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The objective of the study was to estimate genetic and phenotypic trends for first lactation production and reproduction traits in Murrah buffaloes. The information of pedigree and targeted traits of 640 Murrah buffaloes was collected for the period from 1997 to 2020. The first lactation production traits included first lactation milk yield (FLMY), 305 days first lactation milk yield (305FLMY), first lactation length (FLL), first lactation peak yield (FPY) whereas reproduction traits included first service period (FSP), first calving interval (FCI) and first dry period (FDP). Genetic and phenotypic trends were estimated. Phenotypic trends for FLMY, 305FLMY, FLL and FPY exhibited as 36.96 ± 8.58 kg/year, 31.93 ± 8.34 kg/year, 1.47 ± 0.55 days/year and 0.12 ± 0.02 kg/year, respectively and respective genetic trends exhibited as 3.73 ± 1.67 kg/year, 1.94 ± 0.76 kg/year, -0.15 ± 0.07 days/year and 0.01 ± 0.01 kg/year, respectively. It was revealed that there were significant (p < 0.05) and positive phenotypic trends for all production traits while genetic trends were significant (p < 0.05) for FLMY and 305FLMY traits. The phenotypic trends of studied reproductive traits indicated that only FDP trait significantly (p < 0.01) decreased (1.87 days/year) over time. For FSP and FCI traits, nonsignificant (p > 0.05) genetic and phenotypic trends indicated no change over time. This study highlighted potential challenges in maintaining reproductive efficiency alongside productivity improvements in Murrah buffaloes., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

The present study analyses the effect of age at first calving (AFC) on future fertility and productivity in Murrah buffaloes. The data of 314 buffalo heifers of animal farm section, ICAR-CIRB, Hisar were collected over a period of 9 years from 2010 to 2018. The buffalo heifers were categorized into six groups according to the AFC named as 30-35, 36-41, 42-47, 48-53, 54-59 and 60-65 months. The influence of AFC on standard lactation milk (SLMY), peak yield (PY), days in milk (DIM), calving to first service, service per conception, calving to conception interval (CCI) and calving interval till fifth lactation were studied. The study revealed poor productive traits in buffalo heifers calved at younger age (30-35 months) during first parity. The productive value positively corresponded with increase in AFC. During successive lactations, higher mean milk yield (SLMY and PY) was found in groups with 36-41, 42-47 and 48-53 months. The mean number of services per conception was lower in buffalo heifers with 36-41 and 42-47 months following first calving till fifth lactation. Similarly, the said groups had lower mean calving to first service, CCI and CI up to fifth lactation. The survival rate was higher in heifers with AFC 36-41, 42-47, 48-53 and 54-59 months than with AFC 30-35 and 60-65 months. The buffalo heifers with 36-41 and 42-47 months of AFC had higher survival rate and better productive and reproductive traits till fifth parity in the current study. The study concluded that a minimum ideal AFC of 36-41 months yielded the highest productive gain., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

The aim of this research was to assess genetic parameters for first lactation production and reproduction traits in Murrah buffaloes by employing additive and maternal effects. Data on pedigree and specific traits of 640 Murrah buffaloes were gathered from 1997 to 2020. These traits encompassed first lactation milk yield (FLMY), 305-day first lactation milk yield (305FLMY), first lactation length (FLL), first lactation peak yield (FPY), first service period (FSP), first calving interval (FCI) and first dry period (FDP). Genetic evaluations employed six univariate animal models, accounting for both direct and maternal effects, facilitated by THRGIBBS1F90 and POSTGIBBSF90 programs. Fixed factors included in the analysis were period of calving, season of calving and age at first calving. The Bayesian estimates for direct heritability, derived from the most suitable model, were as follows: FLMY: 0.28 ± 0.01, 305FLMY: 0.30 ± 0.01, FLL: 0.19 ± 0.01, FPY: 0.18 ± 0.01, FSP: 0.12 ± 0.01, FCI: 0.14 ± 0.01 and FDP: 0.12 ± 0.01. Maternal effects were found significant, ranging from 5% to 10%, in first lactation traits under Model 2 and Model 5. Additionally, positive and significant genetic and phenotypic correlations were observed among the studied traits. In conclusion, selection based on 305-day first lactation milk yield suggests potential for genetic enhancement in Murrah buffaloes, advocating its inclusion in breeding programmes to bolster early performance. Also, consideration of maternal influences is necessary for genetic progress of animals., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Sub-estrus buffaloes do not exhibit estrus signs despite being cyclic contributing to extended service periods and inter-calving intervals causing significant economic loss. The present study described the effect of synthetic prostaglandin (PGF 2α ) on estrus behaviour, follicular and luteal morphometry, and serum estradiol (E 2 ) and progesterone (P 4 ) profile in sub-estrus buffaloes during the non-breeding season. The incidence of sub-estrus was 38.4% during the non-breeding season. The sub-estrus buffaloes (n = 33) were divided into two groups, viz., Control (n = 16) and PGF 2α treatment (Inj. Cloprostenol 500 μg, i.m., n = 17). Estrus induction response was significantly greater in the treatment (100 vs. 18.75%, p < .001), and a relatively greater proportion of animals conceived in the treatment group (29.41 vs. 6.25%, p = .08). The time elapsed to induction of estrus and insemination following treatment was significantly lower in the treatment group than control. A significant increment in the follicle diameter (9.72 ± 0.45 vs. 13.00 ± 0.45 mm, P < .0001) and serum estradiol (E 2 ) concentration (66.01 ± 11.92 vs. 104.9 ± 13.21 pg/mL, p = .003) observed at the post-treatment period in the PGF 2α treatment group. At the same time, CL diameter was reduced significantly at a higher regression rate in the PGF 2α treated buffaloes than those of control. Of the responded buffaloes, only 30% showed high-intensity estrus attributed to the expulsion of cervico-vaginal mucus (CVM), uterine tonicity, micturition, and mounting response by a teaser bull. From this study, it can be concluded that the administration of PGF 2α could induce estrus in the sub-estrus buffaloes during the non-breeding season. Behavioural changes, along with sonographic observation of POF, regressing CL, and serum E 2 and P 4 concentration would be useful to determine the right time of insemination in sub-estrus buffaloes during non-breeding season., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Sub-estrus is a condition when buffaloes do not display behavioural estrus signs, despite being in estrus and causes a delay in conception and increases the service period. The present study describes the effect of synthetic prostaglandin (PGF 2α ) alone and in combination with trace minerals on the follicular and corpus luteum (CL) dynamics, serum estradiol (E 2 ) and progesterone (P 4 ) concentration correlating estrus response and pregnancy outcome in sub-estrus buffaloes during the breeding season. A total of 50 sub-estrus buffaloes, identified through ultrasonography (USG) examination, were randomly allocated into three groups, viz. T1 (Synthetic PGF 2α , Inj. Cloprostenol 500 μg, i.m, n = 17), T2 (Synthetic PGF 2α  + Trace mineral supplementation, Inj. Stimvet 1 mL/100 kg body weight, i.m., n = 17) and control (untreated; n = 16). Following treatment, 100% of sub-estrus buffaloes were induced estrus in the T1 and T2 groups, while only 18.75% were induced in the control. The CL diameter and serum P 4 concentration were significantly lower at post-treatment, whereas the pre-ovulatory follicle (POF) size and serum E 2 concentration were significantly higher in the T1 and T2 groups as compared to the control (p < .05). The buffaloes of the T2 group had a greater proportion of moderate intensities estrus than those of T1. Moreover, the proportion of buffaloes conceived in the T1 and T2 were 41.2% and 52.95%, respectively. The larger POF diameter and higher serum E 2 concentration were associated with intense intensity estrus and higher conception rate (66.7%) in sub-estrus buffaloes. Similarly, CL regression rate, POF size and serum E 2 concentration were relatively higher in the buffaloes conceived as compared to those not conceived. It is concluded that synthetic PGF 2α in combination with trace minerals induces moderate to intense intensities estrus in a greater proportion of sub-estrus buffaloes and increases the conception rate during the breeding season. Moreover, behavioural estrus attributes correlating follicle and luteal morphometry, serum E 2 and P 4 concentration could be used to optimise the breeding time for augmenting the conception rate in sub-estrus buffaloes., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Oestrus is defined as a period when a female animal exhibits characteristic sexual behaviour in the presence of a mature male. Oestrous manifestation in dairy animals is due to the oestrogen (E 2 ) effect on the central nervous system (CNS). It is a critical issue to be considered on a priority basis. Inefficient oestrous detection reduces the fertility status of the herd. The primary and most reliable indicator of oestrus is standing to be mounted by a bull or another female herd mate, signalling receptivity and the pre-ovulatory state in dairy cattle. Oestrous detection is primarily a management challenge requiring skill and vigilance. To improve the efficiency of oestrous detection in dairy cattle, visual observation is one of the best methods if done three times a day; however, heat detection aids, if combined, give better results. However, techniques like using teaser bulls, tail painting, chin ball markers, ultrasound (USG) examination, hormonal analysis and examination of cervicovaginal mucus (CVM) improve oestrous detection efficiency. Moreover, the changes in production systems have reduced the expression of oestrous behaviour among cows, due to higher oestrogen (E 2 ) metabolism. Therefore, automated systems, such as pedometers, accelerometers and acoustic sensors like infrared thermography (IRT) and image processing, have significantly enhanced reproductive performance by facilitating oestrous detection and optimizing insemination schedules. From this review, we would conclude that oestrous detection alone contributes considerably to the reproductive status of the herd; therefore, applying different methods of oestrous detection reduces the incidence of missed oestrus and improves the fertility status of the herd., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Orexin is a ligand for orexin receptors OX1R and OX2R; it is a neuropeptide with pleiotropic functions, including regulation of reproduction. The current study was carried out to investigate the mRNA expression of the prepro-orexin gene (PPO) and orexin receptors (OX1R and OX2R) in ovarian follicles during different stages of their development in the ovary of water buffalo (Bubalus bubalis) and to determine the role of orexin on oestradiol production. Ovarian follicles were classified into four groups based on size and oestradiol (E 2 ) level in the follicular fluid (FF) as follows: (i) small or F1, (ii) medium or F2, (iii) large or F3, and (iv) dominant/pre-ovulatory follicle or F4. In follicles, the mRNA expression of PPO and OX1R was greater in F3 and F4 follicles in granulosa cells (GC) and theca interna (TI) cells. OX2R expression did not vary among the different follicular stages in GC. Orexin-A and orexin receptors were localized in the cytoplasm of GC and TI, and intensity was higher in F3 and F4 follicles. In addition, we cultured GC and treated them at 0.1, 1.0, and 10 ng/mL orexin-A alone or in the presence of FSH (30 ng/mL) or IGF-I (10 ng/mL) for 48 h. There was a significant (p < .05) increase in oestradiol (E 2 ) secretion and cytochrome P0450 family 19 subfamily A member 1 (CYP19A1) expression from GC at 1.0 and 10.0 ng/mL orexin-A in the presence of 30 ng/mL follicle-stimulating hormone (FSH) or 10 ng/mL insulin-like growth factor-I (IGF-I). In conclusion, the present study provided evidence that the orexin system is expressed in buffalo ovarian follicles, and orexin-A in the presence of FSH and IGF-I has a stimulatory effect on oestradiol secretion from the GC of water buffalo., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

The cryotolerance of semen obtained from Mediterranean buffalo bulls usually is more likely to deteriorate during the summer. To obtain the optimal sperm for fertility, the physiological status and reproductive performance of Mediterranean buffalo bulls in the summer and spring were first analysed by assessing blood serum and seminal plasma samples; then, the lipid profiles of seminal plasma were investigated by LC-MS/MS. The T, T3 and SOD levels of serum and seminal plasma in the spring were significantly higher than in the summer (p < .05). The results suggest that T3 level is positively correlated with semen cryotolerance; sphingolipids are potential markers for semen cryotolerance of Mediterranean buffalo. To our knowledge, this is the first report of targeted lipidomics in semen cryotolerance., (© 2022 Wiley-VCH GmbH.)

Heat stress (HS) during pregnancy adversely affects uterine vascular perfusion and foetal development. L-arginine (L-Arg), a nitric oxide (NO) precursor, has been proven to enhance an organ's vascular perfusion. Therefore, this study aimed to examine the effect of L-Arg administration on the pregnant buffaloes' uterine haemodynamics and uteroplacental thickness under environmental HS conditions. For this purpose, pluriparous (n = 12) HS-pregnant buffaloes (mid-gestation, 180-190 days; temperature humidity index >85) were haphazardly assigned into two groups, either administered a single intravenous (IV) bolus of 5 mg/kg BW of L-Arg Hcl (n = 6; ARG) or received normal saline (0.9%; IV; 25 ml) that served as a control group (n = 6; CON). Combined uteroplacental thickness (CUPT, mm), middle uterine arteries' (MUA) diameter (mm) and uterine Doppler haemodynamics (ipsilateral and contralateral to the pregnant horn) were examined utilizing B-mode and colour Doppler ultrasonography, respectively. The serum levels of progesterone (P4), estradiol 17β (E2), nitric oxide (NO) and total antioxidant capacity (TAC) were assayed using commercial kits. The experimental time points were - 1, 0, 2, 4, 24, 48, 72, 96 and 120 h post-L-Arg administration. The ARG group showed higher (p < .05) CUPT and MUA diameter values starting at 24 h post-treatment and onward than the control buffalo cows. In addition, there were improvements (p < .05) in the blood flow parameters in the ipsilateral MUA after L-Arg treatment evidenced by lower values of pulsatility and resistive indices starting at 48 and 4 h, respectively and onward, and higher values of peak systolic velocity, coloured areas towards the pregnant uterine horn (both 24-72 h). Increases in the NO levels were found during the period between 4 and 120 h in the ARG group compared to the CON group. Moreover, significant increases in the E2 and P4 means were noted in the ARG group, especially at 24 h onward, respectively compared to the CON group. Concerning the TAC status, neither the administration nor the hours affected serum TAC levels in the HS pregnant buffaloes (CON and ARG). In conclusion, L-Arg administration improved uteroplacental thickness and enhanced uterine haemodynamics, NO levels, and steroids production in mid-pregnant buffalo cows under environmental heat stress conditions which could improve foetal growth and development., (© 2022 Wiley-VCH GmbH.)

The aim of the first study was to determine the influence of the calf handling (restricted or full contact) and fenceline bull stimulation on performance of mothers and their calves. The aim of the second study was to determine the relative impact of calf-bull contact, comparing the result of restricted calf and no bull with that of fenceline calf contact and direct bull contact on the performance of mother buffaloes and their calves. In Experiment 1, 24 mother-calf dyads were allocated to three treatments: NCB (no calf-mother-bull contact), RC-FBC (restricted calf and fenceline bull contact) and FC-FBC (fenceline calf and fenceline bull contact). In Experiment 2, another 24 mother-calf dyads were allocated to two treatments: RC-NB (restricted calf and no bull contact) and FC-DBC (fenceline calf and direct bull contact). In Experiment 1, average daily gain (ADG) was greater in FC-FBC than RC-FBC and NCB calves (p < .05). Average dry matter intake (DMI) was greater in FC-FBC than RC-FBC and NCB calves. DMI of buffaloes was greater in FC-FBC than RC-FBC and NCB. Daily milk yield of buffaloes was more (p < .05) in FC-FBC (10.2 ± 0.2 kg) than RC-FBC (8.4 ± 0.2 kg) and RC-FBC (7.8 ± 0.2 kg). There were no statistical differences observed in the interval from calving to first oestrus, first service, service period among three groups. In Experiment 2, ADG's and DMI were greater in FC-FBC calves than RC-FBC (p < .05). DMI of buffaloes was greater in FC-DBC than RC-NBC (p < .05). Daily milk yield of buffaloes was greater (p < .05) in FC-DBC (11.6 ± 0.2 kg) than RC-NBC (8.3 ± 0.2 kg). The first oestrus (51.5 ± 3.1 vs. 103.2 ± 7.2), days to first service (74.8 ± 3.5 vs.112.0 ± 7.6) and service period (78.8 ± 4.8 vs.118.9 ± 8.3 days) were lower (p < .05) in FC-DBC than RC-NBC buffaloes. The fenceline calf-mother contact increased the growth rate, FCR, and feeding efficiency of buffalo calves; reduced some stress responses and improved the productive performance of buffalo mothers. Further, mother-bull contact stimulated the post-partum reproductive performance of buffaloes in fenceline calf contact., (© 2022 Wiley-VCH GmbH.)

This current study aimed for the first time to evaluate the effect of a single intravenous administration of human chorionic gonadotropin hormone (hCG) on the testicular artery haemodynamics measurements (resistance [RI], and pulsatility indices [PI]), plasma steroids (estradiol-17β and testosterone) and nitric oxide (NO) levels in buffalo bulls. Twelve Egyptian buffalo bulls weighted 450 ± 20 kg were randomly divided into the hCG group (n = 6) and injected a single dose with Ovogest (EPIFASI; 5,000 IU, iv), whereas the others (n = 6) were injected with normal saline and served as controls. Doppler evaluation and blood sampling were performed just before the administration (hour 0) and at 1, 2, 4, 6, 8, 10, 24 and 28 hr after administration. Bulls in the control group did not show any alterations in hormonal levels and blood flow parameters (p > .05). In the hCG group, RI was declined (p < .05) in 6 hr post-administration (0.31 ± 0.01 versus 0.45 ± 0.01), while PI was declined (p < .05) later in 10 hr (0.74 ± 0.01 versus 1.23 ± 0.02). Additionally, testicular blood flow was increased (p < .05) 8 hr (42.02 ± 1.02 ml/min/100 g versus 31.34 ± 0.88 ml/min/100 g) after administration. Testosterone and NO levels were (p < .05) increased at 4 and 6 hr post-administration (3.55 ± 0.03 ng/ml versus 2.84 ± 0.01 ng/ml, and 55.32 ± 4.25 µmol/L versus 32.21 ± 1.55 µmol/L), whereas oestradiol levels were elevated (p < .05) in 6 hr (31.25 ± 0.08 pg/ml) only post-administration then declined. In conclusion, the single intravenous administration of hCG triggered many alterations in the supratesticular artery vascularization and hormonal profile that could affect positively on steroidogenesis and testicular function in buffalo bull., (© 2022 Wiley-VCH GmbH.)

The microenvironment in the seminiferous tubules of buffalo changes with age, which affects the self-renewal and growth of spermatogonial stem cells (SSCs) and the process of spermatogenesis, but the mechanism remains to be elucidated. RNA-seq was performed to compare the transcript profiles of pre-pubertal buffalo (PUB) and adult buffalo (ADU) seminiferous tubules. In total, 17,299 genes from PUB and ADU seminiferous tubules identified through RNA-seq, among which 12,271 were expressed in PUB and ADU seminiferous tubules, 4,027 were expressed in only ADU seminiferous tubules, and 956 were expressed in only PUB seminiferous tubules. Of the 17,299 genes, we identified 13,714 genes that had significant differences in expression levels between PUB and ADU through GO enrichment analysis. Among these genes, 5,342 were significantly upregulated and possibly related to the formation or identity of the surface antigen on SSCs during self-renewal; 7,832 genes were significantly downregulated, indicating that genes in PUB seminiferous tubules do not participate in the biological processes of sperm differentiation or formation in this phase compared with those in ADU seminiferous tubules. Subsequently, through the combination with KEGG analysis, we detected enrichment in a number of genes related to the development of spermatogonial stem cells, providing a reference for study of the development mechanism of buffalo spermatogonial stem cells in the future. In conclusion, our data provide detailed information on the mRNA transcriptomes in PUB and ADU seminiferous tubules, revealing the crucial factors involved in maintaining the microenvironment and providing a reference for further in vitro cultivation of SSCs., (© 2021 Wiley-VCH GmbH.)

A female buffalo (Bubalus bubalis) of the Bulgarian Murrah breed aged 1,090 days was observed to give birth to a second newborn (normally developed male) after she had calved (normal female) 49 days earlier. This phenomenon is highly associated with her melatonin treatment within a trial for induction of puberty, the last ear implants being placed approximately 50 days before the assumed date of first mating, to which point the level of progesterone had increased dramatically. Despite none of the matings of the dam was visually witnessed to prove ovulation over an existing gestation, we take the liberty to qualify this phenomenon as superfetation, ruling out the other possible phenomena, namely embryonic diapause as it is highly unlikely to occur in any livestock species, and differentiated development of twin foetuses as it is associated with foetal malformation, which was not observed in this case., (© 2020 Wiley-VCH GmbH.)

Calving ease (CE) is a trait of economic importance that affects animal welfare and farm profitability. The objective of present study was to investigate genetic and environmental factors affecting CE among Primiparous (PP) and multiparous (MP) buffaloes. A total of 9,627 records from 1999 MP and 2,110 PP recorded during the period from 1988 to 2018 were considered. Herd, season of calving, year of calving, birth weight, parity order and gestation length significantly affected CE rate, while age at first calving and sex of calf had no significant effects. Direct and maternal heritabilities of CE in PP and MP were 0.06 and 0.01, respectively. The low heritability of CE indicated that direct selection may not be an effective method to improve CE trait in Egyptian buffalo., (© 2020 Wiley-VCH GmbH.)

The aims of the present study were to determine uterine, vaginal and placental blood flows by Doppler ultrasound cross-buffalo gestation and to evaluate the relationships among reproductive Doppler parameters and serum metabolic parameters as well as oxidative stress. Uterine (UA) and vaginal (VA) arteries were scanned every month, and placentome was scanned from month 4 till 8 in gestation. Time-averaged maximum velocity (TAMV), pulsatility index (PI), resistance index (RI), systolic/diastolic ratio (SD) and arterial diameter (AD) were used for accessing UA and VA hemodynamics. Time-averaged maximum velocity positively correlated with and AD, and both negatively correlated with their PI, RI and SD in UA and VA. TAMV and AD increased constantly in pregnancy, with maximum increase in months 4 and 9. Pulsatility index, RI and AD of UA decreased between months 4 and 9, while PI, RI and AD of VA decreased between months 5 and 9 and then increased in month 10 in pregnancy. Time-averaged maximum velocity of placentome blood flow increased exponentially from months 4 to 8, but decreased at the last two months in pregnancy. Serum lipids were significantly higher in the first month compared to all other months, while glucose was significantly lower in months 9 and 10. Malondialdehyde increased from month 3 till term, but peaked in month 5 and 10. Glutathione and catalase were highest in the first month and remained after. Time-averaged maximum velocity and AD for both UA and VA negatively correlated with serum lipids, glucose, catalase and glutathione, while positively correlated with malondialdehyde and total protein. Thus, increases in uterine blood flow (UtBF), vaginal blood flow (VaBF) and placental blood flow (PaBF) are associated with increased metabolism and oxidative stress in buffalo pregnancy., (© 2020 Wiley-VCH GmbH.)

In Brazil, water buffaloes have been used to produce milk for mozzarella cheese production. Consequently, the main selection criterion applied for the buffalo genetic improvement is the estimated mozzarella yield as a function of milk, fat and protein production. However, given the importance of reproductive traits in production systems, this study aimed to use techniques for identifying genomic regions that affect the age at first calving (AFC) and first calving interval (FCI) in buffalo cows and to select candidate genes for the identification of QTL and gene expression studies. The single-step GBLUP method was used for the identification of genomic regions. Windows of 1 Mb containing single-nucleotide polymorphisms were constructed and the 10 windows that explained the greatest proportion of genetic variance were considered candidate regions for each trait. Genes present into the selected windows were identified using the UOA&#95;WB&#95;1 assembly as the reference, and their ontology was defined with the Panther tool. Candidate regions for both traits were identified on BBU 3, 12, 21 and 22; for AFC, candidates were detected on BBU 6, 7, 8, 9 and 15 and for first calving interval on BBU 4, 14 and 19. This study identified regions with great contribution to the additive genetic variance of age at first calving and first calving interval in the population of buffalo cows studied. The ROCK2, PMVK, ADCY2, MAP2K6, BMP10 and GFPT1 genes are main candidates for reproductive traits in water dairy buffaloes, and these results may have future applications in animal breeding programs or in gene expression studies of the species., (© 2020 Wiley-VCH GmbH.)

Theca cells (TCs) play a key role in follicular growth and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to estrogens needed for oocyte maturation. However, the effects of TCs in the form of conditioned medium on in vitro maturation (IVM) and developmental competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of TC-conditioned medium (TCCM) on maturation efficiency and embryo development of buffalo oocytes after parthenogenic activation (PA). Our results showed that TCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days & 20%) exerted no significant effect on IVM rate (43.06% vs. 44.71%), but significantly (p  < .05) enhanced embryo development (oocyte cleavage, 80.93% vs. 69.66%; blastocyst formation, 39.85% vs. 32.84%) of buffalo oocytes after PA compared with the control group. However, monolayer TC significantly (p < .05) promoted both maturation efficiency (48.84% vs. 44.53%) and embryo development (oocyte cleavage, 80.39% vs. 69.32%; blastocyst formation, 35.38% vs. 29.25%) of buffalo oocytes after PA compared to that in the control group. Furthermore, TCs secreted some testosterone into the conditioned medium, which significantly (p < .05) promoted the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 17β-HSD) in buffalo cumulus-oocyte complexes (COCs). Our study indicated that TCCM (2 days & 20%) did not significantly affect IVM efficiency, but enhanced embryo developmental competence of oocytes after PA principally by stimulating the secretion of testosterone and facilitating estradiol synthesis of buffalo COCs., (© 2020 Wiley-VCH GmbH.)

Although reduced reproductive efficiency during summer has been well documented in buffaloes, the reason for the same is yet to be understood. The present study was conducted to identify the subtle differences in sperm phenotypic characteristics (motility, membrane integrity, acrosome reaction and lipid peroxidation status), oviduct binding ability and expression of fertility-associated genes (AK 1, ATP5D, CatSper 1, Cytochrome P450 aromatase, SPP1 and PEBP1) between winter and summer seasons in buffaloes. Cryopreserved spermatozoa from 6 Murrah buffalo bulls (3 ejaculates/bull/season) were utilized for the study. Real-time quantitative PCR was performed for assessing the expression patterns of select fertility-associated genes. The proportion of motile and membrane intact spermatozoa was significantly higher (p < .05) in winter as compared to summer ejaculates. The proportion of moribund and lipid peroxidized spermatozoa was significantly lower (p < .05) in winter ejaculates as compared to summer. The sperm-oviduct binding index was significantly lower (p < .01) when spermatozoa from summer ejaculates were used as compared to winter ejaculates. The expression of fertility-associated genes did not differ significantly between the two seasons except for PEPB1; the transcriptional abundance of PEPB1 was significantly (p < .05) lower in summer as compared to winter season. It was inferred that buffalo spermatozoa produced during winter season were superior in terms of cryotolerance, membrane and acrosome integrity, lipid peroxidation status and the ability to bind with oviduct explants., (© 2020 Wiley-VCH GmbH.)

Tumour necrosis factor-α (TNF-α) is a cytokine that plays multiple important roles in corpus luteum (CL). Immunolocalization of expression of TNF-α in CL of buffalo was studied in different stages of its development and regression. Corpus luteum of healthy buffaloes (24) was collected from local slaughterhouses and categorized into early (stage I, 1-5 days, n = 6), mid (stage II, 6-11 days, n = 6), late luteal (stage III, 12-16 days, n = 6) and regressing phase (stage IV, 17-20 days, n = 6). In earliest phase of cyclic CL, per cent immunoexpression of TNF-α was significantly (p < .05) lower as compared to all phases with its expression being restricted to few developing luteal cells, usually in neutrophils. A significantly (p < .05) higher number of neutrophils with TNF-α immunoexpression were observed as compared to mid-luteal phase that indicated its role in initiation of angiogenesis at this stage. TNF-α immunoexpression almost doubled in mid-luteal phase, but the number of neutrophils exhibiting TNF-α was significantly (p < .05) lower with respect to all phases of CL. Immunoexpression percentage in late luteal phase increased sharply being significantly (p < .05) higher than earlier two phases of CL. In regressing phase, per cent immunostaining was maximum with highly significant (p < .05) difference as compared to all other stages, observed in all degrading luteal cells, abundant immune cells, that is neutrophils and macrophages which finally led to apoptosis and phagocytosis. Immunoexpression of TNF-α in early luteal phases served its role in initiation of angiogenesis, and its intense expression in regressing phase of CL suggested a shift in its role to apoptosis and structural luteal regression signifying both luteotropic and luteolytic roles in buffalo. This is probably the first study of its kind in buffaloes., (© 2020 Wiley-VCH GmbH.)

Buffalo is considered short-day breeder in tropical and subtropical part of the world and seasonality and photoperiodism impart major influence on its fertility. However, its impact on in vitro embryo production (IVEP) remains elusive. Therefore, this study investigated the effect of seasonal variations and photoperiodism on morphological and molecular parameters of IVEP in buffalo. For this purpose, we conducted two different experiments on the oocytes obtained by aspirating follicles from abattoir derived ovaries. In Exp. I, retrospective analysis was performed for oocyte recovery, blastocyst and hatching rate, during four consecutive seasonal periods (i.e. January-March, April-June, July-September and October-December). In Exp. II, oocytes from peak breeding and non-breeding seasons were subjected to 24 hr in vitro maturation and evaluated for polar body extrusion to assess maturation rate. Results showed that embryo development was markedly low during second quarter (April-June) and maximum during fourth quarter (October-December) of the year; referred as non-breeding and breeding seasons, respectively. Comparative data analysis demonstrated that poor oocyte quality is major reason for lesser efficiency of embryo production during non-breeding season than peak breeding season as suggested by poor oocyte recovery (2.31 ± 0.10 vs. 3.65 ± 0.27) and maturation rate (33.32 ± 2.1 vs. 63.15 ± 7.31). Subsequently, comparative gene expression analysis of blastocysts during peak breeding season significantly upregulated pluripotency gene (OCT-4) and downregulated heat shock protein 90, as compared to non-breeding season. Therefore, it could be divulged from the present study that seasonal variations and photoperiodism have profound effect on oocyte quality and subsequent embryo development. It is recommended to find suitable additives for in vitro maturation that could mitigate seasonal effects., (© 2020 Blackwell Verlag GmbH.)

The study was designed to decipher the inter-relationship between peripheral hormones (kisspeptin and testosterone), sexual behaviour and seminal variables of Murrah buffalo bulls (n = 134). In this study, we recorded that 13%, 37%, 40%, 6% and 4% Murrah buffalo bulls had reaction time of <30, 31-60, 61-180, 181-300 and >300 s, respectively. Further, it was observed that 4%, 85% and 10% buffalo bulls were sexually aggressive, active and dull, respectively, during semen collection. The courtship behaviour was not found to be desirable for the bulls used for the semen collection. Mean of ejaculate volume, sperm concentration and mass motility (0-5 scale) were 3.57 ml, 977.11 million/ml, 2.7, respectively. Correlation studies revealed that the reaction time was positively correlated with courtship behaviour and body weight, and negatively correlated with sexual aggressiveness and sperm concentration. Serum kisspeptin in buffalo bulls, measured for the first time, was found to 3.8 ± 0.7 ng/ml. Serum kisspeptin and testosterone level are negatively correlated to each other and kisspeptin level influenced the sexual behaviour (reaction time, sexual aggressiveness and penile erection) of study bulls. Serum kisspeptin was higher in the buffalo bulls with higher sperm concentration indicating its role in spermatogenesis. In conclusion, for the first time basic information related to sexual behaviour of Murrah buffalo bulls in large population along with its inter-relationship with peripheral hormones (kisspeptin and testosterone) has been documented., (© 2020 Blackwell Verlag GmbH.)

The region-specific expression of seminal proteins in testis and excurrent duct system determines the quality and function of the spermatozoa. In the present study, localization and expression of some of the seminal proteins such as insulin-like growth factor receptor 1β (IGF-1Rβ), phosphatidylethanolamine-binding protein 4 (PEBP4), α-tubulin and tissue factor pathway inhibitor 2 (TFPI2) were carried out in testis, excurrent duct system and spermatozoa of buffalo. IGF-1Rβ was localized in the cells of the seminiferous tubules of the testis, except in primary spermatocytes. The PEBP4 was localized only in the elongated spermatid, whereas α-tubulin and TFPI2 proteins were localized in all cells of the seminiferous tubule including spermatocyte. In the buffalo spermatozoa, IGF-1Rβ, PEBP4, α-tubulin and TFPI2 were localized in the acrosome region, the post-acrosomal region till the tail end, post-acrosome to the entire tail region and the equatorial region, respectively. The study indicates that IGF-1R, α-tubulin and PEBP4 proteins regulate spermatogenesis, whereas TFPI2 may be involved during the zona binding process of the buffalo spermatozoa., (© 2020 Blackwell Verlag GmbH.)

Silent oestrus is an unsurmountable problem in the management of buffalo reproduction. In addressing this issue, we have earlier reported variation in the levels of urinary luteinizing hormone (LH) through the different phases of oestrous cycle with an extended window during the mid-oestrous phase. Based on this report, the present study is designed to assess the salivary LH levels in buffalo during the different phases of oestrous cycle. Bovine LH ELISA kit was used to determine the level of salivary LH. We observed a notable variation in salivary LH levels during the different phases of oestrous cycle. The maximum LH level, 39.07 mIU/ml, observed during oestrus, which was significantly (p < .05) higher than other consecutive phases. Altogether, the results showed a significant (p < .05) fold variation during oestrus compared with other phases. Therefore, the study convincingly shows that salivary LH has the potential of application in development of a modality for non-invasive oestrous detection in buffalo., (© 2020 Blackwell Verlag GmbH.)

This study aimed to examine the teat characteristics in relation to the animal temperament during milking in the Anatolian buffaloes using ultrasonographic, histomorphological and immunohistochemical methods. The teat canal length (TCL), teat wall thickness (TWT), teat cisternal diameter (TCD), teat diameter (TD), teat length (TL), and teat circumference (TC) values in docile (n = 5) and nervous (n = 7) buffaloes were measured at the 0th, 3rd and 6th minute of stimulation. In additional experiments, comparative histomorphology and immunohistochemical examinations of buffalo (n = 7) and cow teats (n = 8) were performed. It was determined that post-stimulation mean TCL values were significantly higher in nervous buffaloes than those of teats in docile buffaloes (p < .05). A significant positive correlation between TCD and TD, TL and TC in both docile and nervous buffaloes was noted (p < .05). Unlike nervous buffaloes where only 3/14 teat canals were open by 3rd minute of milking stimulation, almost all (9/10) teat canals were observed opened in docile buffaloes. There were fewer muscle but more collagen bundles in buffalo teats compared with cow teats. It seems that temperament of animal during milking effects the milking efficiency, and in nervous buffaloes, probably the stimulation alone may not be sufficient for opening of the teat canal and hence achieve complete milking., (© 2020 Blackwell Verlag GmbH.)

This study was designed to investigate the hormonal and metabolic factors associated with placental retention in buffaloes with respect to the roles of oxidative stress biomarkers and serotonin receptors. Blood samples were collected at weeks 3, 2 and 1 pre-partum and at calving from 37 buffaloes; thirty normally dropped their placentae (Non-RFM group) and 7 dropped their placentae after 12 hr post-calving (RFM group). Serum progesterone (P 4 ), oestradiol, cortisol, non-esterified fatty acids (NEFA), beta-hydroxybutyric acid (BHBA), antioxidant/oxidant biomarkers and mineral concentrations were assessed. Histopathology and histochemistry were implemented to detect collagen in foetal placental tissues. Immunohistochemistry for serotonin receptors in placental tissues was performed. Significant elevations in P 4 , cortisol, NEFA, BHBA and oxidative biomarkers concentrations were observed in the RFM group. However, oestradiol, antioxidants and mineral concentrations were significantly lower in RFM buffaloes than Non-RFM group. Histopathological examination revealed degenerative changes and necrosis in retained placental tissue compared with that in normal placental tissues. Serotonin receptors were significantly expressed with collagen condensation in retained placental tissues. Furthermore, inferior reproductive performance was pronounced in the retained group. In conclusion, retained foetal membranes in buffaloes were associated with hormonal imbalance, metabolic perturbation, oxidative stress, serotonin receptor upregulation and markedly reduced fertility indices., (© 2020 Blackwell Verlag GmbH.)

Hypothalamic gonadotropin-releasing hormone (GnRH) controls the activity of hypothalamic-pituitary-gonadal axis and plays a key role in the reproductive performance of animals. In this study, five single nucleotide polymorphisms (SNPs), namely g.991T > C, g.1041T > C g.3424T > C, g.3462C > A and g.3463Inde A, were detected in the GnRH gene of 162 water buffaloes by Sanger sequencing. Each SNP was associated with more than two sperm quality traits of ejaculate volume, sperm concentration, post-thaw sperm motility and sperm abnormality. g.3424T > C and g.3462C > A were related to these four traits and had a remarkable effect on ejaculate volume. The three other SNPs were related to sperm concentration, post-thaw sperm motility and sperm abnormality. Moreover, six haplotypes (H1: TCCAI, H2: CTTC-, H3: TCCCI, H4: CTTA-, H5: CCTA- and H6: CTCC-) composed of five SNPs comprising seven different combined genotypes were generated by linkage disequilibrium analysis. Statistics followed by one-way ANOVA indicated that water buffaloes with the haplotype combination H1H1 had the highest genotypic frequency, and those with the H4H4 haplotype combination had the highest ejaculate volume. The sperm concentration of those with haplotype combination H1H5 was higher than that of the other genotypes. In summary, our study showed a remarkable association between the SNPs of GnRH and sperm quality traits of Chinese water buffalo., (© 2020 Blackwell Verlag GmbH.)

The current study aimed to investigate the effect of testicular artery angioarchitecture on the spermiogram parameters in Egyptian Buffalo bulls. Eight adult buffalo bulls aged between 2 and 8 years were used for semen evaluation. For anatomical studies, the masculine gonads were collected after slaughtering 30 adult bulls and prepared for injection by different masses (Urographine ® , Latex and Epoxy) through the testicular artery. The mass activity of the ejaculate was assessed immediately after collection. The sperm motility in fresh bull ejaculate was more than 80%. The overall mean percentage of sperm abnormalities was <18%. The recorded sperm abnormalities were mostly secondary one including distal protoplasmic droplet, fragmented tail, detached head, detached galea capitis and bent tail. The highest percentage of sperm viability was recorded just after sperm collection (alive > 85%). The results revealed that testicular artery can be divided into three parts (abdominal, funicular and marginal parts) along its course. The coils of the funicular part forming a cone-like structure with its base fixed to the head of the testis. Two epididymal branches to the head and tail of epididymis emanate from the funicular part which continues as pars marginalis on the lateral surface of testis before its division into the lateral and medial testicular arteries on approaching the tail extremity of the testis. The increase in the length of the testicular artery with increase in the size of the testes played a great role in the degree of complexity of the architectural vascular patterns. The degree of complexity is affected by the number of coils formed by the vessel. The increase in the convolutions of the vessel will reduce the speed of blood flow to the gonads. Thus in turn will enabling the thermoregulatory mechanism to work more efficiently and will affect the semen value., (© 2020 Blackwell Verlag GmbH.)

At present, many three-dimensional (3D) culture systems have been reported, improving the oocyte quality of in vitro maturation (IVM), yet the mechanism still needs to be further explored. Here we examined the effects of a new self-made 3D glass scaffold on buffalo oocyte maturation; meanwhile, the underlying mechanism on buffalo oocyte maturation was also detected. Compared to the two-dimensional (2D) glass dish culture, results revealed that the 3D culture can improve the first polar body rate of oocytes, subsequent cleavage and blastocysts rate of parthenogenetic activation embryos (p < .05). The extracellular matrix-related proteins COL1A1, COL2A1, COL3A1, FN and cell connection-related proteins N-cadherin, E-cadherin, GJA1 were found higher in cumulus cells of 3D culture. Moreover, in cumulus cells, proteins of the PI3K/AKT pathway reported being regulated by FN and E-cadherin including PI3K P85 and p-AKT were also higher in 3D culture. Furthermore, proapoptosis proteins P53, BAX, caspase-3 were lower in both cumulus cells and oocytes in 3D culture, while proteins PCNA and BCL2 showed the opposite result. Results also showed that the apoptosis was inhibited, and the proliferation was enhanced in cumulus cells of 3D culture. Finally, the cumulus expansion-related genes HAS2, CD44, HMMR, PTX3, PTGS2 were found higher in cumulus cells of 3D culture. Taken together, the 3D culture could promote oocyte maturation by regulating proteins correlated with the ECM, cell connection and PI3K/AKT pathway, inhibiting the apoptosis of cumulus cells and oocytes, enhancing the proliferation of cumulus cells and the cumulus expansion., (© 2019 Blackwell Verlag GmbH.)

Placenta is formed by a parenchyma rich in extracellular matrix (ECM), and this structure guarantees the proper development of the embryo and placental functioning. Recently, studies have focused on the characterization of ECM in the placenta and foetal membranes of different species. This work aimed to analyse the composition of the ECM and to quantify the types of collagens in its composition. For this, 33 chorioallantoic membranes were used at different gestational ages, which were grouped into five groups. Subsequently, haematoxylin-eosin staining, Masson trichrome and picrosirius were performed for histological analysis. Through the technique of polarized light, it was possible to quantify the total collagen present in the membranes and finally the immunohistochemical technique was performed to verify the presence of collagens and glycoproteins. It was possible to verify that the chorioallantoic membranes have, in all the gestational periods of the initial third of gestation, the same histological structures, being the most significant difference the membrane thickening that occurs gradually during the gestation. However, we notice the appearance of binucleate cells only from group II. In addition, it was verified that a gradual increase of collagen occurs until the group IV, yet from the group V begins to occur a decrease of this protein. In addition, collagen I, collagen III, fibronectin and laminin were present in all membranes. With this, we concluded that the buffalo chorioallantoic membrane presents ECM in constant remodelling at the beginning of gestation and can be used as biomaterial in works on regenerative biology., (© 2019 Blackwell Verlag GmbH.)

The information of twin pregnancy frequencies in buffaloes is scarce in the literature. Evidence even indicates that twin pregnancies are not carried to term, but it does not seem to be the case. In this case report, the event of twin pregnancy in buffaloes is reported as well as its frequency. Twin birth is the presence of two or more embryos in a single gestation. Multiple births are uncommon in buffaloes since the species is uniparous. We studied a population of Murrah buffaloes in which slightly more than 13,000 births were observed. In this study, the frequency of occurrence of twin births in Murrah buffaloes was 0.14%. This low incidence impaired the estimation of genetic parameters. Twins can have advantages and disadvantages depending on the production system, which are discussed in this case report., (© 2019 Blackwell Verlag GmbH.)

The objective of this study was to evaluate the genotype-environment interaction effect on age at first calving in buffaloes. The records were analysed using two approaches: (a) standard animal model and (b) reaction norm model. For the reaction norm analysis, two environmental gradients were formed, using age of first calving or milk yield group contemporary average. The results showed differences in the heritability estimates when using the two approaches. The reaction norm model indicated high heritability in more favourable environments and low magnitude genetic correlations between extreme environments. Based on our findings, we verified the significance of the genotype-environment interaction effect on age at first calving in buffaloes., (© 2019 Blackwell Verlag GmbH.)

With an objective to evaluate the follicular dynamics and vascularity changes in follicles and corpus luteum, the ovaries of cyclic Surti buffaloes (n = 9) were examined daily sequentially by transrectal B-mode and colour flow mode (CFM) ultrasonography starting from the day of oestrus till the onset of next oestrus. Higher proportion of buffaloes evidenced one-wave cycle (66.66%) compared to two-wave cycle (33.34%) with none showing a three-wave cycle. The dominant follicle of the first follicular wave was the ovulatory follicle and persisted for 19.70 ± 0.50 days compared to its persistence for 16.5 ± 1.45 days in a two-wave cycle. The maximum diameter of the ovulatory follicle in a one-wave and two-wave cycle did not differ yet their linear growth rates were significantly lower (p < 0.01) in a one-wave cycle. Colour flow mode examination of follicles revealed that the percentage of follicles with detectable blood flow in the subsequently determined largest follicle (dominant follicle) was not different from that in the second largest follicle before follicle deviation. The blood flow in the dominant follicle increased significantly on the day of oestrus. The mean diameter and blood flow to the corpus luteum (CL) increased linearly and significantly from Day 5 of oestrus till Day 13 after which both parameters started declining. At or around Day 16, there was precipitous fall in the blood supply to the CL and CL diameter that continued declining thereafter to reach the lowest around Day 20 of the oestrous cycle. Rise in plasma progesterone concentrations was synchronous to CL diameter and vascularity and showed significant and positive correlations. It was concluded that Surti buffaloes evidence a preponderance of one-wave follicular growth pattern with a significant increase in the vascularity of ovulatory follicle on the day of oestrus and corpus luteum on Day 13 of the oestrous cycle., (© 2019 Blackwell Verlag GmbH.)

In view of the silent nature of estrus in buffalo, a noninvasive assay kit has long been felt necessary for easy and effective estrus detection. This study was designed to detect estrus in buffalo using a kit formulated in our laboratory based on pheromone compound. Group I: Urine samples collected at estrus phase and group II: randomly collected urine samples were subjected to the test using the kit. No colour developed (i.e., positive reaction) in estrus urine after adding the kit solution. By contrast, pale and/or dark pink colour developed (i.e., negative reaction) in urine from the proestrus and diestrus buffaloes, respectively. Field evaluation of the kit in groups I and II revealed that 60.87% and 71.43% of urine samples were correctly identified as estrus and nonestrus (i.e., proestrus and diestrus), respectively. Therefore, the first of its kind estrus detection kit formulated based on urinary pheromone can as well be used as a simple device to detect estrus in buffalo., (© 2018 Blackwell Verlag GmbH.)

The objectives of this study were to investigate the effect of buffalo oocyte-secreted factors (OSFs) on cumulus cells (CCs) functions, apoptosis and cGMP generation, and whether the direct contact between oocyte and CCs is essential for oocyte-mediated regulation of CCs functions. Buffalo CCs were cultured during IVM within three groups: (a) intact cumulus-oocyte complexes (COCs), (b) CCs cocultured with denuded oocytes (DOs) (CCs + DOs) and (c) CCs monolayer cultured alone (CCsM). After 24 hr of IVM, CCs were harvested for evaluation of the relative mRNA abundance of the genes encoding gap junction (GJA1), glycolysis (PFKP and LDHA), apoptosis (CASPASE-3 and BCL-2) and steroidogenesis (ER-β and PGR) by QRT-PCR, and CASPASE-3 proteins, using western blot. Intracellular cGMP content was also assessed by ELISA. Results showed that the relative abundance of LDHA, PFKP and BCL-2 significantly increased (p < 0.05) in COCs, whereas GJA1 and CASPASE-3 exhibited lower expression (p < 0.05) compared to CCs + DOs and CCsM groups. However, the expression levels of CASPASE-3, both mRNA and protein, were significantly (p < 0.05) downregulated in CCs + DOs compared to CCsM. There was no significant difference in the expression level of PGR and ER-β between the groups. The intracellular content of cGMP was notably (p < 0.05) higher in COCs compared to CCs + DOs and CCsM groups. In conclusion, this study demonstrated, for the first time, that buffalo OSFs protect CCs against apoptosis and stimulate their cGMP production; however, the regulation of cumulus glycolysis and gap junction is confined to those in close contact with the oocyte. Neither OSFs from COCs nor those from DOs have any effect on CCs steroidogenesis., (© 2018 Blackwell Verlag GmbH.)

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label-free shotgun UDMS E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728., (© 2018 Blackwell Verlag GmbH.)

This study was designed to evaluate the dominant follicles development and the estradiol-17β concentrations in non-ovulating and ovulating post-partum buffaloes. Sixteen Bulgarian Murrah buffaloes were submitted to transrectal ultrasonographic examination from the 1st post-partum day until day 50, 3 days apart. The follicular diameter of the different categories of follicles and the ovulations was recorded. The animals were allocated into two groups: I (n = 6) non-ovulating and II (n = 10) ovulating buffaloes. Serum estradiol-17β concentrations on the days for dominant follicle registration were measured by enzyme-linked immunosorbent assay. The results were statistically processed by analysis of variance, non-parametric and correlation analysis. The mean intervals between calving and first dominant follicle detection differed significantly (p < .05) among the groups (19.5 ± 6.2 vs. 13.8 ± 5.1 days), while the mean intervals between registered dominant follicles from two successive waves were comparable. The mean follicular diameters for the same category follicles in both groups were similar. Different estradiol-17β concentrations (p < .05) for the first dominant follicle between non-ovulating (23.5 ± 7.0 pg/ml) and ovulating (33.3 ± 8.4 pg/ml) buffaloes were determined. The cumulative percentages of buffaloes with firstly detected dominant follicle and ovulating animals correlated positively (r ≥ .84; p < .05) to post-partum days. In conclusion, non-ovulating and ovulating post-partum Bulgarian Murrah buffaloes showed differences in the development of the first dominant follicle and estradiol-17β concentrations during the time of dominant follicles detection., (© 2017 Blackwell Verlag GmbH.)

Evaporative cooling during late gestation period improves post-partum reproductive performance in Murrah buffaloes. To prove this hypothesis, sixteen pregnant dry Murrah buffaloes at sixty days pre-partum were selected and divided into two groups of eight animals each. Group 1 of buffaloes (Cooled/CL) was managed under fan and mist cooling during dry period, whereas second group of buffaloes (non-cooled/NCL) remained without the provision of cooling. After parturition, all the animals were managed under evaporative cooling till the end of experimental period. Reproductive performance in cooled (CL) and non-cooled (NCL) groups, respectively, viz. 1st and 2nd ovulation from calving (48.63 ± 2.41, 69.25 ± 2.34 days and 57.75 ± 3.35, 93.63 ± 2.84 days); calving to conception interval (117.88 ± 4.21 days and 117.88± 4.21 days); conception rate (87.5% ± 2.16% and 57% ± 2.26%); and follicular diameter at the time of 1st and 2nd ovulation (14.84 ± 0.16, 15.75 ± 0.13 mm and 12.65 ± 0.13, 13.35 ± 0.11 mm) varied significantly (p < .05). Total peak oestrogen concentration was significantly (p < .05) higher in cooled (26.7 ± 1.32 pg/ml) relative to non-cooled (20.7 ± 1.22 pg/ml) buffaloes. Time from onset of oestrus to ovulation varied significantly (p < .05) in cooled (32 ± 2.22 hr) and non-cooled (40 ± 2.86 hr) buffaloes. The peak progesterone concentration reached to (4.25 ng/ml) in cooled group and (4.16 ng/ml) in non-cooled group after first ovulation., (© 2017 Blackwell Verlag GmbH.)

The objective of the study was to evaluate the efficacy of ovarian response and pregnancy rate in anovular buffaloes following Ovsynch and Ovsynch Plus protocols. Buffaloes (n = 55) were divided into two groups: Ovsynch group (n = 26): GnRH (10 μg, GnRH1) on Day 0, PGF 2 α (25 mg) on Day 7, GnRH (10 μg, GnRH2) on Day 9; Ovsynch Plus group (n = 29): 500 IU equine chorionic gonadotropin (eCG) 72 hr (day -3) prior to Ovsynch protocol, followed by fixed timed artificial insemination (FTAI) 6 and 24 hr after GnRH2 injection in bot groups. Transrectal ultrasonography was performed daily, that is, from day 0 and -3 in Ovsynch and Ovsynch Plus group, respectively for ovarian response and pregnancy diagnosis at day 30 post-insemination. In Ovsynch Plus group, administration of eCG prior to GnRH1 increased (p < .001) the diameter (mm) of dominant follicle (DF) from 10.15 ± 0.26 to 12.23 ± 0.34 within 72 hr of treatment resulting higher ovulatory response to GnRH1. Ovulation after GnRH1 was higher (p < .01) in Ovsynch Plus group (96.6%) than Ovsynch group (61.5%). However, ovulation rate to GnRH2 was similar (p > .05) between groups (Ovsynch group: 76.9% vs. Ovsynch Plus group: 70.0%). Mean DF diameter (mm) that ovulated to both GnRHs was higher (p < .01) than non-ovulated counterparts in both groups (Ovsynch group: 10.80 ± 0.27 vs. 8.47 ± 0.53; Ovsynch Plus group: 11.99 ± 0.24 vs. 9.5 ± 0.63). Pregnancy was established in buffaloes which responded to both GnRHs, irrespective of groups, being higher (p = .52) in Ovsynch Plus group (34.5%) than Ovsynch group (23.1%), though non-significant. In summary, this study showed that eCG inclusion prior to Ovsynch regimen improves ovulatory response in anovular buffaloes during low-breeding season., (© 2017 Blackwell Verlag GmbH.)

This study was designed to investigate the relationship of temperament with oestrus, resumption of ovarian cyclicity and milk yield in post-partum buffaloes. For this study, 102 post-partum Murrah buffaloes were observed for temperament in open paddock and during milking. Based on the temperament score (1-5 score), they were classified into docile, slightly restless, restless, aggressive and nervous. Oestrous behaviour was monitored twice daily in all the buffaloes along with milk yield (weekly), body weight (BW) and body condition score (BCS) at day 0, 30, 60 and 90 post-partum. Based on the temperament score, the number of buffaloes classified as docile, slightly restless, restless and aggressive was 37 (36.27%), 28 (27.45%), 30 (29.41%) and 7 (6.86%), respectively, but none under nervous category. Number of buffaloes resuming cyclicity in various temperaments groups were as follows: docile (n = 35; 94.59%), slightly restless (n = 22; 78.57%), restless (n = 20; 66.67%) and aggressive (n = 7; 85.71%). Temperament was not correlated (p = .128; r=-.152) with oestrous behaviour. But, temperament was correlated (p < .0001; r = .384) with the resumption of cyclicity as docile and slightly restless buffaloes resumed cyclicity (p < .0016) earlier. Temperament of buffaloes was weakly correlated (p = .0017; r=-.306) with milk production with higher (p < .0358) total milk yield in docile (1022 ± 23.75 kg) and slightly restless buffaloes (948 ± 35.86 kg) as compared to other temperament groups. In summary, temperament of post-partum buffaloes was related with resumption of ovarian cyclicity and milk yield, but not oestrous behaviour., (© 2017 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.)

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.)

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 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 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.)

To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor-9 (GDF-9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF-9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen-thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF-9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF-9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF-9 was detected in mural granulosa cells and theca cells of pre-antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF-9. In corpora lutea, GDF-9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF-9 and cultured control that indicated the GDF-9 treatment has no effect on the primordial to primary follicle transition. GDF-9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre-antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF-9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF-9. In conclusion, treatment with GDF-9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity)., (© 2016 Blackwell Verlag GmbH.)

This study aimed to detect the genetic variability in Leptin and Pit-1 genes using polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing also to explore their possible associations with reproductive and productive traits of Egyptian buffaloes. Regarding Leptin gene, three genotypes (AA, AG and GG) were identified with frequency of 0.54, 0.40 and 0.06, respectively, and the genotypes were distributed according to the Hardy-Weinberg equilibrium. Allele A was comparatively higher than G with frequency of 0.74 and 0.26, respectively. For Pit-1 gene, the association could not be performed due to the monomorphism (BB). The results showed that AA genotypes were found to be superior in most of production and reproduction traits. AA genotypes yielded more milk (2332.34 kg, p = 0.04) with higher fat% (6.10, p = 0.004) and fat yield (155.75 kg, p = 0.06), reach peak milk production at 42.19 days and required 2.19 services for conception in comparison with GG genotypes. Birthweight of animals with AA genotype was lesser than with GG genotype (39.35 and 43.67 kg, p = 0.02, respectively). The days open is numerically better in AA genotype animals (99.35 days), but the difference between the three genotypes was non-significant. The distinct significant associations reported in this study suggested that Leptin is reputable candidate genetic marker, which might be used to enhance animals' genetic potential for milk production in conjunction with reproduction., (© 2016 Blackwell Verlag GmbH.)

The presence of Aquaporins 1 (AQP1) and 9 (AQP9), integral membrane water channels that facilitate rapid passive movement of water and solutes, was immunohistochemically detected in the excurrent ducts collected from sexually mature buffalo bulls of proven fertility during the mating (late autumn-winter) and non-mating (late spring to the beginning of autumn) seasons. Furthermore, the research was performed also on the epididymal cauda of a senile buffalo bull with inactive testis. Aquaporins 1 and 9 were immunolocalized at distinct levels. In the efferent ducts, AQP1 immunoreactivity was strongly evidenced at the apical surface of the non-ciliated cells and weakly along the basal membrane of the epithelial cells. The latter reactivity disappeared during the non-mating season. No AQP1 immunoreactivity was detected in the epithelium of epididymis and vas deferens, whereas AQP1 was expressed in the smooth muscle layer of the vas deferens. Aquaporin 1 was present in the blood vessels and in small nerve bundles all along the genital tract. The supranuclear zone of the epididymal principal cells was AQP9 immunoreactive, limited to the corpus and cauda regions, and vas deferens. The samples collected in the two reproductive seasons showed a weaker AQP9 immunoreactivity during the non-mating season. A typical AQP9 immunoreactivity was noticed in the old buffalo examined. The tested AQP molecules showed a different expression pattern in comparison with laboratory mammals, primates, equine, dog and cat. In addition, seasonal differences were noticed which are possibly useful in regard to the comprehension of the morphophysiology of reproduction in the bubaline species, which are still a matter of debate., (© 2016 Blackwell Verlag GmbH.)

The role of melatonin as a protective neurohormone against restoring cyclicity in summer anoestrous animals in photoperiod species has gained wider acceptance. This study was designed to uncover the evidence the slow-release melatonin (MLT) has on initiation of ovarian cyclicity and conception rate (CR) in summer anoestrous buffaloes. Thus, buffaloes diagnosed as summer anoestrous (absence of overt signs of oestrus, concurrent rectal examination and radioimmunoassay for serum progesterone at 10 days interval) were grouped as untreated (Group I, sterilized corn oil, n = 8) and treated (Group II, single subcutaneous injection of MLT @18 mg/50 kg bwt in sterilized corn oil, n = 20). Animals treated and detected in oestrus were artificially inseminated (AI) followed by division into Group III (second dose of MLT on 5th day post-AI, n = 8) and Group IV (no melatonin administration, n = 10). Blood samples were collected at 4 days interval for estimation of serum MLT, progesterone and oestrogen using radioimmunoassay kit. Mean oestrous induction rate (OIR), oestrous induction interval (OII), interoestrous interval (IOI) and CR were estimated. Compared to control, concentration of melatonin was significantly (p < 0.05) higher in treated group ranging from 14.34 ± 1.72 to 412.31 ± 14.47 pg/ml whereas other two hormones did not show any concentration difference. Melatonin-administered buffaloes showed significantly (p < 0.05) higher (90%) OIR with OII of 18.06 ± 1.57 days. Results showed improvement in conception rate in buffaloes administered with post-insemination melatonin. It can be concluded from the study that slow-release melatonin supplementation restored cyclicity in summer anoestrous animals resulting in improvement in conception rate in buffaloes., (© 2015 Blackwell Verlag GmbH.)

In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa., (© 2015 Blackwell Verlag GmbH.)