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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.)

New technologies for detecting pregnancy shortly after mating/insemination and identifying gestational age are essential for speeding up the reproductive cycle and ensuring high reproductive efficiency in livestock farming. Ultrasonography can successfully identify pregnancy and determine gestational age in many domestic animals. On the other hand, many herds of camel and buffalo and flocks of sheep are aware of the day of service, making it difficult to appropriately manage pregnant animals. This study provides a review of the literature on various techniques for ultrasonographically diagnosing pregnancy in camels, buffaloes, and sheep, focusing on the most appropriate times to use each technique, the earliest opportunity to diagnose pregnancy, and the possibility of using various parts of the fetus to create mathematical equations to determine gestational age. Some limitations of ultrasonography in pregnancy diagnosis were identified and significant pregnancy events in dromedaries were discussed, including left-horn and twin pregnancies. The data presented here will prove essential for researchers, farmers, and countries that rely heavily on these animals for providing meat, milk, cosmetics, and other animal products to enhance reproduction and production efficiency., Competing Interests: Declaration of Competing Interest There are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0-5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0-5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (-0.0002 ± 0.07 vs. -0.266 ± 0.09), kurharm0 (-0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0-5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. -0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chilika, a native buffalo breed of the Eastern coast of India, is mainly distributed around the Chilika brackish water lake connected with the Bay of Bengal Sea. This breed possesses a unique ability to delve deep into the salty water of the lake and stay there to feed on local vegetation of saline nature. Adaptation to salinity is a genetic phenomenon; however, the genetic basis underlying salinity tolerance is still limited in animals, specifically in livestock. The present study explores the genetic evolution that unveils the Chilika buffalo's adaptation to the harsh saline habitat, including both water and food systems. For this study, whole genome resequencing data on 18 Chilika buffalo and for comparison 10 Murrah buffalo of normal habitat were generated. For identification of selection sweeps, intrapopulation and interpopulation statistics were used. A total of 709, 309, 468, and 354 genes were detected to possess selection sweeps in Chilika buffalo using the nucleotide diversity (θπ), Tajima's D, nucleotide diversity ratio (θπ-ratio), and F ST methods, respectively. Further analysis revealed a total of 23 genes including EXOC6B , VPS8 , LYPD1 , VPS35 , CAMKMT , NCKAP5 , COMMD1 , myosin light chain kinase 3 ( MYLK3 ), and B3GNT2 were found to be common by all the methods. Furthermore, functional annotation study of identified genes provided pathways such as MAPK signaling, renin secretion, endocytosis, oxytocin signaling pathway, etc. Gene network analysis enlists that hub genes provide insights into their interactions with each other. In conclusion, this study has highlighted the genetic basis underlying the local adaptive function of Chilika buffalo under saline environment. NEW & NOTEWORTHY Indian Chilika buffaloes are being maintained on extensive grazing system and have a unique ability to convert local salty vegetation into valuable human food. However, adaptability to saline habitat of Chilika buffalo has not been explored to date. Here, we identified genes and biological pathways involved, such as MAPK signaling, renin secretion, endocytosis, and oxytocin signaling pathway, underlying adaptability of Chilika buffalo to saline environment. This investigation shed light on the mechanisms underlying the buffalo's resilience in its native surroundings.

Background: The vulnerability of buffalo sperm to cryoinjury necessitates the improvement of sperm cryo-resistance as a critical strategy for the widespread use of assisted reproductive technologies in buffalo., Objectives: The main aim of the present study was to evaluate the effects of different concentrations of rutin and chlorogenic acid (CGA) on buffalo semen quality, antioxidant activity and fertility during cryopreservation., Methods: The semen was collected and pooled from the 3 buffaloes using an artificial vagina (18 ejaculations). The pooled sperm were divided into nine different groups: control (Tris-based extender); 0.4, 0.6, 0.8 and 1 mM rutin (rutin + Tris-based extender); and 50, 100, 150 and 200 µM CGG (CGA + Tris-based extender). Sperm kinematics, viability, hypo-osmotic swelling test, mitochondrial activity, antioxidant activities and malondialdehyde (MDA) concentration of frozen and thawed buffalo sperm were evaluated. In addition, 48 buffalo were finally inseminated, and pregnancy was rectally determined 1 month after insemination., Results: Compared to the control group, adding R-0.4, R-0.6, CGA-100 and CGA-150 can improve total and progressive motility, motility characteristics, viability, PMF and DNA damage in buffalo sperm. In addition, the results showed that R-0.4, R-0.6, CGA-50, CGA-100 and CGA-150 increased total antioxidant capacity, catalase, glutathione peroxidase and glutathione activities and decreased MDA levels compared to the control group. Furthermore, it has been shown that adding 150 µM CGA and 0.6 mM rutin to an extender can increase in vivo fertility compared to the control group., Conclusions: In conclusion, adding rutin and CGA to the extender improves membrane stability and in vivo fertility of buffalo sperm by reducing oxidative stress., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Estrus detection in buffaloes primarily relies on behavioral and physiological signs. Especially during summer, these signs are less prominent to recognize. Thus, estrus detection is a pronounced challenge within the realm of buffalo husbandry, particularly in the summer. Therefore, a simple and accurate estrus detection method is required for buffalo farmers. The observation of fern-like salivary crystallization patterns is one such simple method to detect estrus in buffaloes, bactrian camels, beagle bitches, and cows. However, the exact mechanism for the formation of typical fern-like is not known. We hypothesized that it might be because of the estrus-specific mucins and salts. To test this hypothesis, we prepared the smears by combining different concentrations of mucin type -2 (MUC2) and -3 (MUC3) with sodium chloride (NaCl). Microscopic examination confirmed that fern-like patterns resulted from a combination of the MUC3 and NaCl produced more realistic fern patterns than that of MUC2 or BSA with salt. To predict possible mucin and salt concentration showing natural fern-like patterns at the estrus stage in buffalo saliva, we constructed a guide tree of artificially generated fern-like patterns using an image analysis online tool. This computation analysis revealed that most of the natural buffalo estrus saliva samples showing typical fern-like patterns clustered in the cluster 2 of the guide tree comprising of 13 clusters. In the cluster 2, MUC3 in combination with the salt concentrations of 100, 150, and 250 mM was commonly found in a close proximity to the natural typical fern-like patterns of saliva smear of buffaloes at estrus. Conclusively, the buffalo saliva at estrus is predicted to have a gel-forming heavily glycosylated protein such as mucin along with at least 100 mM of NaCl. RESEARCH HIGHLIGHTS: Glycoprotein and salts combination replicates fern-like pattern of buffalo saliva at estrus. MUC3 and NaCl salt combination produces more realistic fern-like patterns compared with MUC2 or BSA and salt combination. MUC3 with NaCl at 100, 150, and 250 mM consistently resembled natural estrus saliva fern-like patterns. During estrus, buffalo saliva is expected to contain heavily glycosylated mucin and at least of 100 mM NaCl., (© 2024 Wiley Periodicals LLC.)

This study aimed to monitor the mammary health of 37 multiparous Murrah buffaloes through infrared thermography (IRT). Based on the California Mastitis Test (CMT) and milk somatic cell counts (SCC), buffaloes were grouped into healthy (H, n = 16), subclinical mastitis (SCM, n = 10), and clinical mastitis (CM, n = 11). Buffaloes were milked twice daily in the morning (5:00-6:00 AM) and evening (5:00-6:00 PM). Rectal temperature and respiratory rates were recorded, CMT was performed and thermal images of the mammary gland of all the buffaloes were taken before and after each milking. Milk samples were analysed after each milking for SCC, fat, Solids-Not-Fat (SNF), density, protein, lactose, salts, conductivity, and pH immediately in the laboratory from fresh milk samples. The surface temperature of the periocular region of both the eyes, muzzle, flank, and vagina were also taken. Thermal images were used to assess the surface temperature of the udder (USST), teat apex (TAT), teat barrel (TB1T), teat base (TB2T), and teat skin surface (TSST). Eye and USST showed significantly higher temperatures (p < 0.05), whereas skin surface temperatures (SST) of different body parts were non-significant in both SCM and CM animals than buffaloes in the H group. Milk SCC showed a positive correlation with conductivity (r > 0.7), salts, and pH (r < 0.6) and a negative correlation with fat, SNF, density, protein, and lactose. TAT, TB1T, TB2T, TSST, and USST were positively correlated with milk SCC. Receiver Operating Characteristic (ROC) analysis of H and SCM groups showed that USST before milking had optimum sensitivity (Se = 0.80) and specificity (Sp = 0.906) among the various skin temperatures recorded. Thermal images captured during the morning showed higher sensitivity compared to images taken in the evening. Results indicate IRT can be used to monitor the mammary health of buffaloes but using IRT in conjunction with milk SCC can help in the accurate prediction of SCM in dairy buffaloes., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Little is known about the effects of different seasons on the cryopreservation success of buffalo sperm in terms of kinematics and sperm functional parameters., Objective: To study the effect of three seasons (winter, comfort and summer) and cryopreservation on sperm kinematics and functional properties in buffalo bulls., Materials and Methods: Semen ejaculates (n = 90) collected during three seasons i.e. winter (n = 30), comfort (n = 30), summer (n = 30) were evaluated for sperm kinematics and functional properties., Results: Sperm kinematics with respect to total (TM), progressive (PM) and rapid motility (RM) was higher (P < 0.05) in fresh sperm compared to sperm that had been frozen-thawed. Similarly, all kinematic parameters [viz. average path velocity (VAP), straight linear velocity (VSL), curvilinear velocity (VCL), beats cross frequency (BCF), lateral head displacement (ALH), linearity (LIN) and straightness (STR)] were higher (P < 0.01) at the fresh stage. With respect to season, frozen-thawed semen TM (57.67 ± 115 %), PM (50.2 ± 1.15 %) and RM (51.6 ± 1.19 %) were higher (P < 0.01) when using sperm collected during winter. The stage of cryopreservation (i.e., equilibration and freeze-thawing) also showed significant effects (P < 0.01) on mitochondrial superoxide positive status (MSPS), mitochondrial membrane potential (MMP), acrosome status and intra-cellular calcium status., Conclusion: The season of sperm collection and cryopreservation have significant effects on buffalo bull sperm kinematics and functional properties. Doi.org/10.54680/fr24410110612.

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.)

Heat stress profoundly affects the reproductive success of buffaloes, which are vital for the dairy industry due to their unique anatomical and physiological characteristics, necessitating careful evaluation under such conditions. Hence, this guided our search for quantifying heat stress' impact on Mehsana buffaloes using the best THI model and evaluating sires' performance. Fertility records (days open converted to daughter pregnancy rate) were collected in the span of over 24 years, w.e.f. 1989 to 2012. Finally, 3070 records of first lactation cows, daughters of 117 sires from DURDA, Gujarat, India, were used in the analysis. Meteorological data were retrieved from IMD, Pune, to understand the relationship between daughter pregnancy rate (DPR) and heat stress indicators. Several heat stress models were compared based on R 2 , adjusted R 2 , AIC, and BIC values, and the impact of heat stress was quantified. The year was classified into different heat stress zones, viz., Non heat stress zone (NHSZ), Heat stress zone (HSZ), and critical heat stress zone (CHSZ), drawing from the findings of DPR and THI. The THI 4th model based on dry and wet bulb temperature was identified as the best-fit model, and DPR significantly changed (P < 0.01) by 1.14% per unit change in THI value based on the month of calving. The average EBVs of the sires for DPR were found to be 20.78% (NHSZ), 38.09% (HSZ), and 39.08% (CHSZ) using BLUP-SM and 20.78% (NHSZ), 37.30% (HSZ), and 38.87% (HSZ) using BLUP-AM. Subsequently, the optimum sire for each of the zones was prioritized. It is noteworthy that bulls that performed better in NHSZ did not perform as well in HSZ and CHSZ, and vice versa. This supports the possibility of evaluating bulls independently in each heat stress zone., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The study was done to determine additive, maternal and common permanent environmental effects and best-suited model for some production traits using six univariate animal models that differed in the (co)variance components fitted to assess the importance of maternal effect using likelihood ratio test in Murrah buffaloes. Data from 614 Murrah buffaloes related to production traits were collected from history pedigree sheets maintained at the buffalo farm, Department of Livestock Production and Management (LPM), LUVAS, Hisar. The production traits under this study were 305 days milk yield (305DMY), peak yield (PY), lactation length (LL), dry period (DP), lactation milk yield (LMY) and wet average (WA). The heritability estimates were in the range of 0.33-0.44 for 305DMY, 0.25-0.51 for PY, 0.05-0.13 for LL, 0.03-0.23 for DP, 0.17-0.40 for LMY and 0.37-0.66 for WA. Model 1 was considered best for most of the traits, viz., 305DMY, PY, LL, LMY and WA followed by model 2 for DP. Covariance and correlated values within the traits caused inflation of heritability in model 3 and model 6. The maximum covariance between the additive and maternal effect was found in trait LMY, which was 14,183.90 in model 3 and the minimum value was also reported in the same trait for model 6, valued at -3522.37. Multivariate analysis showed that all production traits were moderate to high and positively correlated with each other except for DP, which was low and negative genetic and phenotypic correlated. Spearman's rank correlation coefficients of breeding value among all six models were high and significant, ranged from 0.78 to 1.00 for all the traits except DP, therefore any of the models could be taken into account depending upon the availability of data., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Heat stress is one of the primary environmental factors that harm both the productivity and health of buffaloes. The current study was conducted to estimate the threshold of temperature humidity index (THI) 1 and genetic features for milk yield of first-lactation Mehsana buffaloes using an univariate repeatability test-day model. The data included 130,475 first lactation test-day milk yield (FLTDMY) records of 13,887 Mehsana buffaloes and the daily temperature and humidity. The statistical model included herd test day as fixed effects, days-in-milk (DIM) classes, age of the animal, as well as random factors such as the additive genetic effect (AGE) of animal in general conditions (intercept), AGE of the buffaloes subjected to heat stress (slope), permanent environmental effect of animal in general conditions (intercept), permanent environmental effect of animal under heat stress conditions (slope) and random residual effect. It was expected that the general effects and the heat-tolerance effects would be correlated, represented by the present investigation's repeatability models. The variance components of FLTDMY in the present study were computed using the REML method. The threshold for THI was 78. At the THI below the threshold, the heritability estimated for the FLTDMY trait was 0.29, and the additive genetic variance (AGV) for heat stress conditions was 0. At THI of 83, AGV for heat stress conditions was highest for FLTDMY. The genetic correlation of general AGE to heat-tolerant AGE was -0.40. The results indicated that a consistent selection for milk production, avoiding the thermal tolerance, may diminish the thermal tolerance capacity of Mehsana buffaloes., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This comprehensive review delves into the evolving landscape of assisted reproductive technologies (ARTs) in bovine species, particularly focusing on the pivotal roles of semen additives in the cryopreservation of buffalo and cattle semen. In developing nations, where ARTs are still emerging, these techniques significantly influence bovine reproductive strategies. In contrast, developed regions have embraced them as primary approaches for dairy buffalo and cattle breeding. Semen cryopreservation, while offering advantages like extended storage and genetic propagation, also presents challenges. These include diminished sperm quality due to reactive oxygen species (ROS) production, alterations in sperm structure, and temperature fluctuations. Further, the effect of cryopreservation differs between cattle and buffaloes, with the latter exhibiting poorer semen viability and fertility due to inherent lipid composition susceptibilities. The generation and implications of ROS, especially hydrogen peroxide, contribute significantly to sperm DNA damage and functional impairments. To counteract these challenges, research has intensified on semen additives, aiming to bolster semen quality and protect against oxidative stress-induced damage. As the field advances, the review emphasizes the need for optimized cryopreservation techniques and tailored antioxidant strategies to harness the full potential of ARTs in bovine breeding programs. Doi.org/10.54680/fr24410110112.

Freezing-thawing procedures and semen manipulation for in vitro fertilization induce oxidative stress, which in turn leads to impaired sperm quality. The aim of this study was to evaluate whether incubation of frozen-thawed buffalo semen with olive fruit extracts (OFE), known to contain a high concentration of phenolic antioxidants, would improve semen quality by reducing oxidative stress. Frozen sperm (4 ejaculates/4 bulls/3 replicates) were thawed and diluted to 30 × 10 6 /mL in IVF medium with 0, 72, 143, and 214 μL/mL of OFE, corresponding to 0 (D0-control), 50 (D50), 100 (D100), and 150 (D150) μM hydroxytyrosol. Sperm viability, acrosome integrity, membrane functionality, motility, and sperm kinetics were evaluated immediately after thawing (T0) and after 1 (T1) and 2 h (T2) of incubation at 38.7 °C. Based on the results, sperm biological antioxidant potential (BAP) and ROS levels (ROMs) were assessed in D0 and D100 groups at T1 and T2. To assess the effect of OFE on fertilizing ability, heterologous penetration rates were also evaluated, using bovine abattoir-derived oocytes. The treatment with OFE at all concentrations tested increased (P < 0.05) the percentage of acrosome intact spermatozoa compared to the D0-control at T1, but the effect was more evident (P < 0.01) with D100 (54.5 ± 3.0, 60.5 ± 1.5, 65.2 ± 3.3, and 62.5 ± 1.7, with D0, D50, D100, and D150 OFE, respectively). Total motility, progressive motility, rapid velocity, and progressive velocity decreased (P < 0.05) at T2 only in the D0-control group. The percentage of rapidly progressive sperm and the progressive motility tended to increase (P < 0.10) at T1 and T2, respectively, in D100 compared to D0 (24.7 ± 4.1 vs 16.4 ± 1.6 and 22.8 ± 2.7 vs 17.0 ± 1.2, respectively). The treatment with D100 OFE of frozen-thawed sperm increased (P < 0.05) some kinetic parameters (VAP and WOB). Spermatozoa incubated with D100 OFE exhibited higher (P < 0.01) total and normospermic oocyte penetration rates compared to D0 (86.5 ± 1.4 vs 78.5 ± 0.7, and 70.6 ± 1.5 vs 63.8 ± 1.1, respectively). Additionally, D100 OFE increased sperm BAP concentrations at both T1 and T2, while ROS levels were unaffected. These results suggest that incubating frozen-thawed buffalo semen with OFE is an effective strategy for preserving semen quality and in vitro fertilization ability by enhancing sperm antioxidant capacity., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Buffalo bull semen traits are economically important traits that influence farm fertility and profitability. Genetic improvement of semen characteristics is an important detail of the genetic improvement. This study was conducted to assess the relationship between the breeding values as well as the phenotypic values for semen traits (VOL, MM, LS, AS and CONC) of the Egyptian buffalo bulls. A total of 7761 normal semen ejaculates were collected and characterized at ILMTC laboratory from 26 bulls from 2009 to 2019. For VOL, MM, LS, AS, and CONC, the actual means were 3.89 mL, 62.37%, 60.64%, 3.94%, and 0.67 × 109 sperm/mL, respectively. The prediction of breeding values for semen traits was estimated using a Bayesian procedure. The estimated standardized EBVs and phenotypic values were used in the principal component analysis (PCA). Of five PCs, one PC (PC1) had > 1 eigenvalues that was responsible for 87.19% of the total variation of SEBV, and two PCs had > 1 eigenvalues that were responsible for 59.61% and 21.35% of the total variation of the phenotypic values. Together, PC1 and PC2 accounted for 97.97% of the total variance of SEBV and 80.96% of the total variance of phenotypic values. A graphs of the first two components showed the traits separated into two different directions by group. This indicates each group was under similar genetic influence. Therefore, selection can be done separately for each group without influencing the other. Principal component analysis reduced variables to describe the key information in buffalo semen data., (© 2024. The Author(s).)

Early embryonic mortality resulting from insufficient interaction between the embryo and the uterus leads to the failure of pregnancy in livestock animals. Thus, it is imperative to comprehend the multifaceted process of implantation at molecular levels, which requires synchronized feto-maternal interaction. The in-vitro models serve as valuable tools to investigate the specific stages of implantation. The present study was undertaken to develop a simple method to isolate and culture the primary buffalo endometrial epithelial cells (pBuEECs), followed by proteome profiling of the proliferating cells. Collagenase I was used to separate uterine epithelial cells (UECs) from the ipsilateral uterine horn, and then the cells were separated using a cell strainer. After being seeded on culture plates, UECs developed colonies with characteristic epithelial shape and expressed important markers such as cytokeratin 18 (KRT18), progesterone receptor (PGR), β-estrogen receptor (ESR1), and leukemia inhibitory factor (LIF), which were confirmed by PCR. The purity of epithelial cells was assessed using cytokeratin 18 immunostaining, which indicated approximately 99% purity in cultured cells. The proteome profiling of pBuEECs via high-throughput tandem mass spectrometry (MS), identified a total of 3383 proteins. Bioinformatics analysis revealed enrichment in various biological processes, including cellular processes, metabolic processes, biological regulation, localization, signaling, and developmental processes. Moreover, the KEGG pathway analysis highlighted associations with the ribosome, proteosome, oxidative phosphorylation, spliceosome, and cytoskeleton regulation pathways. In conclusion, these well characterized cells offer valuable in-vitro model to enhance the understanding of implantation and uterine pathophysiology in livestock animals, particularly buffaloes., Competing Interests: Declaration of Competing Interest The authors declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this research article., (Copyright © 2024. Published by Elsevier B.V.)

The aim of this study was to examine the suitability of different growth functions (linear, sinusoidal, Gompertz, Schumacher and Richards) to fit cumulative milk production data from buffalo cows. Cumulative milk production at each day in milk was calculated from two published datasets reporting (i) fortnightly test-day milk yield records of the first lactation of Murrah buffalo that had calved during 1977-2012 and (ii) the first lactation records of Jaffarabadi buffalo collected from history-cum-pedigree registers for each quinquennium between 1991 and 2010. Each function was fitted to the lactation curves using nonlinear regression procedures. The Richards and sinusoidal equations provided the smallest root mean square error values, Akaike's and Bayesian information criteria and, therefore, the best fit for the cumulative lactation curves for milk yield. The Richards equation appeared to provide the most accurate estimate of the cumulative milk production at peak milk yield. Sinusoidal and flexible classical growth functions are appropriate to describe cumulative milk production curves and estimate lactation traits in buffalo.

The present study was conducted to localize the immunoexpression of VEGF-A (Vascular Endothelial Growth Factor) and von Willebrand factor (vWF) in corpora lutea of healthy buffaloes (24) collected from local slaughterhouses. CL collected were categorized into early (stage I, 1-5 days, n = 6), mid (stage II, 6-11 days, n = 6), late luteal phase (stage III, 12 to 16 days, n = 6) and regressing phase (stage IV, 17 to 20 days, n = 6). The percent positive immunostaining for VEGF-A was significantly (p < 0.05) higher in mid-luteal phase than the other three stages of CL. However, it was higher in early luteal phase as well indicated intense angiogenesis in both early and mid-luteal phases. The number of capillary endothelium expressing vWF was significantly (p < 0.05) highest in mid-luteal phase among all the phases. However, in late luteal phase, the percent area positive for VEGF-A immunostaining was reduced but it was significantly (p < 0.05) higher than corpus albicans phase. Thus, in regressing phase or corpus albicans, it was lowest and reduced considerably. However, in late luteal phase, the number of capillaries with vWF immunoexpression reduced significantly (p < 0.05) but it was lowest in corpus albicans phase. Therefore, the immunotaining pattern for VEGF-A and vWF concluded that there was a spositive linear correlation between the two, that is, as the VEGF-A expression was increased, the number of vWF positive capillaries also increased and vice versa. The VEGF-A expressed by the luteal parenchyma in different stages of development and regression of corpus luteum was thus observed to be involved in promoting the angiogenesis and luteal cell proliferation as supported by vWF expressed by endothelium of proliferating capillaries in buffalo corpus luteum throughout the estrous cycle., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Conventional induction protocol (CIP) of calving in buffaloes employs the intramuscular (IM) administration of dexamethasone (40 mg) and cloprostenol sodium (500 μg). If there is no progression in terms of cervical dilatation, then a second dose of cloprostenol sodium (500 μg) is administered intramuscularly. This protocol possesses certain demerits: (1) a wide range of response time intervals, and (2) increased risk of fetal membrane retention. Considering the cervix as a caudal continuation of the myometrium with its own contractile potential, and the limitations of CIP, we developed intracervical (IC) drug administration route in buffaloes. The proposed technique was evaluated for its use in a total of 22 cases of incomplete cervical dilatation in uterine torsion-affected buffaloes (IC-14 and IM-8). In addition to CIP, the IC group received an intracervical injection of cloprostenol sodium (500 μg) at the start of the experiment whereas the IM group received an extra intramuscular dose of cloprostenol sodium (500 μg) either after 24 h or when no progression in cervical dilatation is noticed. Surprisingly, the average response time during the experiment in the IC group was 5.8 h shorter (p < 0.000) than in the IM group (IC-5.7 ± 0.17 h vs. IM-11.9 ± 0.74 h). The duration from calving to fetal membrane expulsion (IC-12.8 ± 0.60 h vs. IM-17.5 ± 1.40 h; p < 0.002) and incidence of retention of fetal membrane were also less in the IC group (57.1% vs. 87.5%). The proposed intracervical drug administration potentiates cervical dilatation and can be regarded as a safe, effective, and feasible technique for attaining reliable results., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The portio-vaginalis uteri (PVU) and its mucus secretion have shown an essential role in conception. A significant endeavour to improve buffaloes' reproductive efficiency is the investigation of their basic reproductive pattern, which provides a reference for applications in breeding and pregnancy. The present study aimed to evaluate the anatomical and histological alterations in PVU regarding to the vaginal artery (VA) hemodynamic at luteal and early pregnant stages in buffalos. Egyptian live buffaloes (n = 16) and fresh genitals (n = 25) of mature buffalo were used. Different luteal and early pregnant stages were macroscopically identified with the shape and mucosal colouration with discharges of the PVU. Histological examination showed a significant difference in area % of alcian blue and periodic acid Schiff positive granules which considered an indication for presence of acidic and neutral mucins respectively in the epithelial cells of PVU mucosa which increased in pregnant stage than in other luteal stages. VA assessment demonstrated an increase in luminal diameter and thickness of tunica muscularis in pregnant stage than other stages (P < 0.05). Middle uterine (MUA) and VA arteries peak velocity point (PSV mm/sec) were elevated (P < 0.05) in pregnant stage, with a marked reduction in both resistance and pulsatility indices (RI and PI), and ratio of systolic /diastolic (S/D). Positive correlation was detected between VA. PSV and, MUA. PSV (r = 0.87), but a negative relation was detected with VA. S/D (r = -0.77), VA.PI (r = -0.89), VA. RI (r = -0.97), MUA. S/D (r = -0.94), MUA. PI (r = -0.85), and MUA. RI (r = -0.88). Doppler indices were negatively corrected with the VA. PSV (r = -0.68). It was concluded that there was a significant alterations in histological features of the cervical PVU at different physiological stages (luteal and early pregnant) in buffalos in relation to the MUA and VA hemodynamic pattern and that hypotheses can be established regarding the female cyclicity that affected by both arteries hemodynamics change., (© 2023. The Author(s).)

The existing treatise targeted to compare the effects of adding different nano-emulsions essential oils (olive, flaxseed, and grapeseed oils) in freezing extender on semen quality and freezability in buffalo. Nano-emulsions were prepared from olive, flaxseed, and grapeseed oils and characterized for their sizes and shapes. Semen extended in four tubes were supplemented with 0 (control) and 3.5% nanoemulsion oils, including olive (NEO), flaxseed (NEFO) and grape seed oils (NEGSO) respectively. NEGSO resulted in the highest (p < 0.05) membrane integrity, vitality, progressive motility (P-motility) of sperm compared to the other groups in post-thawed buffalo bull semen (at 37 °C for 30 s). The addition of NEGSO had the best results for membrane integrity, progressive motility, and vitality of sperm after incubation (at 37 °C and 5% CO 2 for 2 h). A superior (p < 0.05) value of total antioxidant capacity in frozen-thawed spermatozoa was monitored in all supplemented groups as relative to the control. The values of malondialdehyde (MDA) and nitric oxide (NO) were lower (p < 0.05) in NEGSO group compared with other groups. Both NEO and NEFO exhibited the same results for MDA, and NO levels (p > 0.05). All supplemented groups exhibited lower hydrogen peroxide levels (p < 0.05) as relative to the un-treated group. The lowest (p < 0.05) caspase 3 levels were verified in NEGSO treatment, followed by NEFO and NEO treatments. Post-thawed sperm showed ultrastructural damages in the control group, and theses damages were attenuated or resorted by the NEGSO, NEFO and NEO supplemented to freezing extender. In consequences with in vitro results regarding the sperm attribute, a greater pregnancy rate (92%) was observed in NEGSO group as compared with NEFO (88%), NEO (76%) and CON (68%) groups. Our findings demonstrate that NEGSO (3.5%) could be used as a new strategy in enhancing sperm functionality, potential fertility and reducing the oxidative damage and apoptosis markers. This could be significantly applicable for sperm physiology cryopreservation in the milieu of assisted reproduction systems., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This study investigated the beneficial effects of relaxin on cryotolerance of buffalo spermatozoa and reproductive hormones during low breeding season. Collected semen was diluted in five aliquots with relaxin addition (0.25 mg/mL, 0.50 mg/mL, 0.75 mg/mL, 1 mg/mL, and control). After gentle dilution (37°C), cooling (4°C, 2 h), equilibration (4°C, 4 h), and packaging (straws, polyvinyl French, 0.5 mL), frozen (cell freezer), and thawed (37°C, 30 s) for analysis. Blood samples were collected at different time intervals i.e., -60, -30 and 0 min (pre-dose) and 30, 60, 90, 120 and 150 min (post-dose) from a jugular vein. This study manifest that adding relaxin (1 mg/ mL) in freezing medium ameliorates sperm motility, functionality (%), and seminal plasma total antioxidant capacity (TAC, μM/L) than control during low breeding season. Furthermore, we found that relaxin supplementation at 1 mg/mL significantly improves seminal plasma ATP concentrations (nmol/million) than control, 0.25 mg/mL, and 0.50 mg/mL, and fertility (control, and 0.75 mg/mL). Further, relaxin injection significantly improves plasma T, LH and IGF-1 levels (150 and 120 min vs. -60, and - 30), and FSH, KP, and GnRH concentrations (150 min vs. -60), during low breeding season. Taken together, this study revealed that relaxin ameliorates motility, functionality, and fertility of buffalo spermatozoa. Moreover, relaxin injection (1 mg/mL) improves essential reproductive hormones levels in buffalo signifying its importance in the field of reproductive physiology. Further studies are required to determine the exact mechanism of action of relaxin in enhancing semen quality, fertility and reproductive hormones., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

This study was conducted to compare the scrotal skin dynamics of camel and buffalo bulls regarding thermoregulation, season and testosterone. Scrotal skin and blood samples (n = 8 each) were collected during breeding (October to March) and non-breeding seasons (April to August). Tissue slides were prepared by the paraffin embedding technique followed by hematoxylin and eosin (H&E) staining and were analysed by ImageJ®. Relative humidity and temperature were recorded in different seasons to calculate the thermal humidity index (THI). Serum testosterone level was estimated through radioimmunoassay (RIA). The data were analysed by applying ANOVA, the THS test was used as a post-ANOVA interface and the correlation coefficient was also calculated among season, testosterone and skin parameters. The results showed that skin thickness, season and THI were negatively correlated with testosterone levels. In the neck, body and apex, the papillary layer was thicker in buffalo bull compared to camel bull during the breeding season; however, the reticular layer followed a reverse trend in the apex when THI was low. In the scrotal neck during the non-breeding season, hair follicle (HF) length and density were recorded high in camel bull compared to buffalo bull when THI was elevated. The density of HF followed the otherwise trend in the apex during the breeding season. The density of SG was found highest in all regions of camel bull as compared to buffalo bull and showed direct relation with THI. To conclude, this study delineates how the histo-dynamics of scrotal skin varies and accommodate itself with testosterone and THI over the different season in camel and buffalo., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Despite significant progress in vitro maturation (IVM) and in vitro culture (IVC) of oocytes and embryos, their developmental competence remains low. To address this issue, we used buffalo oocytes as a model system to investigate the effects and mechanisms of oxygen concentration on IVM and IVC. Our findings demonstrated that culturing buffalo oocytes with 5% oxygen significantly enhanced the efficiency of IVM and developmental competence of early embryos. Immunofluorescence results suggested that HIF1α played a critical role in these progresses. RT-qPCR results showed that maintaining a stable expression of HIF1α in cumulus cells with 5% oxygen concentration enhanced glycolysis, expansion, and proliferation abilities, up-regulated the expression of development-related genes, and suppressed apoptosis level. Consequently, it improved the maturation efficiency and quality of oocytes, leading to improve developmental capacity of buffalo early embryos. Similar outcomes were also observed when embryos were cultured with 5% oxygen. Collectively, our study provided insights into the role of oxygen regulation during oocytes maturation and early embryo development, and could potentially improve the efficiency of human assisted-reproduction technology., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest exist., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: According to reports, the majority of domesticated species exhibited uterine torsion. It was occasionally noted as a cause of dystocia in buffaloes. The uterus might twist more frequently late in pregnancy because of certain animal traits. The current research monitored the clinical findings and laboratory assays associated with uterine torsion cases in pregnant buffalo-cows through comparing between normal labored buffalo-cows (Norm-Lab gr ; n = 20), mechanically corrected uterine torsed animals without medicament interference (UtrTors gr ; n = 160), and mechanically corrected uterine torsed animals with medicament interference (UtrTors-Med gr ; n = 40) through focusing on placental characterization, calves body weight, milk constituents and milk somatic cell count (SCC) in normal labored buffaloes and uterine torsed ones. Through clinical and laboratory investigations of these buffaloes (N = 220) had been conducted 3 times; 7 h pre-calving and post calving (Post uterine correction) i.e. 48 and 96 h. Uterine torsion prevalence parameters, placental characterization, calves body weight, milk constituents and milk somatic cell counts were evaluated in normal labored buffaloes and uterine torsed ones., Results and Conclusions: The study concluded pre-calving remarkable variations in clinical findings, leukogram picture, calf birth weight and some placental characterization parameters between Norm-Lab gr and each of UtrTors gr and UtrTors-Med gr whereas these variations disappeared post-partum as a result to either only mechanical correction or mechanical correction plus medicaments interference. No pre-or post-calving significant changes between UtrTors gr and UtrTors-Med gr except for the abnormal clinical findings were more representative in UtrTors-Med gr than those in UtrTors gr particularly pre-calving. The applied pre-calving therapeutic regimen including dexamethasone-prostaglandin-receptal combination had a powerful potential efficacy that induced vaginal delivery of calves in UtrTors-Med gr as well as prepartum mechanical correction of torsed uterus approved higher efficacy in UtrTors gr . The applied prepartum mechanical correction of torsed uterus and/or pre-calving therapeutic regimen as well as subsequent post-calving, post uterine correction applied medicament treatment accelerated rapid recovery of affected buffalo-cows through achieving rapid restoring of their physiological parameters. Buffalo-cow's milk composition, milk pH and milk SCC were not affected whereas no significant variations were reported between Norm-Lab gr , UtrTors gr and UtrTors-Med gr ., (© 2023. The Author(s).)

It is a known fact that cryopreservation initiates premature capacitation in spermatozoa during the cryopreservation process. Protein tyrosine phosphorylation is a landmark of cascade reaction accountable for capacitation or capacitation-like changes in spermatozoa. Therefore, our hypothesis was to test an inhibitor (H89) that reversibly inhibits the cascade reaction responsible for capacitation during the cryopreservation process but does not hamper normal capacitation and fertilizing ability of sperm. For this, sixteen ejaculates were collected from Murrah buffalo bulls (n = 4). Each ejaculate was divided into four equal aliquots and diluted in an egg yolk-based semen dilutor supplemented with 0, 2, 10, and 30 μM concentrations of H89 and cryopreserved. Interestingly, H89 reduces cholesterol efflux from spermatozoa and protects spermatozoa from membrane damage during the cryopreservation process. H89 did not prevent lipid peroxidation of the sperm membrane. H89 reduced intracellular calcium concentration in spermatozoa in a dose-dependent manner, but tyrosine phosphorylation reduction was observed in the 2 and 10 μM H89 groups. The CTC assay revealed that the percentage of uncapacitated spermatozoa in different treatment groups increases in a dose-dependent manner. In the in vitro capacitation medium, the effect of H89 is abolished and spermatozoa underwent normal capacitation, but H89-treated spermatozoa attached to zona pellucida in large numbers compared to untreated spermatozoa. In conclusion, H89 does not only inhibit tyrosine phosphorylation of spermatozoa but it reduces cholesterol efflux and calcium influx, and ultimately reduces capacitation-like changes during the cryopreservation process., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that would prejudice the impartiality in conducting the experiment and publishing the manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.)

Introduction: Inhibin DNA vaccine has already been proven to improve the fertility of animals. This study aimed to investigate the effects of a novel Anti-Müllerian hormone (AMH)-Inhibin (INH)-RF-amide-related peptides (RFRP) DNA vaccine on immune response and reproductive performance in buffalo., Methods: A total of 84 buffaloes were randomly divided into four groups and nasally immunized twice a day with 10 ml of either AMH-INH-RFRP DNA vaccines (3 × 10 10 CFU/ml in group T1, 3 × 10 9 CFU/ml in group T2, and 3 × 10 8 CFU/ml in group T3) or PBS (as a control) for 3 days, respectively. All animals received a booster dose at an interval of 14 days., Results: ELISA assay revealed that primary and booster immunization significantly increased the anti-AMH, anti-INH, and anti-RFRP antibody titers in the T2 group compared with that in the T3 group. After the primary immunization, the antibody positive rate was significantly higher in the T2 group than that in the T3 group. In addition, ELISA results indicated that concentrations of E2, IFN-γ, and IL-4 were significantly higher in the antibody-positive (P) group compared to the antibody-negative (N) group. In contrast, there was no significant difference in the concentrations of P4 between the P and N groups. Ultrasonography results revealed a highly significant increase of 2.02 mm in the diameter of ovulatory follicles in the P group compared to the N group. In parallel, growth speed of dominant follicles was significantly higher in the P group than that in the N group (1.33 ± 1.30 vs 1.13 ± 0.12). Furthermore, compared to N group, the rates of oestrus, ovulation, and conception were also significantly higher in the P group., Conclusion: The novel AMH-INH-RFRP DNA vaccine improves the proportion of oestrus, ovulation, and conception in buffalo by promoting the production of E2 and the growth of follicles., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Zhao, An, Ahmad, Niu, Zhang, Nie, Tang, Liang and Yang.)

Sperm harbours a wide range of proteins regulating their functions and fertility. In the present study, we made an effort to characterize and quantify the proteome of buffalo bull spermatozoa, and to identify fertility associated sperm proteins through comparative proteomics. Using high-throughput mass spectrometry platform, we identified 1305 proteins from buffalo spermatozoa and found that these proteins were mostly enriched in glycolytic process, mitochondrial respiratory chain, tricarboxylic acid cycle, protein folding, spermatogenesis, sperm motility and sperm binding to zona pellucida (p < 7.74E-08) besides metabolic (p = 4.42E-31) and reactive oxygen species (p = 1.81E-30) pathways. Differential proteomic analysis revealed that 844 proteins were commonly expressed in spermatozoa from both the groups while 77 and 52 proteins were exclusively expressed in high- and low-fertile bulls, respectively. In low-fertile bulls, 75 proteins were significantly (p < 0.05) upregulated and 176 proteins were significantly (p < 0.05) downregulated; these proteins were highly enriched in mitochondrial respiratory chain complex I assembly (p = 2.63E-07) and flagellated sperm motility (p = 7.02E-05) processes besides oxidative phosphorylation pathway (p = 6.61E-15). The down regulated proteins in low-fertile bulls were involved in sperm motility, metabolism, sperm-egg recognition and fertilization. These variations in the sperm proteome could be used as potential markers for the selection of buffalo bulls for fertility., (© 2023. The Author(s).)

Apelin is an adipose tissue-derived hormone with many physiological functions, including the regulation of female reproduction. It acts through an orphan G protein-coupled receptor APJ/APLNR. The present study aimed to investigate the expression of apelin and its receptor APJ in the ovarian follicles and corpus luteum (CL) and the role of apelin on steroidogenesis and cell survival. Ovarian follicles were classified into four groups based on size and estradiol (E 2 ) level in the follicular fluid as follows: (i) F1 (4-6 mm; <0.5 ng/mL) (ii) F2 (7-9 mm; 0.5-5 ng/mL) (iii) F3 (10-13 mm; 5-40 ng/mL) and (iv) F4 (dominant/pre-ovulatory follicle) (>13 mm; >180 ng/mL). The corpora lutea (CL) were categorized into early (CL1), mid (CL2), late luteal (CL3), and regressing (CL4) CL stages. Expression of apelin increased with follicle size, with significantly greatest in the dominant or pre-ovulatory follicle (P < 0.05). Expression of APJ was greater in large and dominant follicles than in small and medium follicles (P < 0.05). In CL, the mRNA and protein abundance of apelin and apelin receptor was greater during mid (CL2) and late luteal (CL3) stages as compared to early (CL1) and regressing (CL4) stages (P < 0.05). Both the factors were localized in granulosa and theca cells of follicles and small and large luteal cells of CL. The pattern of the intensity of immunofluorescence was similar to mRNA and protein expression. Granulosa cells were cultured in vitro and treated at 1, 10, and 10 ng/mL apelin-13 either alone or in the presence of the follicle-stimulating hormone (FSH) (30 ng/mL) or insulin-like growth factor-I (IGF-I) (10 ng/mL) for 48 h. The luteal cells were treated with apelin-13 at 1, 10, and 100 ng/mL doses for 48 h. Apelin treatment at 10 and 100 ng/ml significantly (P < 0.05) increased E 2 secretion, cytochrome P450 aromatase or CYP19A1 expression in GC. In luteal cells, apelin at 10 ng/mL and 100 ng/mL significantly (P < 0.05) increased progesterone (P 4 ) secretion and HSD3B1 expression. In GCs, apelin, either alone or in combination, increased PCNA expression and inhibited CASPASE3 expression suggesting its role in cell survival. In conclusion, this study provides novel evidence for the presence of apelin and receptor APJ in ovarian follicles and corpora lutea and the stimulatory effect on E 2 and P 4 production and promotes GC survival in buffalo, suggesting the role of apelin in follicular and luteal functions in buffalo., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Semen dilution and cryopreservation alter the homogeneity of seminal plasma, resulting in a non-physiological redox milieu and consequently poor sperm functionality. Considering the concentration-specific bimodal action of nitric oxide (NO) in the regulation of sperm functions, cryopreservation media supplemented with optimized concentrations can improve the semen attributes. The present study aimed to evaluate the effect of adding an optimized concentration of sodium nitroprusside (SNP) and N-nitro-L-arginine methyl ester (L-NAME) in an extender on in vitro semen quality. An aliquot of semen samples (n = 32) from Murrah buffalo bulls (n = 8) was divided into control (C) and treatment (T-I: SNP in extender at 1 µmol/L; T-II: L-NAME in extender at 10 µmol/L). Fresh semen quality parameters showed no significant difference at 0 h except for the structural integrity in the T-II group. Post-thaw semen quality parameters and sperm kinematics using computer-aided sperm analysis (CASA) revealed significantly higher (p < 0.05) cryoresistance in the treatment groups. Viability, acrosome integrity, and membrane integrity were significantly higher (p < 0.05) in both treatment groups; however, the results were pervasive in T-II. Lower abnormal spermatozoa were observed in both T-I and T-II. SNP supplementation led to a significant rise (p < 0.05) in NO, whereas L-NAME reduced the NO concentration in post-thawed samples, which was directly correlated with different sperm functionality and associated biomarkers viz. total antioxidant capacity (TAC) and thiobarbituric acid reactive substance (TBARS). It was concluded that the cryopreservation media supplemented with SNP and L-NAME at 1 µmol/L and 10 µmol/L, respectively, lower the cryo-damage and improve post-thaw seminal attributes., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The present study aimed to investigate the Doppler indices and mRNA transcripts of hormone receptors in relation to the response of dilatation therapy in incomplete cervical dilatation (ICD) associated with uterine torsion in buffaloes. Out of 36 successfully detorted uterine torsion cases, eight buffaloes revealed a fully dilated cervix, while the remaining 28 had ICD, and subjected to dilatation therapy (500 μg cloprostenol + 2 mg estradiol benzoate + 80 mg valethamate bromide + 50 IU oxytocin + 250 mL calcium borogluconate). The responses of dilatation therapy were assessed in 26 buffaloes as one died, and one could not follow up. Doppler indices of middle uterine arteries on trans-rectal ultrasound were evaluated pre- and 30-60 min post-detorsion. Cervical tissue biopsies were collected from 16 buffaloes to study mRNA transcripts of hormone receptors. The duration, degree, location of uterine torsion, fetal viability, consistency of the cervix, relaxation of pelvic ligaments, udder engorgement, and gestation length were also recorded to evaluate the response of dilatation therapy. The 73.08% (19/26) buffaloes responded to the therapy with a duration ranging from 2 to 56 hrs (18.41 ± 4.11). The significantly increased blood flow volume (BFV) and time-average peak velocity (TAP) while the significantly reduced resistive index (RI) and pulsatility index (PI) in an ipsilateral middle uterine artery (MUA) at post-detorsion were observed in dilation therapy responded than the not-responded group. The mRNA transcripts of estradiol receptors-α (ESR1), prostaglandin receptors (PTGFR), and oxytocin receptors (OXTR) were upregulated by 7.47, 6.63, and 8.72-fold in the ICD group, respectively. The Doppler indices along with duration of illness, location of uterine torsion, consistency of the cervix, and udder engorgement can be used to predict the response of dilatation therapy in ICD associated with uterine torsion. The upregulated mRNA expression of ESR1, PTGFR and OXTR is mandatory for success of dilatation therapy., Competing Interests: Declaration of interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.)

We investigated the effect of different types of mother-young contacts (fenceline, restricted and no contact) on the health, growth performance, behaviour and physiological state of Murrah buffalo calves. A total of 24 calves were allocated to three groups of 8: NCM (no calf-mother contact), RCM (restricted calf-mother contact) and FCM (fenceline calf-mother contact). At three months of age, the FCM calves had a higher average body weight (58.6 ± 1.5 kg) than the RCM (52.8 ± 1.3 kg) and NCM (53.6 ± 1.3 kg) calves ( P < 0.05). The average daily gain (ADG) at three months of age was greater in FCM (0.6 ± 0.1 kg/d) than RCM (0.5 ± 0.1 kg/d) and NCM (0.5 ± 0.1 kg/d) calves ( P < 0.05). The mean immunoglobulin G (IgG) concentrations were significantly greater on d 7, 28, 42 and d 56 of sampling ( P < 0.05 for all comparisons) in FCM than RCM and NCM calves. The mean cortisol levels were highest in the NCM calves followed by RCM and the lowest values were observed in FCM calves on d 0, d 7, d 28, d 42 and d 56, respectively ( P < 0.05, for all recorded days). The diarrhoea score and eggs per gram of faeces (EPG) were significantly greater in the NCM group compared to the RCM and FCM groups. The time spent in cross sucking, licking inanimate objects and self-licking was greatest ( P < 0.05) in NCM calves, followed by RCM calves, and was almost non-existent in the FCM group on all the recorded weeks. Time spent in backward ear position was greater ( P < 0.05) in FCM than RCM and NCM calves. The average time spent in forward ear position was greatest ( P < 0.05) in the RCM calves followed by NCM with the lowest time observed in FCM calves. The mean duration of time spent by calves in the asymmetrical and axial ear position differed significantly ( P < 0.05) among NCM, RCM and FCM calves, P < 0.05. The full and fenceline buffalo calf-mother contact system showed higher growth rates, lower levels of stress responses, oral stereotypies and eggs per gram of faeces. In addition, ear postures may be used as a dependable, and reliable measure of positive, low arousal emotional states.

Sperm mitochondrion is one of the major susceptible organelles that get damaged during cryopreservation. The study aimed to minimize mitochondrial dysfunction and oxidative stress during sperm cryopreservation using mitochondria-specific antioxidants. For this, semen was collected from five buffalo bulls (3 ejaculates/bull). The ejaculates were diluted in an low-density lipoprotein-based extender and divided into four equal aliquots. Mitochondria-targeted antioxidant (MitoQ) was added at a final concentration of 0 (control), 0.02, 0.2 and 2 μM separately in each aliquotes and cryopreserved. The addition of MitoQ at a concentration of 0.02 μM improved post-thaw sperm motility, plasma membrane integrity and able to sustain sperm motility for a longer time. To investigate MitoQ's effects on mitochondrial function, we measured mitochondrial membrane potential (MMP) using JC-1 dye, superoxide production using Mitosox assay, and lipid peroxidation by TBARS assay. The supplementation of 0.02 μM MitoQ in the extender prevented the significant reduction of MMP and reduced superoxide production resulting in lower lipid peroxidation of sperm plasma membrane after cryopreservation. Further, we found that a higher concentration of MitoQ decreases MMP and increases mitochondrial superoxide production. In conclusion, MitoQ @ 0.02 μM can alleviate oxidative stress by regulating mitochondrial functionality in spermatozoa during cryopreservation., (© 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.)

Background: Dissolved oxygen (DO) in semen dilutor may lead to the production of reactive oxygen species (ROS) and buffalo sperm may become more prone to deleterious effects of ROS due to the presence of high amounts of polyunsaturated fatty acids (PUFAs) in their membranes., Objective: To study the correlation between dissolved oxygen level, antioxidants and oxidants in semen diluted with partially deoxygenated extender at various stages of cryopreservation., Materials and Methods: Each semen sample was split into two aliquots viz., Aliquot I [diluted with Extender I (control: without deoxygenation)] and Aliquot II [diluted with Extender II: partially deoxygenated by liquid nitrogen (LN) flushing], which were diluted, filled in straws, cryopreserved and evaluated post-thaw., Results: The DO levels (P < 0.05) decreased significantly after LN flushing of the extender and they increased significantly (P < 0.05) in post-thaw semen. The progressive motility, viability, hypo-osmotic swelling response, acrosomal integrity, glutathione peroxidase (GPx), total antioxidant capacity (TAC) and superoxide dismutase (SOD) decreased significantly (P < 0.05) in both control and treated semen after thawing. SOD and TAC were positively correlated in semen treated with normal extender at the pre-freeze stage; however, in semen treated with partially deoxygenated extender, no correlation was found between SOD and TAC at the pre-freeze stage. ROS and total TAC were negatively correlated in semen treated with partially deoxygenated extender at the pre-freeze stage; however, no correlation was found between ROS and TAC in control semen., Conclusion: The partial deoxygenation of extender affects the correlation between sperm quality parameters, antioxidants, and oxidants during different stages of semen cryopreservation. doi.org/10.54680/fr22310110712.

Background: The discrepancy between the endogenous antioxidants concentrations and free radicals results in oxidative stress and cellular injury., Objective: To appraise the usefulness of Rosemarinus officinalis (RO) aqueous extract in protecting buffalo spermatozoa during freezing / thawing process., Materials and Methods: Qualifying ejaculates from four well-restrained bulls were evaluated initially and then diluted in a freezing medium supplemented with RO-0.0, RO-0.5 %, RO-1.0%, RO-2.0 %, and RO-4.0 %, cooled to 4 degree C in 2 h, equilibrated for 4 h at 4 degree C, packed in straws, and cryopreserved, and thawed at 37 degree C for 30 s followed by evaluation., Results: We found that freezing medium supplemented with RO-2.0 % improves progressive motility (%) compared to the control. Similarly, a lower rate of apoptosis-like changes (%) was recorded with RO-4.0 % than the control, RO-0.5 % and RO-1.0 %. This response was accompanied by an increment in viable spermatozoa. Semen samples supplemented with RO-2.0 % and RO-4.0 % displayed higher TAC (total antioxidant capacity, uM per L) and ATP (nmol/million) content than the control. In addition, semen samples supplemented with RO-2.0 % displayed lower concentrations of ROS (reactive oxygen species, 10 4 RLU/20 min/25 million) than the control and RO-0.05 %. Also LPO (lipid peroxidation, uM per L) with RO-2.0 % and RO-4.0 % was lower than the control., Conclusion: The inclusion of rosemary aqueous extract ameliorates motility features, structural and functional parameters, viability, TAC and ATP content of bull sperm. Conversely, the inclusion of rosemary aqueous extract alleviates apoptosis-like changes, ROS and LPO in comparison to the control. Further studies are required to determine the mechanism of action of rosemary aqueous extract in ameliorating semen quality and fertility of buffalo spermatozoa. doi.org/10.54680/fr22210110712.

The present investigation evaluated the effect of microclimate modification on feed intake, growth performance, and hemato-biochemical profile of Murrah buffalo (Bubalus bubalis) heifers during summer. Twenty-four buffalo heifers, between 15 and 20 months of age with an average body weight of 363.75 ± 11.27 kg, were randomly assigned to 4 groups based on their age and body weight. The heifers of the T0 (control) group were kept under the shed without any cooling treatment, while the animals in group T1 were tied with a cooling jacket. Buffalo heifers of group T2 were subjected to a cooling jacket with forced ventilation and animals in group T3 were treated with intermittent sprinkling (10 min., at 2 h intervals) and forced ventilation between 9.00 AM and 6.00 PM during the experiment. The ambient temperature inside the shed and core body temperature were reduced in groups T1, T2, and T3 compared to T0. Heifers had higher dry matter intake when subjected to cooling treatments T1, T2, and T3, whereas water intake was reduced in T2 and T3 groups. The animals in T2 and T3 groups attained higher average daily weight gain, while the feed conversion was better in the T3 group compared to T0. The hematological measures such as hemoglobin, total erythrocyte count, and total leucocyte count were found higher in T0. The serum glucose, sodium, and potassium levels increasedand alkaline phosphatase activity decreased in groups T1, T2 and T3 when compared with T0. It can be concluded that the provision of intermittent sprinkling and cooling jacket in combination with forced ventilation could improve the microclimate, which in turn could enhance the performance of Murrah heifers during hot summer days in the tropics., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Cryopreservation is an important technique for the long-term storage of semen for artificial insemination (AI). Buffalo spermatozoa are sensitive to cryopreservation procedures because of the presence of a high amount of polyunsaturated fatty acids in the plasma membrane., Objective: To study the effect of different concentrations of BHT on the quality of Murrah buffalo bull semen for low-dose cryopreservation., Material and Methods: Semen was collected from four high fertile Murrah buffalo bulls (6 ejaculates each) using an artificial vagina. A total of 24 ejaculates were collected from each bull twice a week using an artificial vagina. Every sample was split into four parts: Control without additives; and three treatments with BHT at 0.5 mM, 1 mM or 2 mM. Semen was cryopreserved at low-dose sperm cryopreservation of 20, 15, 10 and 5 million sperm per aliquot after supplementation of BHT. Semen samples were evaluated for fresh, pre-freeze and post-thaw stages., Results: There was a significant increase (p<0.05) in sperm quality parameters, such as progressive motility (%), viability (%), HOST response (%), acrosome integrity (%) and post-thaw motility, with the addition of 0.5-1 mM BHT., Conclusion: The addition of BHT in Murrah buffalo semen improves the low dose cryopreservation quality in a dose-dependent manner. doi.org/10.54680/fr23110110612.

Leydig cells (LCs) are testosterone-generating endocrine cells that are located outside the seminiferous tubules in the testis, and testosterone is fundamental for retaining spermatogenesis and male fertility. In buffalo, adult Leydig cells (ALCs) are developed by immature Leydig cells (ILCs) in the postnatal testes. However, the genes/pathways associated to the regulation of testosterone secretion function during the development of postnatal LCs remains comprehensively unidentified. The present study comparatively analyzed the transcriptome profiles of ILC and ALC in buffalo with significant differences in testosterone secretion. Differentially expressed genes (DEGs) analysis identified 972 and 1,091 annotated genes that were significantly up- and down-regulated in buffalo ALC. Functional enrichment analysis showed that cAMP signaling being the most significantly enriched pathway, and testosterone synthesis and lipid transport-related genes/pathways were upregulated in ALC. Furthermore, gene set enrichment analysis (GSEA) shows that cAMP signaling and steroid hormone biosynthesis were activated in ALC, demonstrating that cAMP signaling may serve as a positive regulatory pathway in the maintenance of testosterone function during postnatal development of LCs. Protein-protein interaction (PPI) networks analysis highlighted that ADCY8, ADCY2, POMC, CHRM2, SST, PTGER3, SSTR2, SSTR1, NPY1R, and HTR1D as hub genes in the cAMP signaling pathway. In conclusion, this study identified key genes and pathways associated in the regulation of testosterone secretion function during the ILC-ALC transition in buffalo based on bioinformatics analysis, and these key genes might be deeply involved in cAMP generation to influencing testosterone levels in LCs. The results suggest that ALCs might increase testosterone levels by enhancing cAMP production than ILCs. Our data will enhance the understanding of developmental mechanism studies related to testosterone function and provide preliminary evidence for molecular mechanisms of LCs regulating spermatogenesis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We explored different aspects of buffalo spermatozoa during cryopreservation. The meta-data comprised of 285 studies, published from January 2008 to March 2020. A free web tool CADIMA as well as PRISMA 2009 Flow Diagram were used for carrying out this study. The inter-reviewer agreement among studies allocated was satisfactory for criteria A (selection bias), B (performance bias), C (detection bias) and D (attrition bias), respectively. India led the percent (%) research ladder with 34.4, followed by Pakistan (29.5), Egypt (12.3), Iran (7.7), Italy (5.6), Indonesia (3.2), China (2.1), Brazil (1.4), Thailand (1.1), Philippines and Bulgaria (0.7 each), Saudi Arabia, Turkey, Vietnam, and USA (0.4 each). Among four categories of studies, Group-1 evaluated only supplements/additives/media in the freezing semen extender (n = 191/285; 67.02%); Group-2 conducted in vivo fertilization (n = 62/285; 21.75%) and Group-3 conducted in vitro fertilization/ cleavage rate/penetration rate/ blastocyst yields (n = 28/285; 9.82%) with their specific cryodiluents/media, respectively. Group-4 conducted different experimental supplements/additives/media and carried out both in vitro and in vivo fertilization simultaneously (n = 4/285; 1.40%). Conventional spermatozoa cryopreservation was reported by 51.9% studies followed by programmable fast freezing by 20.7% studies. A few leading extender types included BioXcell (3.9%); Soyamilk-skim (3.5%); and Andromed (2.1%). The study also describes French straws for semen filling, cooling temperatures, extension time, equilibration time, cryopreservation stages, thawing temperatures, seasons, thawing time, and stains used during semen evaluation assays. The study concludes that the research on spermatozoa cryopreservation of buffalo is largely conducted at quality level and a need of applying these findings for evaluation of fertility potential (in vivo and in vitro) is indispensable for effective genetic improvement., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Missing an estrus event is an economic problem in buffaloes because of lack of a simple and accurate estrus identification method. Saliva, a non-invasive fluid available every time, showed typical fern-like crystallization patterns at early estrus in buffaloes. However, to implement this salivary ferning based estrus identification method in the field conditions, the present study, for the first time, validated this method in four buffalo population samples (PS) representing four different field scenarios, an organized herd (PS1, 10 buffaloes monitored daily for a year (149 estrus events)), artificial insemination (AI) centers (PS2, 114 buffaloes brought for AI), induced estrus condition (PS3, 44 buffaloes) and farmers' doorsteps (PS4, 275 random buffaloes with unknown reproductive history and no estrous signs). Totally, 582 saliva samples were collected from 443 buffaloes. Salivary smears were observed under a simple microscope and/or a less expensive (< 1USD) paper microscope, Foldscope. On the basis of salivary fern-like patterns, the proportions of estrus identification were significantly different among PS. Specifically, the proportions in the PS1 (0.74, P < 0.0001) and PS4 (0.08, P < 0.05) were significantly higher than their population proportion estimates, 0.5 and zero, respectively. Therefore, this estrus identification method is much useful in the PS1 and PS4, the practical field scenarios requiring an accurate estrus prediction method, compared to the PS2 and PS3. Especially, this method is 91 % confirmatory to detect early estrus in PS4. Therefore, salivary ferning is a useful technique to identify early estrus in buffaloes in the field conditions at farmers' doorsteps., Competing Interests: Declaration of Competing Interest The authors declare no competing or financial interests., (Copyright © 2021 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Background: Heat stress in tropics is generally associated with significant economic losses resulting from reduced performance, morbidity, and mortality of livestock. To avoid serious consequences of heat stress, it is imperative to better understand the physiological responses and biochemical changes under the state of altered body homeostasis during different seasons of the year., Objectives: This study aimed to evaluate the seasonal dynamics of physiological, oxidative and metabolic responses of lactating Nili-Ravi buffaloes to the tropical climate of South China., Methods: Physiological responses including rectal temperature (RT), body surface temperature (BST) and respiratory rate (RR) along with serum biochemical and antioxidant parameters of 20 lactating Nili-Ravi buffaloes were evaluated during different seasons of the year., Results: Higher temperature-humidity Index (THI) during the summer season (>80) resulted in a significant increases in RR and BST as compared to the winter season. Higher oxidative stress was observed in the summer season as revealed by significantly higher MDA while lower serum antioxidant enzyme (TAC, GSH-Px, SOD and CAT) contents. Moreover, serum cortisol was also significantly higher in summer and autumn. The levels of growth hormone and ACTH were also significantly (P < 0.05) lower in summer and autumn as compared to other seasons. The negative association of THI with physiological and antioxidant parameters was observed while it was positively associated with serum MDA and cortisol levels., Conclusions: Our study revealed moderate heat stress in lactating buffaloes in the summer season which calls for attention to avoid economic losses and animal welfare issues., (© 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)