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Mastitis remains a major challenge in the dairy sector, causing significant economic losses and impacting animal welfare globally. Early detection of sub-clinical (SCM) and clinical mastitis (CM) is crucial for effective management, particularly in varying environmental conditions. The current study aims at thermal imaging of the udder, teat, and short milking tube (SMT) of the milking machine across milking phases and seasons to assess SCM and CM via infra-red thermography (IRT) in a sub-tropical organised farm of Karnal, India. The udder health status of 98–117 lactating Holstein Friesian crossbred (HFCB) cows was screened monthly using IRT and further accessed using the California Mastitis Test (CMT) and somatic cell count (SCC). The thermal image analysis revealed a difference (p

Aim: The present study was carried out on semen ejaculates of 15 Karan Fries (KF) bulls maintained at Artificial Breeding Research Centre, National Dairy Research Institute, Karnal, India with an objective to evaluate the relationship between the conventional and fluorescent based semen quality analysis of the bulls. Materials and Methods: A total of 96 ejaculates were collected from 15 KF (Holstein Friesian [HF] crossbred) bulls. Semen were evaluated for color, volume, mass activity (MA) and percentage of individual motility (IM), sperm concentration, percent live spermatozoa, hypo-osmotic swelling test and acrosome integrity, chromatin integrity, sperm viability, and membrane integrity. Data were analyzed using SPSS software package for descriptive analysis. The correlation between rankings of sires based on conventional and fluorescent semen parameters were calculated by Spearman’s rank correlation coefficient. Results: The average ejaculates volume (ml), sperm concentration (106/ml), MA, IM (%), live (%), morphological abnormalities (%), host (%), acrosome integrity (%), chromomycin A3 (CMA3) (%), SYBR-PI (%), and fluorescent isothiocyanate-peanut agglutinin (FITC-PNA) (%) were 4.57±0.36, 1162.98±97.93, 2.95±0.09, 60.8±1.22, 71.41±2.10, 9.31±1.15, 65.5±1.81, 86.6±1.59, 3.53±0.43, 65.39±2.23 and 74.47±2.53, respectively. Rank correlations were found to be significant for SYBR-PI and FITC-PNA with most of the parameters evaluated by conventional methods. Overall, among conventional criteria, IM revealed ranking of bulls almost similar to that of fluorescent criteria. Conclusion: Overview of our results indicated that, among conventional criteria, MA and IM revealed ranking of bulls almost similar to that of fluorescent criteria.

Aim: The study was conducted at Artificial Breeding Research Centre, NDRI, Karnal, to determine the sexual behavior and its relationship with semen quality parameters in Sahiwal breeding bulls. Materials and Methods: A total of 63 ejaculates were collected from six adult Sahiwal bulls (age ~47 mo and bwt ~466 kg), to study the relationship of sexual behavior and semen quality. The degree of association between different variables was estimated by Pearson’s correlation coefficient method. Results: The results depicted that, sexual aggressiveness showed significantly high positive correlation with libido score (LS) and sexual behavior score (SBS). Reaction time (RT) and total time taken in mounts (TTTM) had a significant negative correlation with LS and SBS. Penile erection score and penile protrusion score (PPS) both had a significant positive correlation with ejaculatory thrust score, mating ability score, and SBS. Results of correlation among seminal attributes and with sexual behavior depicted that ejaculate volume had positive significant correlation with initial progressive motility (IPM), sperm concentration (SCON), head abnormality, total abnormality, hypo-osmotic swelling test (HOST), acrosomal integrity (AI) whereas, mass activity had positive significant correlation with IPM, SCON, non-eosinophilic spermatozoa count (NESC), HOST, AI, RT and TTTM and IPM had positive significant correlation with SCON, NESC, HOST, AI, and TTTM, whereas and HOST had positive significant correlation with AI. Among seminal attributes, SCON had a positive significant correlation with PPS where as head abnormalities had a positive significant correlation with RT and TTTM. Conclusion: It can be concluded that the relationship of sexual behavior and semen quality parameters are reflecting that the sexual behavior of individual bulls is important to harvest good quality and quantity of semen as desired type of sexual preparation can be provided.

Aim: To study the effect of preputial washing on bacterial load, preservability and semen quality in Murrah buffalo bulls Materials and Methods: A total of 36 collections of three Murrah buffalo bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal, were collected at weekly intervals from each bull without preputial washing and latter ejaculates from same bull with preputial washing by infusing normal saline (0.85%), KMnO4 (0.02%) and savlon (2.0%) to first, second and third bull, respectively. The microbial load and semen quality were evaluated during different hours of storage at refrigerated temperature (0, 24 and 48 h) and after thrawing of cryopreserved (at −196°C) semen. Results: The results of preservation of semen at refrigerated temperature showed that bacterial load was markedly lower in ejaculates of bulls subjected to preputial washing. Semen preserved at refrigerator temperature and cryopreserved, the effect of washing solution was significant for individual motility (IM), non-eosiniphilic count, hypo-osmotic swelling reactivity (HOST), total plate count (TPC) and acrosome integrity. KMnO4 was found to be the best in lowering bacterial load, sperm abnormalities and in improving semen quality such as motility, non-eosinophilic count, HOST and acrosome integrity even up to 48 h of preservation and cryopreserved semen. Effect of duration of preservation and stage of cryopreservation was also significant for IM, non-eosiniphilic count, HOST, sperm abnormalities and acrosome integrity. Conclusion: Overall the results suggested that preputial washing with KMnO4 solution improved the semen quality and reduced microbial load of Murrah buffalo bull’s semen preserved at refrigerated temperature and cryopreservation.

Aim: The present study was carried out to analyze the disposal rate in different age groups of Karan Fries (KF) males in National Dairy Research Institute herd. Materials and Methods: Records on 1740 KF crossbred bulls born during the period 1997-2012 were collected with an objective to ascertain the effect of genetic and non-genetic (Period of birth and season of birth) factors on the disposal pattern of KF males. The percent of animals disposed from the herd due to mortality and culling was calculated by proportion using descriptive statistics. The data were subjected to Chi-square test to test the difference due to different factors. Results: Overall disposal rate for the different age groups of 0-1 m, 1-2 m, 2-3 m, 3-6 m, 6-18 m, 18 m-3 year and 3-5 year were calculated as 17.9, 16.3, 14.2, 25.8, 49.0, 37.6 and 51.65%, respectively. In the age groups, 3-6 m, 6-18 m and 3-5 year, effect of periods of birth were found to be statistically significant (p1 month, culling was the primary cause.

Aim: The present study was carried out to analyze mortality pattern in Murrah buffalo males in National Dairy Research Institute herd. Materials and Methods: Records of 1029 Murrah buffalo males born during the period 1997-2012, at NDRI, Karnal, were analyzed to study mortality pattern in different age groups. The percent of animal disposed from the herd due to different reasons was calculated by proportion using descriptive statistics. Results: The overall percent mortality in 0-1m, 1-2m, 2-3m, 3-6m, 6-18m, 18m-3 yr and >3 year age groups were 17.49, 5.99, 3.34, 5.42, 6.35, 2.59 and 3.93, respectively. The main cause of mortality in 0-1, 1-2, 2-3 and 6-18 m of age groups was respiratory problems (6.61%, 1.79%, 1.06% and 1.97% respectively). Main causes of mortality in age group 3-6 months were general debility (1.57%) followed by respiratory and digestive problems (1.22%), miscellaneous (0.87%), liver and urinary problems (0.17%). Mortality could not reveal any consistent trend across different seasons and period of birth. General debility (1.29%) followed by toxemia, liver problems and miscellaneous reasons were main causes of mortality in age group 18 m – 3 yr of age. In males above three years of age, general debility (2.81%) followed by respiratory problems (1.12%) were the main causes of mortality. Conclusion: Intensive health care and management is required for young male buffalo calves especially in hot humid months of rainy season and cold inclement weather conditions in winter months to minimize mortality due to respiratory and digestive problems.

The present study was planned to understand the effect of scrotal surface temperature on semen quality and Infrared thermography as a tool for breeding soundness evaluation of Zebu bulls. The thermal imaging of the scrotum revealed significant differences in temperatures from the neck to the ventral portion of the scrotum. The average neck and ventral temperature of the scrotum was 34.2±0.62°C and 31.22±0.53°C, respectively. The average gradient temperature of the scrotal surface (neck-ventral) was 2.95±0.30°C with a range of 1.2-5.7°C. The MA of bull semen (3.12±0.04 vs. 1.89±0.13), live sperm (73.82±0.88% vs. 59.42±1.58%), HOST (66.67±1.52% vs. 55.49±1.14%) and sperm concentration (1226.36±18.80 vs. 942.19±23.47m/ml) were significantly more in good semen quality bulls than poor semen quality bulls. Total sperm abnormality was significantly lesser in bulls having a larger scrotal temperature gradient. The scrotal skin thickness was significantly more in poor semen quality bulls than good semen quality bulls (5.70±0.12 vs. 5.07±0.13). The scrotal skin thickness was found highly correlated (r-0.77) with the scrotal temperature gradient. So it can be concluded that scrotal surface temperature is related to the semen quality of bulls, and thus the thermal imaging can be used for breeding soundness evaluation of Zebu bulls.

Low sperm numbers in artificial insemination (AI)-doses are being used widely to make the best use of high genetic value bulls as well as sex-sorted semen. Sperm concentration needed for AI to obtain reasonable fertility, taking genetic value of bull and numerous others components into consideration is one of the essential constituents for successful AI breeding program. However, low sperm concentrations in AI-doses lead to reducing post-thaw viability. The reduction in viability of low sperm doses may be affected by fresh semen volume, sperm number and seminal plasma level at final dilution. Reduction in quality and fertility of low sperm doses is one of the limitations for their use in successful AI programme. Sperm number per AI required to achieve optimum fertility is one of the main crucial things to AI industry, and numerous efforts have been made in this regard. Due to great variability among bulls, sperm number per AI could be a limiting factor in achieving acceptable fertility values. Fertility of low sperm doses may vary among bulls, and non-return rates (NRRs) with low sperm doses may be determined by fertility level of bull. On the basis of individual bulls, sperm numbers in AI doses needed to be adjusted to reduce the variations in NRRs among bulls. Utilizing high fertile bulls for low sperm doses with acceptable non-return rates (NRRs) may be a way to cover a large number of bovines under AI in countries like India. Deposition site within the uterine horn may alter non return rates following inseminations with low sperm doses. Following deep-uterine inseminations, acceptable pregnancies may be achieved with low sperm doses and even if ovulation side is unknown.

The objective of the study was to establish the characteristics of pedometer activity in crossbred cows (20) and to determine the relationships between the pedometer activity and the serum concentrations of periestrual hormones. Animals in oestrus were detected by trained personnel based on oestrus behaviour by visual observation twice daily. Further oestrus was detected by pedometer activity and it was confirmed by serum progesterone and estradiol concentration. Individual animal daily activity data were collected and transformed into hourly activity in Excel sheet and arranged for statistical analysis. The relationship of pedometer activity with E2, E2: P4 and progesterone level on the day of oestrus was determined. Pearson correlation analysis was performed using the statistical package SPSS (SPSS Inc. USA). Activity count per hour (ACPH) increased from 248.51±22.46, 312.51±37.16, 323.52±49.24 and 423.42±47.77 from -3 day, -2 day and -1 day of proestrus, respectively to estrus day. After estrus, the activity decreased from 313.76±46.62 and 271.36±36.06 on the 1st day and 2nd day of metestrus, respectively. A similar trend was observed for serum E2 and E2: P4 which increased from 3 days before estrus to reach a peak level on the day of estrus (31.40±2.34 pg/ml) and declined after that to the basal level (2 pg/ml) after estrus. Contrary to this, P4 concentration showed a decreasing trend from –3 day proestrus to estrus (0.46±0.05) and then a gradual increase after estrus day. ACPH showed positive correlation with estradiol concentrations (r = 0.34; P = 0.0779) and E2:P4 ratio (r=0.50; P=2.077) but, negative correlation with progesterone concentration on day of oestrus (r=–0.73; P=2.74). In conclusion, our results suggested that the concentration of E2, E2:P4 ratio and P4 concentration during proestrus and on the day of estrus are the important factors contributing the behavioral manifestation of estrus in terms of ACPH in crossbred cows.

This article presents a review pertains the laser irradiation effects and its possible mechanisms of action on spermatozoa functions in domestic animals. To improve artificial insemination, laser is sensitive and cost effective technique, when compared to other conventional methods. Laser may have both positive and negative effects on spermatozoa functions. Since the effects of light are mediated by reactive oxygen species, and the levels of these reactive oxygen species following irradiating spermatozoa with laser may be responsible for determining the effects of laser on sperm. Dose of laser may be regarded as of great significance and this dosage of laser may be responsible for determining its effects on spermatozoa. Optimum dosage of laser for improving seminal attributes may vary among various species and this need to be standardized in each of them. The beneficial effects include improving sperm livability, acrosomal integrity, hypo-osmotic swelling response, mitochondrial function and computer-aided sperm analysis parameters. The increase in cytochrome c oxidase activity, ATP levels and mitochondrial membrane potential, in laser irradiated cells may be responsible for enhanced sperm quality parameters. Improving fertility with laser irradiated spermatozoa has been reported in few species like boar and need to be elaborated in other species. In conclusion laser may be regarded as an easy, cheap and time saving technology for improving artificial insemination; in addition, laser may have various potential applications in the field of reproductive biotechnology as well as in livestock farms and veterinary polyclinics.

The study was planned to overcome the poor libido problem through biostimulation on eight healthy and adult Sahiwal breeding bulls not donating semen at Artificial Breeding Research Centre, ICAR-NDRI, Karnal, Haryana. The bulls were first taken as control and their sexual behaviour was recorded by CCTV video camera. The bulls were biostimulated by applying estrous specific mucus on the rump region of dummy prior to semen collection. Out of 8 bulls, 6 bulls (75%) started donating good quality semen after application of estrous specific mucus. The 6 bulls produced quality semen having optimum ejaculate volume, mass activity, initial progressive motility, concentration, pH, non eosinophilic spermatozoa count, total morphological abnormality, HOST, acrosomal integrity, SYBR-14PI and CMA3. There was no significant difference observed in semen quality and sexual behaviour between cervical mucus and synthetic nasal spray treated groups. All sexual behaviour parameters significantly improved in both the treatment group as compared to control. Thus biostimulation by cervical mucus is effective for quality semen production besides better sexual performance of bulls.

The present study was conducted at Artificial Breeding Research Centre, to ascertain the influence of genetic (3 genetic groups of Holstein Friesian and Tharparkar crosses and pure Tharparkar) and non genetic (Periods i.e.,1997– 2000, 2001–2004, 2005–2008 and 2009–2012; seasons i.e., winter, summer, autumn and rainy; age group i.e., 2–2.5 yrs, >2.5–3 yrs and >3 yrs) factors on semen production performance of Karan Fries (KF) and TP breeding bulls. Overall percent of males reserved for breeding was 16.67% in KF and 22.73% in TP. Out of males reserved for breeding, 58.97% KF and 20% TP males produced semen. Only 64.9% produced freezable semen in KF bulls whereas in TP bulls, all males which donated semen produced freezable quality semen. Periods of birth showed significant effect on % age at first successful collection (AFSC) and (P18 months of age). For both KF and TP, overall least square means of AFSC, AFSF, ALSF (age at last successful freezing), ALSC (age at last successful collection), SPP (semen production period), FSPP (freezable semen production period) and AD (age at disposal) were 879.21, 1077.65, 1659.87, 1425.53, 831.82, 775.44 and 1329.38 days, respectively. Effect of period of birth was significant for all the traits except AFSC. Season of birth had no significant effect on all the traits while genetic groups and age at disposal have significant effect on AFSC and AFSF, respectively.

Sub-fertility is one of the major concerns in crossbred bulls as 42.98% bulls are getting culled due to sub- fertility problems therefore, a study was planned on 18 Karan Fries bulls producing poor quality semen or not mounted. They were randomly grouped into 3 treatment groups of 6 animals each and 2 normal bulls were kept as control. Sexual behavior and semen quality were studied during pre-therapy (1 month), therapy (90 days) and post- therapy (90 days) period. Data were analyzed using ANOVA. During therapy period, first group treated with 10 g of herbal product orally, second group with 0.0082 mg of GnRH intramuscularly at 10 days interval and third group with 80 ppm of zinc for 90 days for each group. The results depicted that herbal and GnRH treatment significantly reduced the reaction time, whereas herbal, GnRH treatment and zinc supplementation significantly improved the libido score and reduced per cent of non-mounts during the therapy and post-therapy period. The volume of semen and level of testosterone improved in all treatments groups. Herbal, GnRH treatment and zinc supplementation increased the average number of good and medium quality ejaculates/month/bull and maximum efficiency achieved during the treatment period. There was increase in percent live sperm and decrease in abnormality in herbal treated and zinc supplemented group. Therefore, it can be concluded that GnRH therapy, herbal treatment and zinc supplementation will be effective to overcome the sub-fertility problem in crossbred bulls.

To improve the quality of poor ejaculates, sephadex (FS) and sephadex with ion-exchangers (FS+IE) filtration were used to examine split samples of 18 ejaculates of 6 Karan Fries bulls at various stages of cryopreservation. Data were analyzed using analysis of variance. All semen quality traits improved in both the filtration and further superiority of FS+IE column was observed due to its better efficacy of trapping immotile, dead and abnormal spermatozoa as well as there was decrease in sperm concentration, tail and total abnormalities. The initial, prefreeze and post-thaw motility, viability, sperm cells with intact plasma membranes and sperms with normal acrosomes of FS+IE filtrates were significantly better than the nonfiltered and FS filtered semen samples during different stages of cryopreservation. Therefore, it can be concluded that better quality semen can be harvested from poor ejaculates through FS+IE and FS filtration to improve fertilization potential of the spermatozoa.

Poor male fertility significantly affects dairy production, primarily due to low conception rates (CR) in bulls, even when cows are inseminated with morphologically normal sperm. Seminal plasma is a key factor in evaluating the fertilizing ability of bull semen. The extracellular vesicles (EVs) in seminal plasma contain fertility-associated proteins like SPAM1, ADAM7, and SP10, which influence sperm function and fertilizing potential. This study aimed to assess EV-associated fertility proteins in bulls with varying fertility levels and to investigate the effects of seminal plasma EVs (SPEVs) from high-fertility (HF) bulls on the spermatozoa of low-fertility (LF) Sahiwal bulls. Seminal plasma was isolated from the fresh semen of Sahiwal bulls, with four bulls classified as high fertility and three as low fertility. Fertility-associated proteins such as SP10, ADAM7, and SPAM 1 were highly expressed in SPEVs of high-fertility (HF) bulls. PKH26 dye-labeled SPEVs demonstrated significant uptake by spermatozoa at pH 6.8 with ≥ 4 h of co-incubation. Exposure of HF SPEVs to low-fertility (LF) spermatozoa reduced acrosome response, capacitation, and reactive oxygen species (ROS) formation. Our findings suggest that protein repertoires in SPEVs influence sperm activities such as motility, acrosome response, and ROS production. Supplementing LF spermatozoa with HF SPEVs could enhance their functional characteristics, highlighting these proteins as potential resources to modulate cattle bull sperm fertilizing ability., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: The animal study was approved by the Institutional Animal Ethics Committee (IAEC), National Dairy Research Institute, Karnal. The study was conducted in accordance with the local legislation and institutional requirements. The study was conducted in compliance with ARRIVE guidelines., (© 2025. The Author(s).)

Sperm motility is the prime functional attribute for semen quality and fertility of the bull. However, the bull's age directly affects the semen quality, and the bull's fertility and productive life decline with age. Even though research on age has been conducted in the past, it is still unclear how old a bull should be maintained at artificial insemination centers. The minimum standard protocol (MSP) states that a bull has an 8-years productive lifespan, yet it has been shown that some bulls older than 8 years have produced freezable ejaculates. It has yet to be established what the bull's ideal productive life is under the tropical climate of India without affecting in-vitro sperm functions. For the validation of the maximum age of a bull up to that it could be utilized in the artificial insemination centers, cut-off values of oxidative stress in freezable semen ejaculates were used. The current study was done at the Artificial Breeding Research Centre, ICAR-NDRI, Karnal, Haryana, India. A total of eighteen Sahiwal breeding bulls were divided into three groups: Young (2-4 years), Adult (> 4-8 years), and Older (> 8-11 years). Three ejaculates were taken from each bull; thus, a total of 18 ejaculates were utilized in each group for semen examination. Semen quality of Young bulls was poor compared with adult and older bulls. Seminal parameters such as dead, moribund, apoptotic, reacted acrosome, and protamine deficient sperms were higher in older bulls than in adult bulls; however, the values were non-significant. Less difference was found in the semen parameters among adult and older bulls. This indicates that the semen quality of older Sahiwal bulls was optimum, freezable, and can be utilized for up to 11 years (Average 9.5 years)., Competing Interests: Declarations. Animal ethics and Consent to participate declarations: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Bull fertility is a multi-factorial trait and is affected by many factors, such as nutrition, genetics, and epigenetics. Superior quality male germplasm with high genetic merit helps to improve the livestock production trait. To achieve the target of livestock production, the availability of superior male germplasm is a great concern. In developing countries, there is a gap between the highly fertile frozen semen doses produced and the highly fertile frozen semen doses required. Improving the quality of existing low-fertile semen from high genetic merit bulls seems to play a promising role in filling this gap. Seminal exosomes are extracellular vesicles secreted by the epithelial cells of the testis, epididymis, and accessory sex glands such as the prostate gland. They contain a cargo of bioactive molecules such as proteins, nucleic acids and various metabolites. These molecules are transferred to the spermatozoa during its maturation and help in sperm capacitation, maturation, acrosome reaction, and fertilisation. Studies reveal that seminal exosomes help to improve the sperm functionality of low-quality sperm. Identification of the molecular profile of exosomes of bulls with proven fertility and their addition in an extender containing low-fertile semen may help to ameliorate the sperm quality of low-fertile semen, which may eventually aid in generating quantities of highly fertile ejaculates., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

In India, where dairy production leads globally, infrared thermography (IRT) and short milking tube thermography specifically are vital for managing mastitis. Therefore, the present study focuses on thermal imaging of the udder and short milking tube (SMT) of the milking machine during the peak milking process of Sahiwal cows and Murrah buffaloes during winter, summer, rainy and autumn seasons to identify sub-clinical (SCM) and clinical mastitis (CM) cases using the Darvi DTL007 camera. The udder health was assessed using the California Mastitis Test, Somatic Cell Count (SCC) and IRT throughout the year. Log 10 SCC and thermogram analysis revealed a difference (p < 0.01) between healthy, SCM, and CM cases during different seasons in both breeds. Further results showed an increase (p < 0.01) in SMT thermograms of SCM and CM cases compared to healthy quarters in Sahiwal cows during winter, summer, rainy, and autumn were 4.26 and 7.51, 2.37 and 4.47, 2.20 and 3.64, 2.90 and 4.94 ºC, respectively and for Murrah buffaloes were 3.56 and 5.55, 2.70 and 3.81, 1.72 and 3.10, 3.14 and 4.42ºC, respectively. The highest degree of increase in milking udder skin surface temperature and SMT of SCM and CM cases compared to healthy quarters was observed during the winter and the least during the rainy season. Thus, regardless of the seasons examined in this study, SMT thermograms effectively assessed SCM and CM., (© 2024. The Author(s) under exclusive licence to International Society of Biometeorology.)

Mastitis is a global concern in the dairy sector, demanding innovative solutions for effective management for quality lifetime milk production. In this study, infrared thermography (IRT) as a non-invasive technology was integrated into routine farm activities for continuous health monitoring of animals. For 30 days, we systematically monitored the udder health status in 40 Sahiwal cows (160 quarters), employing IRT along with the California Mastitis Test (CMT). We also assessed somatic cell count (SCC), microbial identification, and milk quality parameters of representative samples. The thermal imaging data was analyzed, considering both backward propagation from the 0 th day to the -10th day and forward propagation from the 0 th day to the +10th day. Our findings revealed that on the 0 th day, the mean temperatures of the udder surface skin temperature (USST) and teat skin surface temperature (TSST) exhibited differences (p < 0.05) between the quarters affected by sub-clinical mastitis (SCM) and clinical mastitis (CM) in comparison to the healthy quarters, with the highest degree of difference observed. The observed temperature differences between CM and SCM quarters compared to healthy ranged from 1.8 to 3.62 °C and 0.98 to 3.23 °C for USST, and from 1.68 to 3.16 °C and 0.56 to 2.32 °C for TSST, respectively. Furthermore, our observations indicated that both udder and teat quarters responded differently to mastitis. A temperature rise of 1.37 °C in SCM quarters and 1.75 °C in CM quarters was observed between the -10th and -8th day relative to day 0, with the increase being more pronounced in the morning hours. Also, a notable temperature surge occurred during the -2nd and -1st days relative to the 0 th day. The log 10 SCC values and milk quality parameters significantly differed (p < 0.05) between mastitis-affected and healthy samples. In addition, Staphylococcus spp. was identified as the predominant mastitis-causing pathogen in the bacteriological identification conducted in this study. Therefore, IRT efficiently assesses the initiation point of udder infection in Sahiwal cows, aiding in effective udder health management., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T. K. Mohanty reports financial support was provided by Indian Council of Agricultural Research, New Delhi, India. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sahiwal cattle, known for their high milk yield, are propagated through artificial insemination (AI) using male germplasm, largely contingent on semen quality. Spermatozoa, produced in the testes, carry genetic information and molecular signals essential for successful fertilization. Seminal plasma, in addition to sperm, contains nano-sized lipid-bound extracellular vesicles (SP-EVs) that carry key biomolecules, including fertility-related miRNAs, which are essential for bull fertility. The current study focused on miRNA profiling of SP-EVs from high-fertile (HF) and low-fertile (LF) Sahiwal bulls. SP-EVs were isolated using size exclusion chromatography (SEC) and characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Western blotting detected the EV-specific protein markers TSG101 and CD63. The DLS analysis showed SP-EV sizes of 170-180 nm in HF and 130-140 nm in LF samples. The NTA revealed particle concentrations of 5.76 × 10 10 to 5.86 × 10 11 particles/mL in HF and 5.31 × 10 10 to 2.70 × 10 11 particles/mL in LF groups, with no significant differences in size and concentration between HF and LF. High-throughput miRNA sequencing identified 310 miRNAs in SP-EVs from both groups, with 61 upregulated and 119 downregulated in HF bull. Further analysis identified 41 miRNAs with significant fold changes and p-values, including bta-miR-1246, bta-miR-195, bta-miR-339b, and bta-miR-199b, which were analyzed for target gene prediction. Gene Ontology (GO) and KEGG pathway analyses indicated that these miRNAs target genes involved in transcription regulation, ubiquitin-dependent endoplasmic reticulum-associated degradation (ERAD) pathways, and signalling pathways. Functional exploration revealed that these genes play roles in spermatogenesis, motility, acrosome reactions, and inflammatory responses. qPCR analysis showed that bta-miR-195 had 80% higher expression in HF spermatozoa compared to LF, suggesting its association with fertility status ( p < 0.05). In conclusion, this study elucidates the miRNA cargoes in SP-EVs as indicators of Sahiwal bull fertility, highlighting bta-miR-195 as a potential fertility factor among the various miRNAs identified., 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 © 2024 Chauhan, Kashyap, Chera, Pal, Patel, Karanwal, Badrhan, Josan, Solanki, Bhakat, Datta and Kumar.)

Backgroud: Before fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm's ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction., Results: In this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P < 0.05) higher DLD abundances. Herein, we optimised the MICA concentration to inhibit DLD function, spermatozoa were treated with MICA in time (0, 1, 2, 3, 4, and 5 h) and concentrations (1, 2.5, 5, and 10 mmol/L) dependent manner. Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration, which was used for further experimentation in HF and LF. Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly (P < 0.05) higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa. The kinematic parameters of the spermatozoa such as percent total motility, velocity parameters (VCL, VSL, and VAP) and other parameters (BCF, STR, and LIN) were also decreased in MICA treated spermatozoa in comparison to the control (capacitated) spermatozoa., Conclusions: The present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation, which subsequently reduces their capacity to fertilize., (© 2024. The Author(s).)

Mastitis is a global production disease that needs an intelligent solution to tackle effectively. Infrared Thermography (IRT) is a non-invasive technology that could be incorporated into routine day-to-day farm activities to monitor the health status of the animals. In this study, the udder health status was routinely monitored for 30 days among 41 Murrah buffaloes via IRT and the California Mastitis Test (CMT). Further, somatic cell count (SCC), microbial identification, and milk quality parameters were also estimated for representative samples. The thermal imaging data obtained was tabulated and back propagated from the 0th day to the -10th day and front propagated from the 0th day to +10th day for all the udder quarters. Results revealed that on the 0th day, the mean of udder skin surface temperature (USST) and teat skin surface temperature (TSST) showed a difference (p < 0.05) in the sub-clinical mastitis (SCM) and clinical mastitis (CM) affected quarters to the healthy quarters, and their degree of difference was the highest. The indication of infection was signaled during the -9th to -5th day to th e 0th day in SCM and CM cases. There was a steep increment in the temperature from -2nd and -1st day to the 0th day of infection. Sometimes, some quarters show an increment in temperature due to mastitis during morning hours but recover by evening milking due to the animal's innate immune system. Thus, the initiation period in which the udder gets assaulted is crucial in the early assessment of SCM by monitoring temperature change using IRT., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sub-clinical mastitis exhibits a higher prevalence in dairy goats than clinical mastitis, necessitating the adoption of non-invasive diagnostic techniques such as infrared thermography (IRT) to detect this economically significant production disease in the dairy sector. Accordingly, this study aims to employ IR imaging of the udder and teat quarters of lactating crossbred goats (Alpine × Beetal and Sanen × Beetal) across various seasons, utilising IRT, to discern cases of sub-clinical (SCM) and clinical mastitis (CM). Over a year, 100-110 lactating crossbred dairy goats underwent consistent IRT screenings, followed by a comprehensive evaluation of udder health status using the California mastitis test and somatic cell count (SCC). The receiver operating characteristic (ROC) analysis was conducted to establish the cut-off values for different thermographic parameters in this study. The results revealed that the SCC increased significantly (p < 0.01) in healthy, SCM, and CM milk samples across the seasons. The analysis of ROC revealed a comparatively higher sensitivity, specificity, and accuracy for udder thermograms during SCM than CM and vice versa for teat thermograms. IRT analysis reflected a difference (p < 0.01) in the udder and teat thermograms among quarters of healthy, SCM, and CM in summer, winter, autumn, and rainy seasons. A significant increase (p < 0.01) in udder thermograms was observed for quarters affected with SCM and CM relative to healthy, with an increase of 1.89 and 2.94 °C in winter, 0.85 and 1.63 °C in summer, 0.73 and 1.41 °C in rainy, and 1.33 and 2.38 °C in autumn, respectively. Similarly, for teat thermograms it was 1.79 and 2.81 °C in winter, 0.76 and 1.41 °C in summer, 0.70 and 1.37 °C in rainy, and 1.09 and 1.93 °C in autumn, respectively. Therefore, regardless of the seasons examined in this study, IRT proves to be an effective and supportive tool for early mastitis detection in lactating crossbred dairy goats., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Buffalo bulls are backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of dairy farms. Seminal plasma harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility-associated proteins which contribute to fertilizing potential of spermatozoa. In this study, we explored size, concentration, and complete proteome profiles of SP EVs from two distinct fertility groups to uncover proteins influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified EVs were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging from 146.5 to 258.7 nm in high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200 nm, and concentrations varying from 2.84 to 6.82 × 10 11 and 3.57 to 7.74 × 10 11 particles per ml in HF and LF bulls, respectively. No significant difference was observed in size and concentration of seminal EVs between two groups. We identified a total of 1,862 and 1,807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1,754 proteins were common in both groups and about 87 proteins were highly abundant in HF group while 1,292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. Additionally, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins, protein disulfide-isomerase A4 and gelsolin, are associated with sperm-oocyte fusion and acrosome reaction, respectively, and their immunolocalization on spermatozoa may indicate that these proteins are transferred through EVs. Our evidences support that proteins in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, our investigation indicates that SPEVs possess crucial protein repertoires that are essential for enhancing sperm fertilizing capacity., 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 © 2024 Badrhan, Karanwal, Pal, Chera, Chauhan, Patel, Bhakat, Datta and Kumar.)

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.)

Mastitis is a global threat that challenges dairy farmers' economies worldwide. Sub-clinical mastitis (SCM) beholds the lion's share in it, as its visible clinical signs are not evident and are challenging to diagnose. The treatment of intramammary infection (IMI) demands antimicrobial therapy and subsequent milk withdrawal for a week or two. This context requires a non-invasive diagnostic tool like infrared thermography (IRT) to identify mastitis. It can form the basis of precision dairy farming. Therefore, the present study focuses on thermal imaging of the udder and teat quarters of Murrah buffaloes during different seasons to identify SCM and clinical mastitis (CM) cases using the Darvi DTL007 camera. A total of 30-45 lactating Murrah buffalo cows were screened out using IRT regularly throughout the year 2021-22. The IMI was further screened using the California mastitis test. The thermogram analysis revealed a significant difference (p < 0.01) in the mean values of the udder and teat skin surface temperature of Murrah buffaloes between healthy, SCM, and CM during different seasons. The mean values of udder skin surface temperature (USST) during different seasons ranged between 30.28 and 36.81 °C, 32.54 to 38.61 °C, and 34.32 to 40.02 °C among healthy, SCM, and CM-affected quarters. Correspondingly, the mean values of teat skin surface temperature (TSST) were 30.52 to 35.96 °C, 32.92 to 37.55 °C, and 34.51 to 39.05 °C, respectively. Further results revealed an increase (p < 0.01) in the mean values of USST during winter, summer, rainy, and autumn as 2.26, 4.04; 2.19, 3.35; 1.80, 3.21; and 1.45, 2.64 °C and TSST as 2.40, 3.99; 2.28, 3.26; 1.59, 3.09; and 1.68, 2.92 °C of SCM, CM-affected quarters to healthy quarters, respectively. The highest incidence of SCM was observed during autumn and CM during winter. Henceforth, irrespective of the seasons studied in the present study, IRT is an efficient, supportive tool for the early identification of SCM., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Extensive dilution of cattle semen with tris-based extender compromises certain sperm kinetic and functional traits following cryopreservation., Objective: To study sperm functions of buffalo bulls under high dilution rates., Materials and Methods: Twenty-four ejaculates were harvested twice a week from four buffalo bulls, and diluted to sperm concentrations of 80, 60, 40 and 20 million/mL. Diluted samples were filled in straws, equilibrated at refrigeration temperature for 4 h, and frozen in liquid nitrogen. Frozen sperm samples were thawed for evaluation of kinetic and functional attributes., Results: Compared to 20 million/mL (million/mL) sperm sample, the total motility, progressive motility and rapid motility were reduced (P < 0.05) in 5 million/mL sample. The proportion of live sperm were significantly (P < 0.05) higher in 10, 15 and 20 million/mL samples than in 5 million/mL sample. The percentage of moribund sperm, dead sperm, and sperm with lipid per oxidation increased significantly (P < 0.05) in 5 million/mL sample., Conclusion: The reduction of sperm concentrations to < 10 million/mL affects post-thaw Buffalo sperm kinetic and functional attributes.. https://doi.org/10.54680/fr24110110712.

"India is the world's leading producer of milk" and demands a non-invasive diagnostic tool like infrared thermography (IRT) to identify the costliest production disease, mastitis. It can form the basis of precision dairy farming. Therefore, the present study focuses on thermal imaging of the udder and teat quarters of Sahiwal cows during different seasons to identify subclinical (SCM) and clinical mastitis (CM) cases using the Darvi DTL007 camera. A total of 24-69 lactating Sahiwal cows were screened out using IRT regularly throughout the year. The intramammary infection status was further assessed using the CMT. The receiver operating characteristic analysis was carried out to develop the current study's cut-off for various thermographic parameters. The incidence for SCM and CM ranged from 26.47 to 38.75% and 17.83-22.79%, respectively during different seasons in Sahiwal udder quarters. The thermogram analysis revealed a significant difference (p < 0.01) in the mean values of the udder and teat surface temperature of Sahiwal cows between healthy, SCM, and CM during different seasons. The mean values of udder skin surface temperature (USST) during different seasons ranged between 29.07 and 36.91 °C, 31.51 to 37.88 °C and 32.42 to 38.79 °C among healthy, SCM, and CM-affected quarters, and correspondingly, the mean values of teat skin surface temperature (TSST) were 28.28 to 36.77 °C, 30.68 to 37.88 °C and 31.70 to 38.73 °C, respectively. Further results revealed an increase (p < 0.01) in the mean values of USST during winter, summer, rainy, and autumn as 2.44, 3.35; 0.97, 1.88; 1.06, 1.83; 1.29, 2.39 °C and TSST as 2.4, 3.42; 1.11, 1.96; 1.21, 2.19, 1.3, 2.4 °C of SCM, CM-affected quarters to healthy quarters, respectively, in Sahiwal cows. Thermograms showed a strong positive correlation with the CMT scores of SCM, CM cases, and healthy samples. Henceforth, irrespective of the seasons studied in the present work, IRT is an efficient, supportive tool for the early identification of subclinical mastitis., Competing Interests: Declaration of Competing Interest “None.”, (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In the present study, thermal images of the short milking tube of the milking machine representing four udder quarters independently attached to a milking animal, along with pre-milking and post-milking udder and teat thermograms, were taken using a hand-held digital infrared thermal camera (DarviDTL007) during morning milking of lactating Murrah buffaloes (n = 132) to assess the mastitis status. California mastitis test (CMT) and somatic cell count (SCC) of milk samples were carried out to screen the udder quarters as healthy, subclinical (SCM), and clinical mastitis (CM). The thermograms revealed an increase (p < 0.05) of 2.19 and 3.72ºC in the mean values of short milking tube (SMT) surface temperature among SCM and CM quarters compared to healthy quarters, respectively. The mean values of udder skin surface temperature (USST) for pre-milking, milking, and post-milking of SCM and CM compared to healthy quarters showed an increase (p < 0.05) of 2.17, 1.96, and 1.61ºC and 3.11, 2.88, and 2.73ºC, respectively. Similarly, compared to healthy quarters, the mean values of teat skin surface temperature (TSST) for pre-milking and post-milking of SCM and CM showed an increase (p < 0.05) of 2.12 and 1.66ºC and 3.07 and 2.45ºC, respectively. Also, CMT and SCC results showed a strong positive correlation (r = 0.68-0.91, p < 0.01) with all the thermographic parameters. Thus, thermograms of SMT alone can be used as an efficient detection tool in assessing SCM among Murrah buffaloes., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)