nifedipine and SKF96365 (25 μM) on uteri of non-pregnant and pregnant (early and mid) animals. The DRCs were also significantly shifted towards right in the presence of Y-27632 (10 μM), GF109203X (5 μM) and Pyr3 (10 μM) on uteri of non-pregnant and early pregnancy stage buffaloes while only in the presence of U-73122 (1 μM) on uteri of mid-pregnancy stage buffaloes. Our finding suggest that and L-type Ca 2+ channels, IP3-RyR-gated, and store-operated calcium channels including transient receptor potential channel (TRPC) pathways play significant role in mediating oxytocin-induced contractions in myometrium of pregnant and non-pregnant buffaloes. SERCA plays major role only during early-pregnancy while functional role of protein kinase C (PKC), Rho-kinase and TRPC3 pathways decreased and role of G-protein coupled receptor-phospholipase C (GPCR-PLC) pathway increased with advancement of pregnancy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study was conducted to determine the effect of immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis on ovarian responses and fertility in cross-bred buffaloes. A total of 134 cross-bred buffaloes were divided into four groups: groups T1 (n = 34), T2 (n = 35) and T3 (n = 31) were nasal immunized twice a day with 10 ml of 1 × 10 10  CFU/ml of the C501 (pVAX-asd-IS) vaccine for 5, 3 and 1 day, respectively. Group C (n = 34) was nasal immunized with 10 ml PBS for 5 days. All animals were immunized twice with an interval of 14 days and administered with 200 μg of a GnRH analogue on day 28, 0.5 mg PGF 2α on day 35 and 200 μg of the same GnRH analogue on day 37. TAI was performed at 18 and 24 hr after the second GnRH treatment. Fourteen days after primary immunization, C501 (pVAX-asd-IS) elicited significant immune responses, and anti-inhibin IgG antibody titres in group T1 were significantly higher (p < .01) than groups T3 and C. After the second GnRH treatment, the growth speed of the dominant follicles in group T1 was significantly faster (p < .05) than groups T3 and C. The number and diameter of large follicles (≥10 mm) as well as ovulatory follicles in group T1 were the greatest in all groups, resulting in a greater conception rate in buffaloes with positive anti-inhibin antibodies. These results demonstrate that immunization with the C501 (pVAX-asd-IS) vaccine, coupled with the Ovsynch protocol, could be used as an alternative approach to improve reproductive performance in cross-bred buffaloes., (© 2016 Blackwell Verlag GmbH.)

The misuse of recombinant bovine somatotropin (rbST) to increase milk yield involves buffalo not just cows. Screening methods to identify rbST-treated cattle have already been proposed. However, there have been no studies on prolonged periods with a high number of animals. In this study, we developed a new enzyme-linked immunosorbent assay (ELISA) to measure the serum responsiveness towards rbST, based on an acid-stripping procedure and relatively simple integral calculation dilution curves. We also applied the analysis to 640 serum and 96 milk samples collected from 16 buffalo treated with rbST and 16 controls, over a period of approximately three months. Its suitability as a screening method, in compliance with EU law, was also assessed. A bi-factorial approach was also evaluated, including the measurement of insulin-like growth factor 1 concentration in serum. Results showed that our ELISA could be used on its own for screening purposes, without the need to assess other biomarkers. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The aim of this study was to investigate the reproductive and productive performance of pure Egyptian (PE) buffaloes and their crosses with Italian buffaloes. In this study, 2969 dairy buffaloes were used (1599 PE; 615 F 1 crosses, 50% PE and 50% Italian buffaloes; and 755 backcross [BC], 75% PE and 25% Italian buffaloes). When compared to PE, the BC and F 1 had a significantly lower incidence of calving difficulty (odds ratio [OR] = 0.18, p < .0001 and .34, p < .0001, respectively) in conjunction with a lesser incidence of stillbirth (OR = 0.06, p < .0001 and 0.43, p < .0001, respectively). Backcross buffaloes were also noted to have a lower rate of not conceived after first insemination (OR = 0.74, p < .0001) in comparison with PE. The cross F 1 buffaloes were superior in terms of productive traits, whereas PE buffaloes were best in most of the reproductive traits (days of non-pregnant p = .001, service per conception p < .0001 and calf weight p = .01). Although the study results appeared to indicate towards a contrary association between milk production and reproductive traits, BC was similar to F 1 in terms of production and similar to PE in terms of reproductive traits. Thus, the farmers should be encouraged to breed BC animals in their herds for enhancing milk yield to meet with the market demands., (© 2016 Blackwell Verlag GmbH.)

The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study examines the differential role of calcium signaling pathway(s) in histamine-induced uterotonic action during early and mid-pregnancy stages in buffaloes. Compared to mid pregnancy, tonic contraction, amplitude and mean-integral tension were significantly increased by histamine to produce myometrial contraction during early pregnancy with small effects on phasic contraction and frequency. Although uterotonic action of histamine during both stages of pregnancy is sensitive to nifedipine (a L-type Ca 2+ channels blocker) and NNC55-0396 (T-type Ca 2+ channels blocker), the role of extracellular calcium seems to be more significant during mid-pregnancy as in this stage histamine produced only 9.38±0.96% contraction in Ca 2+ free-RLS compared to 21.60±1.45% in uteri of early pregnancy stage. Intracellular calcium plays major role in histamine-induced myometrial contraction during early pregnancy as compared to mid pregnancy, as in the presence of cyclopiazonic acid (CPA) Ca 2+ -free RLS, histamine produced significantly higher contraction in myometrial strips of early-pregancy in comparison to mid-pregnancy (10.59±1.58% and 3.13±0.46%, respectively). In the presence of U-73122, the DRC of histamine was significantly shifted towards right with decrease in maximal effect (E max ) only in early pregnancy suggesting the predominant role of phospholipase-C (PL-C) in this stage of pregnancy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present study investigated the combined effect of fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGF-A) on estradiol (E 2 ) secretion and relative abundance of mRNA for aromatase enzyme (CYP19A1), proliferating cell nuclear antigen (PCNA) and BCL-2 associated X protein (BAX) in cultured buffalo granulosa cells (GCs). Follicles were isolated and classified into four groups based on size and E 2 concentration in follicular fluid (FF): Small, 4-6mm diameter, E 2 <0.5ng/ml; Medium, 7-9mm, E 2 =0.5-5ng/ml; Large, 10-13mm, E 2 =5-40ng/ml; Preovulatory (PFs), >14mm, E 2 >180ng/ml. The GCs of PF were cultured in 24 well cell culture plates and allowed to become 75-80% confluent. Then cultured GCs were treated with FGF2 (200ng/ml) and VEGF-A (100ng/ml) separately and in combination for three incubation periods (24, 48 and 72h). Estradiol secretion was greater in GCs treated with FGF2+VEGF-A compared to FGF2 or VEGF-A at all incubation periods and was greatest (P<0.05) at 72h of incubation. The relative abundance of CYP19A1 and PCNA mRNA were relatively consistent with the amount E 2 secretion. In contrast, the relative abundance of Bax mRNA was less in GCs treated with the combination of FGF2 and VEGF-A as compared to either FGF2 or VEGF-A alone and the least concentration (P<0.05) was at 72h of incubation. Findings with use of immunocytochemistry of cells treated with these factors were consistent to the relative abundance of mRNA transcript for the factor. The present findings indicate that FGF2 and VEGF-A may function in a synergistic manner to promote steroidogenesis and survival of cultured buffalo GCs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The use of artificial insemination (AI) in buffalo ( Bubalus bubalis ) is limited by poor ovarian activity during the hot season, seasonal qualitative patterns in semen, low resistance of sperm cells in the female tract, difficulties in estrus detection, and variable estrus duration. Although AI procedures are commonly used in bovine, use of AI has been limited in buffalo. In the zootechnical field, different studies have been conducted to develop techniques for improvement of fertilizing ability of buffalo spermatozoa after AI. In this study, for the first time, the use of alginate encapsulation and cryopreservation of buffalo spermatozoa is described, and the same procedure was performed with Holstein Friesian ( Bos taurus ) semen. Results obtained from in vitro analyses indicate that the encapsulation process does not have detrimental effects (compared to controls) on quality parameters (membrane integrity, progressive motility, path average velocity) in either species. Similarly, there were no detrimental effects after cryopreservation in either species. The fertilizing potential of encapsulated and cryopreserved semen was evaluated after AI in 25 buffalo and 113 bovine females. Pregnancy rates were not affected in either species. The results of this study show proof of concept for the use of frozen semen controlled-release devices in buffalo.

Background: Catalase enzyme is usually distributed in mammalian seminal plasma, where it decomposes hydrogen peroxide into water and oxygen and enhances sperm survivability., Objective: To evaluate the effect of catalase (0, 100, 200 or 300 IU/ml) added in tris-citric acid (TCA) based extender on motion characteristics, viability and DNA integrity of bubaline spermatozoa at post dilution (PD) and post thawing (PT) stages of cryopreservation., Materials and Methods: Collection of semen was done in four Nili-Ravi bulls with an artificial vagina (42 degree C). Qualified semen samples from each bull were further subdivided into four aliquots for dilution with the experimental TCA extender containing either 0.0 (T1), 100 IU (T2), 200 IU (T3) or 300 IU (T4) catalase (activity12660 U/mg)., Results: At PT, mean computer progressive motility, average path velocity, straight line velocity, curvilinear velocity, visual motility and DNA integrity were higher (P < 0.05) in catalase fortified treatment groups as compared with control. Regarding plasma membrane integrity and supra-vital plasma membrane integrity, at PT the mean values were higher (P < 0.05) in T4 as compared with control. At PD and PT, mean acrosomal integrity of buffalo bull spermatozoa was higher (P < 0.05) in T4 group as compared with control., Conclusion: Addition of catalase at a concentration of 300IU/ml in TCA cryodiluent improved the freezability of water buffalo spermatozoa.

The cryopreservation of germ cells is a major tool for the propagation of animals with desired genetic traits. Although cryopreservation of spermatozoa in some animals is effective, its effectiveness is variable. For example, cryopreservation efficiency of buffalo bull spermatozoa remains very poor. In this study, we evaluated sperm DNA damage and ultrastructure in buffalo bull spermatozoa vitrified in the presence or absence of cholesterol-loaded cyclodextrins (CLC). Our results showed that cryopreserved buffalo spermatozoa had elevated levels of deteriorated plasma and mitochondrial membranes, which are the likely causes of DNA damage after vitrification. Accordingly, the levels of the activity of Alanine Aminotransferase (ALT), Alkaline phosphatase (ALP) and Aspartate Aminotransferase (AST) were also elevated following exposure of buffalo bull spermatozoa to a cycle of freezing-thawing. Importantly, supplementation of Tris-Egg Yolk-Glucose (TEYG) extender with (CLC) improved the quality of buffalo spermatozoa following cryopreservation. This protective effect of CLC is likely due to decreasing mitochondrial and plasma membrane deterioration with subsequent inhibition of DNA damage. These results suggest that cholesterol loss is the likely reason for poor semen quality in buffaloes following cryopreservation, and provide evidence that manipulating lipid content during cryopreservation is a promising strategy to improve the quality of buffalo semen., (Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.)

Previous studies suggested a close relationship between ruminant gut microbes and the mammary gland. In this study, shotgun metagenomic sequencing was used to reveal the differences in the intestinal microbiome potentially related to milk components in Murrah buffaloes and Chinese Holstein cattle. A PCoA based on the weighted Unifrac distances showed an apparent clustering pattern in the structure of intestinal microbiota between buffalo and cattle. We could attribute the structural difference to the genera of Sutterella, Coprococcus and Dorea. A further analysis of microbial functional features revealed that the biosynthesis of amino acids (including lysine, valine, leucine and isoleucine), lipopolysaccharide biosynthesis and cofactor/vitamin biosynthesis were enriched in the buffalo. In contrast, dairy cattle had higher levels of pyruvate metabolism and carbon fixation in photosynthetic organisms. A further correlation analysis based on different milk components and the typical microbiome uncovered a significant positive correlation between milk protein and the microbial biosynthesis of amino acids, which was also positively correlated in the genera of Parabacteroides, Dorea and Sutterella. This study will expand our understanding of the intestinal microbiome of buffalo and cattle as representative ruminants, as well as provide new views about how to improve the production and nutritional qualities of animal milk., Competing Interests: The authors declare no competing financial interests.

This study investigates for the first time mRNA pregnancy-associated glycoprotein 2 (PAG-2) expression in blood cells during early pregnancy in water buffalo. The PAGs constitute a large family of glycoproteins expressed in the outer epithelial layer of the placenta in eutherian species. All PAGs are not concomitantly expressed throughout pregnancy; some of them are expressed in the earlier phases, whereas others appear later and are expressed over a shorter period. Twenty-one lactating buffaloes were analyzed-17 females were synchronized with PRID and artificially inseminated (AI), whereas four females were synchronized but not inseminated (control group). Blood was collected at Days 0, 18, 28, 40, and 75 from AI (AI = Day 0). Expression of PAG-2 mRNA in blood samples was measured with real-time polymerase chain reaction. Pregnancy diagnosis was performed on Day 28 (D28) and Day 40 (D40) after AI by ultrasonography (US) and by PAG-1 RIA method. The females diagnosed pregnant at D28 and confirmed pregnant at D40 were defined as D28(+)D40(+) group; the females diagnosed pregnant at D28 but not confirmed pregnant at D40 were defined as D28(+)D40(-) group; and the females that were diagnosed as nonpregnant on either days were defined as D28(-)D40(-) group. PAG-2 mRNA at Day 0 was not observed in any groups. The D28(+)D40(+) group showed the highest expression, starting on Day 18 and increasing progressively up to Day 75. PAG-2 mRNA was also expressed on Day 18 in both D28(+)D40(-) and D28(-)D40(-) groups, but their levels were lower than those of D28(+)D40(+) group and almost constant over time. PAG-2 mRNA was never detected in the control group. The significant difference in the expression of PAG-2 mRNA between the D28(+)D40(+) group and the D28(-)D40(-) group, starting from Day 18, suggests that these animals might have conceived, but have experienced early embryonic loss; therefore, the PAG-2 mRNA was still present in blood circulation although at lower levels, as found in the D28(+)D40(-) group. In conclusion, this study shows that PAG-2 mRNA can be detected in peripheral maternal blood cells earlier than circulating PAG-1 molecules and could be useful for studies on early pregnancy and embryonic mortality., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study was primarily designed to evaluate the effect of different concentrations of ultraviolet (UV)-C-irradiated chicken egg yolk plasma (EYP; v:v; 10%, P1; 15%, P2; 20%, P3) or 20% (v:v) of whole chicken egg yolk (WCEY) in tris-citric acid (TCA) extender on water buffalo sperm quality during cryopreservation (postdilution, PD; postequilibration, PE; post-thawing, PT). Also the effect of best evolved concentration of UV-C-irradiated EYP in extender on in vivo fertility of buffalo spermatozoa was evaluated. At PE and PT, computer-assisted sperm analysis progressive motility (PM, %) was significantly higher in P3 compared with P1 and WCEY. Rapid velocity (RV, %) was higher (P < 0.05) in P3 compared with P1 and WCEY during cryopreservation (PD, PE, and PT). Average path velocity (μm/s) and straight line velocity (μm/s) were higher (P < 0.05) in P2 and P3 than WCEY at PE and PT. The decline percentage (%, longevity) in PM and RV was lower (P < 0.05) in P3 compared with WCEY during 2 hours incubation under in vitro condition at PT. Supravital plasma membrane integrity (%) was higher (P < 0.05) in P2 and P3 compared with control at different stages (PE and PT). Mitochondrial transmembrane potential (%) was higher (P < 0.05) in P2 and P3 compared with P1 and WCEY at different stages (PD and PT). Percentage of viable sperm with intact acrosome, and sperm DNA integrity (%) were higher (P < 0.05) in P2 and P3 compared with WCEY at PT. The in vivo fertility rate (%) was significantly higher with P3 compared with WCEY (76.61 vs. 64.49). In conclusion, WCEY (20%) can be replaced with UV-C-irradiated chicken EYP (20%) in TCA extender for cryopreservation of water buffalo spermatozoa., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recent strong selection for dairy traits in water buffalo has been associated with higher levels of inbreeding, leading to an increase in the prevalence of genetic diseases such as transverse hemimelia (TH), a congenital developmental abnormality characterized by absence of a variable distal portion of the hindlimbs. Limited genomic resources available for water buffalo required an original approach to identify genetic variants associated with the disease. The genomes of 4 bilateral and 7 unilateral affected cases and 14 controls were sequenced. A concordance analysis of SNPs and INDELs requiring homozygosity unique to all unilateral and bilateral cases revealed two genes, WNT7A and SMARCA4, known to play a role in embryonic hindlimb development. Additionally, SNP alleles in NOTCH1 and RARB were homozygous exclusively in the bilateral cases, suggesting an oligogenic mode of inheritance. Homozygosity mapping by whole genome de novo assembly also supported oligogenic inheritance; implicating 13 genes involved in hindlimb development in bilateral cases and 11 in unilateral cases. A genome-wide association study (GWAS) predicted additional modifier genes. Although our data show a complex inheritance of TH, we predict that homozygous variants in WNT7A and SMARCA4 are necessary for expression of TH and selection against these variants should eradicate TH.

The aim of this study was to compare estradiol benzoate (EB) and GnRH for the induction of ovulation in a TAI protocol in buffalo during the nonbreeding season. In experiment 1, 141 buffaloes (56 cows and 85 heifers) received an intravaginal P4 device (1.0 g) plus EB (2.0-mg, intramuscular [im]) at random stage of the estrous cycle (Day 0). On Day 9, the P4 device was removed, and buffaloes were given PGF 2α (0.53-mg im sodium cloprostenol) plus eCG (400-IU im). Buffaloes were then randomly allocated to one of three groups and treated as follows: EB24 (n = 47), EB (1.0 mg im) 24 hours after P4 device removal; EB36 (n = 50), EB 36 hours after P4 device removal; GnRH48 (n = 44), GnRH (10 μg im buserelin acetate) 48 hours after P4 device removal. Ultrasound examinations were performed on Day 0 to ascertain ovarian follicular status, Day 9 to measure follicular diameter, and from Day 11 to Day 14 (every 12 hours for 60 hours) to establish the time of ovulation. There were no significant differences between EB24, EB36, and GnRH48 for diameter of the ovulatory follicle (13.1 ± 0.3, 13.7 ± 0.3, and 13.7 ± 0.3 mm; P = 0.26) and ovulation rate (78.7%, 82.0%, and 84.1%; P = 0.93). When compared with heifers, cows had a greater diameter of the dominant follicle on Day 9 (10.3 ± 0.3 and 8.6 ± 0.2 mm; P = 0.0001), diameter of the ovulatory follicle (14.1 ± 0.3 and 13.1 ± 0.2 mm; P = 0.01), ovulation rate (91.1% and 75.3%; P = 0.02), and interval from P4 device removal to ovulation (76.3 ± 1.3 and 72.5 ± 1.4 hours; P = 0.05). In experiment 2, 511 buffaloes (354 cows and 157 heifers) were assigned to the same treatments described in experiment 1 (EB24, n = 168; EB36, n = 172; and GnRH48, n = 171), and all animals were submitted to timed artificial insemination (TAI) 64 hours after P4 device removal. Pregnancy diagnosis was undertaken 30 days after TAI. There were no significant differences between EB24, EB36, and GnRH48 for pregnancy rate (45.2%, 43.0%, and 49.7%; P = 0.46), and the pregnancy rate did not differ (P = 0.31) for cows (47.5%) and heifers (42.7%). The findings from the two experiments indicated that EB (24 or 36 hours) and GnRH (48 hours) induce comparable follicular responses, ovulation, and pregnancy rates in buffalo cows and heifers. Although there were some differences in the follicular responses between cows and heifers, the pregnancy rate to TAI was nonetheless similar., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this experiment was to determine the effect of dried rumen digesta pellet levels on feed intake, digestibility, rumen ecology, and blood metabolites in swamp buffalo. Four 2-year-old male swamp buffalo with an initial body weight (BW) of 150 ± 10.0 kg were randomly assigned according to a 4 × 4 Latin square design to receive four levels of dried rumen digesta pellets (DRDPs). The dietary treatments were supplementation of DRDP at 0, 50, 100, and 150 g dry matter/day, respectively. Total feed intake was significantly different among treatments (p < 0.05) and was highest in the 150 g/day DRDP supplement (2.68 kg/day). Intakes of neutral detergent fiber (NDF) and acid detergent fiber did not affect DRDP levels, while intakes of organic matter and crude protein (CP) were altered significantly when 150 g of DRDP was used (p < 0.05). Buffalo fed with DRDP at 150 g/day had the highest CP and NDF digestibility (p < 0.05). DRDP supplementation did not affect rumen pH, and temperature and the concentration of ruminal ammonia-nitrogen and blood urea nitrogen were not altered among the treatments. The mean value of fungal zoospores in the buffalo was significantly different among treatments and was highest in supplementation with DRDP at 150 g. The mean value of propionic acid was significantly different at various levels of DRDP; it was highest in the group fed with 150 g DRDP (p < 0.05). Thus, supplementation of DRDP at 150 g improved feed use and increased fungal zoospore population. In addition, DRDP feeding is recommended, since it has positive economic impacts and helps control environmental pollution.

A feeding trial with 18 Murrah buffalo heifers (238 ± 63 kg; 1.7 ± 0.4 years old) was conducted for 57 days, and feed intake and body weight were recorded followed by 7 days of urine and feces collection. Heifers were grouped into low (RFI-I) and high (RFI-II) feed efficiency groups by regressing DMI (g head -1  day -1 ) on BWG (g head -1  day -1 ) and mid-test MBW (kg). RFI-II heifers had slightly higher DMI (g head -1  day -1 and expressed per unit of BW, %BW, MBW, mid-test BW, and mid-test MBW) and all associated nutrient intakes, BWG, feed efficiency traits (FCR, CPCR), digestibility of nutrients, and retentions of crude protein and fat over RFI-I heifers. The corresponding intake values (kg head -1  day -1 ) for RFI-I versus RFI-II were the following: DMI (6.73 ± 0.56 vs 6.51 ± 0.50), CPI (0.99 ± 0.008 vs 0.97 ± 0.07), DOMI (5.06 ± 0.47 vs 5.11 ± 0.42), and TDNI (4.95 ± 0.47 vs 5.00 ± 0.42). BWG (669 ± 52 vs 776 ± 47 g head -1  day -1 ), feed efficiency traits (FCR: 10.32 ± 1.16 vs 8.77 ± 1.04 and CPCR: 1.52 ± 0.17 vs 1.30 ± 0.16), N balance (38 ± 9 vs 45 ± 8 g N head -1  day -1 ), and protein and fat retentions (239 ± 57 vs 282 ± 51 g CP head -1  day -1 and 430 ± 6 vs 494 ± 59 g fat head -1  day -1 ) of RFI-I were less than RFI-II heifers, respectively. Based on the predicted RFI values, RFI-II buffalo heifers could be selected for feed efficiency.

The aim of this study was to evaluate differences in efficiency of feed utilisation between buffalo calves with low and high residual feed intake (RFI) by comparing feed intake, nutrient digestibility, growth traits and blood metabolites. Eighteen male Murrah buffalo calves (aged 4-6 months; 70 ± 1.0 kg body weight) were fed ad libitum with a total mixed ration for 120 d. Based on linear regression models involving dry matter intake (DMI), average daily gain (ADG) and mid-test metabolic body size, calves were assigned into low and high RFI groups. The RFI varied from -0.33 to +0.28 kg DM/d with an average RFI of -0.14 and 0.14 kg DM/d in low and high RFI calves, respectively. Calves had a mean DMI of 1.9 and 2.4 kg/d and an ADG of 0.5 and 0.6 kg/d in low and high RFI groups, respectively. Low RFI calves ate 19.0% less DM each day and required significantly less metabolisable energy for maintenance compared with high RFI calves (12.5 vs. 16.7 MJ/d). Nutrient digestibility and nitrogen balance did not differ among low and high RFI calves. In more efficient animals (low RFI calves) higher (p < 0.05) plasma level of growth hormone, insulin-like growth factor-1 (IGF-1), triiodothyronine (T3) and lower concentration of thyroxin hormone were detected. No significant differences in levels of insulin, hydroxyproline, plasma and urine creatinine, total protein and albumin between high and low RFI groups were found. Blood metabolites showed significant (p < 0.05) differences at initial and final stages of study in both groups. At final stage of study, RFI showed negative correlations with growth hormone, IGF-1, T3, urine creatinine and albumin. Low RFI buffalo calves are more efficient in feed utilisation and the differences in blood metabolites are probably due to differences in feed intake and body metabolism.

The present study assessed sperm functional characteristics in the frozen-thawed semen of buffalo bulls and estimated their relationship with field fertility. Frozen semen samples from three different freezing operations each from nine Murrah buffalo bulls were used for the assessment of different sperm functions related to fertilizing potential. Bulls were classified into high (n = 2), medium (n = 5), and low (n = 2) fertile based on adjusted field fertility. The sperm functions estimated included membrane integrity using carboxyfluorescein diacetate-propidium iodide, acrosome reaction status using fluorescein isothiocyanate peanut agglutinine, status of apoptosis using Annexin-V, protamine deficiency using Chromomycin A 3 , membrane stability using Merocyanine 540 and lipid peroxidation status using 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene. The relationship between the proportion of live acrosome-intact spermatozoa and fertility was positive and significant (r = 0.59; P = 0.001). The proportion of moribund spermatozoa showed a significantly negative correlation with fertility (r = -0.50; P = 0.008). Similarly, the relationship of spermatozoa with unstable membrane (r = -0.51; P = 0.007), necrotic (r = - 0.42; P = 0.028), early necrotic (r = -0.42; P = 0.031), and apoptotic spermatozoa (r = -0.39; P = 0.046) with bull fertility was negative and significant. The correlation between the protamine-deficient spermatozoa and fertility was negative, but not significant. Among different combinations of tests, live acrosome-intact spermatozoa and lipid peroxidation status of spermatozoa revealed high positive correlation with buffalo bull fertility (adjusted R 2  = 0.73, C[p] = 0.80). These preliminary findings may help in developing tools for assessing fertility of buffalo bulls, once validated in more animals., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the relationship between the ovarian follicular response at the start of the OVsynch-fixed-time artificial insemination (TAI) program and pregnancy outcome in the Mediterranean river buffalo. The study involved 116 pluriparous buffaloes at 118 ± 65 days in milk maintained under commercial conditions on a buffalo dairy enterprise in southern Italy. Synchronization of the stage of the estrous cycle in the Ovsynch-TAI program involves an injection of GnRH on Day 0 (GnRH1), PGF 2α on Day 7, and GnRH on Day 9 (GnRH2). Timed artificial insemination is carried out on Day 10. Ovarian ultrasound examinations were undertaken on days 0, 2, 7, 9, 10, and 11 to record total follicles, the dimensions of follicles greater than 0.75 cm, dimensions of the corpus luteum (CL), and the occurrence of ovulation. Blood concentrations of P4 were measured on days 0 and 10 and 10 days after TAI. Pregnancy status was determined on days 27 and 45 after TAI to calculate late embryonic mortality. The data were analyzed by ANOVA and multiple logistic regression. A total of 70 of 116 (60.4%) buffaloes showed ovulation after GnRH1. Buffaloes that ovulated after GnRH1 had a smaller (P < 0.05) area of the ovulatory follicle on Day 0 compared with buffaloes that did not ovulate (1.08 ± 0.1 and 1.29 ± 0.1 cm 2 , respectively). The former buffaloes also had a larger (P < 0.05) CL area (1.81 ± 0.1 and 1.51 ± 0.1 cm 2 , respectively) and greater (P < 0.05) blood concentrations of P4 (1.84 ± 0.2 and 1.34 ± 0.2 ng/mL, respectively) on Day 0. A larger (P < 0.05) proportion of buffaloes that ovulated after GnRH1 showed regression of the CL after the injection of PGF 2α on Day 7 compared with buffaloes that did not ovulate (68 of 69 [98.6%] and 27 of 32 [84.4%], respectively). Buffaloes that ovulated after GnRH1 also had a greater (P < 0.05) ovulation rate after GnRH2 than buffaloes that did not ovulate (65 of 70 [92.8%] and 37 of 46 [80.4%], respectively). Buffaloes that ovulated after GnRH1 had a higher (P < 0.01) pregnancy rate than buffaloes that did not ovulate, both at Day 27 (53 of 70 [75.7%] and 14 of 46 [30.4%], respectively) and Day 45 (46 of 70 [65.7%] and 10 of 46 [21.7%], respectively). The incidence of late embryonic mortality tended to be less for buffaloes that ovulated after GnRH1 than buffaloes that did not ovulate (7 of 53 [13.2%] and 4 of 14 [28.6%], respectively). The present findings have demonstrated a strong direct relationship between the ovarian follicular response at the start of an OVsynch-TAI program and pregnancy outcome in the Mediterranean river buffalo., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P 4 ) secretion and mRNA expression of phosphotidylinositide-3kinase-protein kinase B (PI3K-AKT), phosphoinositide-dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real-time RT-PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P 4 secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p < .05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p < .05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P 4 secretion was highest (p < .05) with 100 ng/ml at 72 hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p < .05) and BAD along with caspase 3 were lowest (p < .05) at 100 ng/ml at 72 hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P 4 secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions in buffalo CL., (© 2016 Blackwell Verlag GmbH.)

The present study was aimed at determining changes in milk yield and composition along with the plasminogen-plasmin system of milk, plasma hormones, and metabolites of buffaloes during hot dry season (air temperature range 39.7 to 44.8 °C) under two different management systems. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals accessed benefit of mist and fan cooling from 9:30 a.m. to 5:00 p.m., while control group animals were devoid of it. Duration of experiment was 6 weeks. Under mist and fan cooling system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as rectal temperature, respiration rate, pulse rate, and forehead and middorsal temperatures were significantly (P < 0.05) reduced compared to control, which subsequently resulted higher milk yield by 4.44 % (P < 0.001). Analysis of milk samples revealed higher concentration of plasminogen (7.99 vs 6.27 μg/ml; P < 0.01) and β-casein (1.09 vs 0.92 g/dl; P < 0.001) and lower plasmin level (0.178 vs 0.194 μg/ml; P < 0.05) in buffaloes under the treatment group compared to that under the control. Plasma glucose level was higher (P < 0.001) by 21.08 %, whereas cortisol, norepinephrine, and NEFA levels were lower (P < 0.001) by 19.19, 15.38, and 11.41 %, respectively, in treatment animals. However, exposure of buffaloes to cooling system did not alter composition and calcium content of milk, GH, and epinephrine level in plasma. Hence, it may be concluded that provision of cooling system during summer was effective to minimize environmental stress and improve milk production by manipulation of the PG-PL system in buffaloes.

Improving the quality of in vitro maturated buffalo oocytes is essential for embryo production. We report here the effects on microtubules and microfilaments in oocytes and embryo development that result from treating buffalo oocytes with the phosphodiesterase 3 (PDE3) inhibitor cilostamide. Addition of 20μM or 50μM cilostamide for 24h during in vitro maturation showed no differences in the percentage of oocytes arrested at the germinal vesicle (GV) stage. When 20μM cilostamide was added to the pre-maturation culture for 6h, 12h or 24h and continued for another 24h without cilostamide, oocytes resumed meiosis, but with significantly lower (P<0.01) maturation rates in the 24h group than that in the other two groups. During oocyte maturation in vitro, no microtubules were detected before GV breakdown (GVBD). After GVBD, microtubules combined with condensed chromatin to form the meiotic metaphase spindle. Microfilaments covered a thick area around the cellular cortex and overlying chromosomes. Cilostamide had no effects on microtubules and microfilaments in metaphase II oocytes, and there were no significant differences in the rates of cleavage, blastocyst formation and number of blastocyst cells between oocytes treated pre-maturation with inhibitor for 6h and those of the control group (P>0.05). In summary, cilostamide reversibly arrested the resumption of meiosis without any adverse impact on the dynamic changes in microtubules and microfilaments in buffalo oocytes and their in vitro developmental capacity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The effects of l-cysteine in extender on antioxidant enzymes profile during cryopreservation, post-thaw quality parameters and in vivo fertility of Nili-Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of l-cysteine (0.0, 0.5, 1.0, 2.0 and 3.0 mm) and frozen in 0.5-ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P < 0.05) at pre-freezing and post-thawing in extender containing 2.0 mm l-cysteine as compared to other groups. Post-thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm s -1 ), straight line velocity (μm s -1 ), curvilinear velocity (μm s -1 ), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0 mm l-cysteine as compared to other groups (P < 0.05). The fertility rates (59 versus 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mm of l-cysteine than in the control. In conclusion, the addition of 2.0 mm l-cysteine in extender improved the antioxidant enzymes profile, post-thaw quality and in vivo fertility of Nili-Ravi buffalo bull spermatozoa., (© 2016 Blackwell Verlag GmbH.)

Sperm selection techniques have been developed to get sperm suspensions enriched in motile and functional cells. Studies show that selection before cryopreservation improves post-thaw quality of cryopreserved sperm but information on buffalo bull sperm is scarce. The study was aimed to 1) perform a comparative analysis of sperm selection procedures; Swim-Up (SU), Sephadex™-G15 Filtration (S-G15) or Glass Wool Filtration (GWF) for total and motile cell recovery, 2) to assess the impact of sperm selection prior to cryopreservation on sperm quality (motility, morphology, cell membrane and normal apical ridge, viability and livability, chromatin integrity) and sperm functionality (Embryo Cleavage after IVF with selected sperm) in post-thawed suspensions of buffalo bull sperm. Semen was collected from 5 Nili Ravi buffalo bulls maintained at the Semen Production Unit Qadirabad, District Sahiwal, Pakistan. Ejaculates were divided into four aliquots for SU, S-G15 and GWF and control. After sperm selection, total and motile sperm recovery was highest in GWF samples (total sperm=84.08±8.39%; motile sperm=80.42±3.57%). An improvement (P<0.05) in all post-thaw parameters was observed in S-G15-selected sperm and, in some parameters in GWF-filtered sperm suspensions compared to control. The highest (P<0.05) embryo cleavage rate (%) was achieved with frozen-thawed sperm selected with S-G15 prior to cryopreservation (44.72±4.18) compared to control (21.98±3.00). In conclusion, post thaw sperm quality was improved after sperm selection from fresh buffalo bull semen through S-G15 and GWF procedures compared to SU and control while, the fertility rate (cleavage rate) was improved with sperm processed using the S-G15 procedure., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Overwintering larvae of the beetle Dendroides canadensis produce potent antifreeze proteins to inhibit inoculative freezing and promote supercooling. We hypothesized that addition of Dendroides canadensis recombinant antifreeze proteins (DAFPs) in the extender will improve the quality and fertility of cryopreserved Nili-Ravi buffalo (Bubalus bubalis) sperm. The study was divided into two parts: (1) Evaluation of the effect of DAFPs on the quality of frozen-thawed buffalo bull sperm and (2) Examination of the fertilizing ability of frozen-thawed buffalo bull sperm. Semen was collected from three bulls using an artificial vagina (42 °C). Qualifying ejaculates from each bull were divided into four aliquots and diluted (at 37 °C, 50 × 10(6) sperm/mL) in tris-citric acid extender containing DAFP (at 0.1, 1.0, and 10 μg/mL), and the sperm were evaluated for important characteristics relative to a control without DAFP. D canadensis recombinant antifreeze proteins at any of the three concentrations did not affect sperm progressive motility or plasma membrane integrity (PMI), either before or after the semen was cooled to 4 °C in 2 hours. However, after 24 hours of cryostorage at -196 °C, followed by thawing at 37 °C for 30 seconds, sperm progressive motility and PMI were higher (P < 0.05) in extender containing DAFP at 10 μg/mL compared with control. The in vitro-fertilizing ability of cryopreserved (-196 °C) sperm supplemented with DAFP (10 μg/mL) was slightly higher (P = 0.098) compared with control, as assessed through in vitro cleavage rate of in vitro matured buffalo oocytes. Also, the in vivo fertility rate was evaluated by inseminating 100 buffaloes (50 inseminations per extender) 12 hours after standing heat. The fertility rate of cryopreserved buffalo bull sperm in terms of positive pregnancy at 90 days after insemination was clinically higher but remained statistically nonsignificant in extender containing DAFP at 10 μg/mL (52.0%) compared with control (43.8%). In conclusion, supplementation of 10 μg/mL of DAFP in the extender improved the motility and PMI of Nili-Ravi buffalo sperm after freeze-thawing, and yielded numerically higher, although statistically nonsignificant, in vitro cleavage, and in vivo fertility rate., (Copyright © 2016. Published by Elsevier Inc.)

The aim of the present study was to demonstrate the modulatory role of leptin on bubaline granulosa cells (GCs) and luteal cells (LCs) functions using an in vitro cell culture system and to establish a cross talk between leptin and insulin-like growth factor-1 (IGF-1). GCs were collected from group IV follicles (>13 mm size) and LCs from mid-luteal phase corpus luteum and were grown in serum-containing media supplemented with leptin at three different dose rates (0.1, 1, and 10 ng/mL) and time durations (24, 48, and 72 hours). We evaluated the production and secretion of estradiol (E2) and progesterone (P4) using RIA and the mRNA expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 aromatase (CYP19A1), sterol regulatory element-binding protein 1 (SREBP1), steroidogenic factor-1 (SF1), anti-apoptotic gene PCNA, pro-apoptotic gene caspase 3 and endothelial cell marker, Von Willebrand factor (vWF), using quantitative real-time polymerase chain reaction. The results depicted a direct inhibitory action of leptin on GCs steroidogenesis in a time-dependent manner (P < 0.05), whereas in the presence of IGF-1 the inhibitory effect was reverted. Furthermore, leptin augmented both cellular proliferation (PCNA) and apoptosis (caspase 3). On the other hand, in LCs, leptin alone showed an apparent stimulatory effect on steroidogenesis (P < 0.05); however, in the presence of IGF-1, an antagonistic effect was witnessed. Moreover, leptin had an inhibitory effect on apoptosis while promoted cellular proliferation and angiogenesis. These findings were further strengthened by immunocytochemistry. To conclude, these observations for the first time reported that in buffaloes leptin has a direct dose-, time-, and tissue-dependent effect on ovarian steroidogenesis, angiogenesis, and cytoprotection, and furthermore, it can regulate the effect of systemic factors like IGF-1. Hence, this in vitro study provides an insight into the putative roles of leptin alone and its interactions in vivo., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The present study investigated the expression and localization of angiopoietin (ANPT) family members in buffalo ovarian follicles of different size. It also looked at the role of ANPTs in estradiol secretion and mRNA expression of phosphoinositide-3-kinase-protein kinase B signaling pathway cellular proliferation (phosphoinositide-dependant kinase and protein kinase B [AKT]) and proapoptotic (BAD) factors with caspase 3 in cultured buffalo granulosa cells (GCs). The mRNA and protein expression of ANPT-1 was greatest (P < 0.05), whereas ANPT-2 was reduced (P < 0.05) in preovulatory follicles as compared to F1 follicle. Tyrosine kinase with immunoglobulin-like and EGF-like domains 1 transcripts and protein expression did not change in all follicular groups, whereas tyrosine kinase with immunoglobulin-like and EGF-like domains 2 mRNA was highest (P < 0.05) in theca interna but not GC layer of preovulatory follicle. All members of ANPT family were localized in GC and theca interna showing a stage specific immunoreactivity. Cultured GCs were treated with ANPT-1 and ANPT-2 separately at doses of 1, 10, and 100 ng/mL and in combination at 100 ng/mL for three incubation periods (24, 48, and 72 hours). Estradiol secretion was highest (P < 0.05) at 100 ng/mL at 72 hours of incubation when GCs were treated with either protein alone. The mRNA expression of phosphoinositide-dependant kinase and AKT was highest (P < 0.05), and BAD with caspase 3 was lowest (P < 0.05) at 100 ng/mL at 72 hours of incubation, when cultured GCs were treated separately with each protein or in combination. The immuoreactivity of AKT, pAKT, and pBAD were maximal, whereas BAD was minimal with 100 ng/mL at 72 hours when cultured GCs treated with either protein alone. The findings indicate that ANPTs are expressed in a regulated manner in buffalo ovarian follicle during different stages of development where they may promote steroidogenesis and GC survival through autocrine and paracrine actions., (Copyright © 2016 Elsevier Inc. All rights reserved.)