Molecular insights into antimicrobial resistant Staphylococcus aureus strains: A potential zoonosis of goat origin.

Antimicrobial-resistant (AMR) Staphylococcus aureus (S. aureus) strains have attained global attention due to their life-threatening zoonotic nature. Being a member of ESKAPE group, S. aureus has an ability to escape the biocidal action of antimicrobial drugs. The current study investigated the prevalence and molecular characterization of methicillin-resistant S. aureus (MRSA), ß-lactam-resistant S. aureus (BRSA), aminoglycoside-resistant S. aureus (ARSA), tetracycline-resistant S. aureus (TRSA), and fluoroquinolones-resistant S. aureus (FRSA) associated with goat subclinical mastitis (SCM). Furthermore, the antimicrobial resistance and susceptibility profile of various antibiotics and non-antibiotics (NSAIDs, nisin, N-acetylcysteine, vitamin-C) along with their possible role in modulating the antibiotic resistance of MDR isolates was also investigated. A total of 768 goat milk samples were subjected to California mastitis test for SCM followed by bacteriological and molecular characterization of S. aureus. Moreover, in-vitro susceptibility of resistant antibiotics, non-antibiotics, and their combination against MDR S. aureus were conducted through well diffusion and broth microdilution assays. The results depicted that 55.47 % and 26.82 % of milk samples were positive for SCM and S. aureus, respectively. The molecular assay confirmed 35.92 % of isolates as MRSA, 45.63 % as BRSA, 50.49 % as ARSA, and 32.52 % but no isolate was confirmed as FRSA on molecular basis. The multidrug resistance was observed in 62.13 % and 47.09 % isolates, respectively. Molecular characterized MDR S. aureus revealed high homology of study isolates with the isolates of neighboring countries like India, Korea, Iran, and China. Antimicrobial susceptibility trials on well diffusion assay showed higher efficacy of different non-antibiotics with resistant antibiotics as penicillin with ketoprofen and gentamicin with flunixin meglumine while oxytetracycline with N-acetylcystiene. The synergy testing by checkerboard assay revealed synergistic activity of penicillin with ketoprofen, gentamicin with flunixin meglumine, and oxytetracycline with N-acetylcysteine. The current study highlighted the emergence and spread of AMR S. aureus strains from goat SCM and provided insights into possible drug repurposing of various non-antibiotics to modulate the multidrug resistance of S. aureus which will be helpful in devising the therapeutic options and control measures for this pathogen.

On-farm epidemiology, virulence profiling, and molecular characterization of methicillin-resistant Staphylococcus aureus at goat farms.

Antimicrobial resistance (AMR) becomes a challenging issue that limits the therapeutic options for both veterinary and public health professionals. The current study aimed to investigate the on-farm epidemiology, antibiotics resisting profiling, virulence analysis, and molecular detection of methicillin-resistant Staphylococcus aureus (MRSA) at the caprine-human interface. A total of 768 goat milk samples and 94 skin swabs from farm personnel were collected from 30 goat flocks and processed for isolation of S. aureus. The study isolates were confirmed as MRSA based on the oxacillin and cefoxitin disc diffusion test and the presence of mecA gene. MRSA isolates of goats and human origin were characterized and further evaluated for the presence of virulence genes responsible for intramammary infections and public health hazards. The results revealed 26.82 % and 27.79 % goat milk samples and human samples positive for S. aureus, respectively. A higher MRSA prevalence of 35.92 % and 10.71 % was found in goat and human isolates respectively. The phylogenetic analysis revealed a lesser extent of homology in mecA gene of S. aureus isolates at the caprine-human interface. Moreover, this study revealed strong evolutionary connection between the study isolates and MRSA isolates of Pakistani cattle and buffalo while the in-silico protein analysis showed that all sequences have the same protein motifs resembling penicillin binding protein 2a. The risk factors analysis revealed that teat length, drainage system, hygienic measures during milking, use of teat dip, teat injury, and veterinary services were significantly associated with subclinical mastitis in goats. A total of 43.24 % of local MRSA isolates showed multi-drug resistance (MDR). The isolates showed higher resistance to oxytetracycline followed by gentamicin and vancomycin while moxifloxacin, and linezolid were among the susceptible antibiotics. Local MRSA isolates carried virulence markers (nuc and coag genes) and biofilm-associated icaA (43.24 %) and icaD (29.73 %) genes which are responsible for the intramammary infection. The local isolates also carried the virulence genes of public health concern including the enterotoxin C (sec) gene (24.3 %), enterotoxins B (seb) gene (5.41 %), and enterotoxin D (sed) gene (2.7 %). Enterotoxins A (sea) and E (see) genes were not detected in any isolate. The study concluded that MRSA is an emerging and prevailing pathogen in dairy goats with a high potential to transmit to associated human beings. The presence of a variety of virulence factors as well as the associated antibiotic resistance makes MRSA a potential threat at animal-human interface and thus demands further research.

Effects of extender type and storage time on sperm quality parameters of Kail ram semen stored at 5 °C.

The study was designed to evaluate the effect of different extenders and storage times on sperm quality parameters of extended Kail ram semen. Semen was collected from five adult Kail rams using an artificial vagina. Semen samples with >70% total sperm motility were pooled, diluted with Tris (TR), sodium citrate (SC), and skim milk (SM)-based extenders, and stored at 5 °C. Sperm motility and kinematics, viability, and plasma and acrosomal membrane integrity were assessed every 24 hrs for 120 hrs. Sperm longevity was estimated by placing semen in a water bath at 37°C to assess sperm kinematics. Storage time as the main effect had a negative (P < 0.05) impact on sperm quality parameters. The percentages of total motile (TM), progressive motile (PM), rapid, and medium progressive (MP) motile sperm were similar at 24 hrs of storage for the three extenders. However, semen extended in TR had a higher (P < 0.05) percentage of TM, PM, rapid, and MP sperm compared to SM and SC extenders at 48 hrs of storage. The sperm kinematics (VCL, VSL, VAP, and ALH) of progressive sperm were similar for all three extenders up to 72 hrs of storage. In addition, semen extended in TR had a higher (P < 0.05) percentage of sperm with intact plasma and acrosomal membranes at a storage time of 48 hrs. At 37 °C, the percentage of TM and PM sperm was higher (P < 0.05) in the TR extender than in the SM and SC extenders at 60 minutes and beyond. In conclusion, the Kail ram sperm extended in TR and stored at 5 °C maintained better quality after 48 hrs storage than sperm extended in SM and SC extenders. At 37 °C, sperm extended in TR also retained better quality parameters at 60 min and afterward than sperm extended in SM and SC.

Effects of Ferula asafetida, closantel, albendazole, oxfendazole, and ivermectin against Haemonchus contortus in goats and sheep.

BACKGROUND: Haemonchosis is a fatal disease of small ruminants caused by the parasite Haemonchus contortus (H. contortus). The most common drugs used in the treatment of H. contortus include albendazole, oxfendazole, and ivermectin. However, as previously reported in the treatment of haemonchosis, these medicines have acquired drug resistance problems over time. Interestingly, natural plant compounds have demonstrated promising effects in the treatment of H. contortus. Therefore, the current study evaluated the effects of plant extract, Ferula asafetida, against common drugs such as albendazole, oxfendazole, ivermectin, and closantel for the treatment of haemonchosis in small ruminants. METHODOLOGY: The current study was conducted on different small ruminant farms in Kasur District, Punjab, Pakistan. The positive animals (n = 720) after coprological examination were selected in this study and divided into two major groups (n = 360 goats and n = 360 sheep). Further, animals were divided into five treatment groups (A-E) and one control group with no treatment (F). Albendazole, oxfendazole, ivermectin, closantel, and Ferula asafetida were administered orally to groups A-E, respectively. The eggs per gram feces (EPG) were determined through the McMaster technique on days 0, 7th, and 14th of treatment. RESULTS: The results showed a significantly higher efficacy of closantel and Ferula asafetida against H. contortus in both goats (100% and 70%; p < 0.05) and sheep (99% and 87%; p < 0.05), respectively. No correlation was observed between EPG reduction with age and gender in both goats and sheep. CONCLUSION: Allopathic drug closantel and herbal drug, Ferula asafetida, have been proved an effective dewormer against H. contortus in small ruminants.

Molecular epidemiology of Coxiella Brunetii in small ruminants in Punjab, Pakistan: a novel reporting analytical cross sectional study.

Coxiella burnetii, an intracellular zoonotic bacterium, causes query (Q) fever in ruminants. Its role has never been elucidated in small ruminants from Pakistan. The current study is designed to (a) determine the prevalence of coxiellosis in small ruminants, (b) evaluate the association of various potential risk factors and biomarkers in the occurrence of Coxiella burnetii, (c) and determine phylogeny and genetic variability of its various isolates identified during the study. For this purpose, 320 blood samples from sheep (n = 160) and goats (n = 160) were collected from 9 Union Councils of district Kasur, Punjab, and processed for DNA extraction. C. burnetii was confirmed by amplification of IS1111 transposase gene with an amplicon size of 294 bp. The results showed that the overall positive percentage of C. burnetii is 36.87% (sheep: 46.9% and goats: 30%). The phylogenetic tree was also constructed which described the possible origin of this pathogen from environment. Besides, after translation into amino acid, the resultant alignment showed several unique changes at position numbers 18 and 27 in the isolates from goats and at 27 and 66 from those of sheep. These mutations can have major impact on the infectious characteristics of this pathogen. Furthermore, different potential risk factors and clinical biomarkers like age, tick infestation, abortion, mastitis, and infertility were also studied and found that these are significantly (p < 0.05) associated with the occurrence of coxiellosis. It is concluded from the study that C. burnetii is endemic in small ruminants in Punjab, Pakistan. The outcomes of this study are alarming for scientific community as well as for policy makers because coxiellosis is an emerging threat to both humans and animals in this region due to its interspecies transmission ability.

Prevalence of Toxoplasma gondii oocysts through Copro-PCR in cats at Pet Center (UVAS), Lahore, Pakistan.

Toxoplasmosis is a major zoonotic disease of warm-blooded animals caused by Toxoplasma gondii. Cats are the only definitive host and they excrete environmentally resistant T. gondii oocysts in their faeces. Coproscopy was used to detect oocysts of enteric coccidians and then Copro-PCR was employed to test specifically for T. gondii in 470 cat samples. The prevalence of T. gondii oocysts was 2.3% (11/470) based on PCR. We observed 15 (3.2%) of 470 samples positive for coccidian oocysts by microscopy. The presence of Copro-DNA of T. gondii was found significantly higher (p<0.05) in males than females. We tested 11 samples of T. gondii oocysts in which 9 samples were from coccidian oocysts positive samples and 2 samples from negative faecal samples. Our results showed that PCR is the reliable method for the detection of faecal oocysts of T. gondii in cats as compared to microscopy. As per our knowledge, ours is first study for Copro-PCR prevalence of cats' T. gondii oocysts excretion in Pakistan.

Epidemiological study of common diseases and their risk factors in camels in South Punjab, Pakistan.

Bacteriological study of mastitis along with common blood protozoan diseases were studied in dromedary camels in Cholistan, Dera Ismail Khan and Rahim Yar Khan districts in South Punjab, Pakistan. For this purpose 300 camels were sampled randomly at different common grazing and watering point. For study of blood parasites clinically suspected and apparently healthy camels, 150 each, were sampled. An overall prevalence of 15%and 5% was recorded for trypanosomiasis and Anaplasmosis respectively. Trypanosoma evansi was identified with 280 bp product on polymerase chain reaction test. There was significant (P < 0.05) decline in the values of total erythrocyte counts, hemoglobin concentration, packed cell volume, serum total proteins and albumin while erythrocyte sedimentation rate was increased in infected camels as compared to healthy ones. Aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and alkaline phosphatase were also significantly increased in blood protozoan the infected animals. Milk samples for bacteriology were collected from healthy lactating camels (n = 100). Information about different risk factors were gathered on designed performa. Subclinical mastitis on surf field test was recorded in 42% camels while 2% cases of clinical mastitis were recorded. Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Bacillus cereus and. Corynebacterium kutscheri were isolated with characteristic beta and alpha hemolysis patterns. Chi-square analysis showed significant difference as p < 0.05 among various species of bacteria (χ2 = 21.649, P-Value = 0.0001, df = 3). Antibiogram showed Gentamicin, Norfloxacin, Oxytetracycline as most effective therapy for mastitis in camel.

Use of physiological biomarkers in diagnosis along with field trials of different trypanisidal drugs in camels of Cholistan desert.

The point prevalence of trypanosomiasis with different physiological biomarkers along with evaluation of the most responsive trypanosidal drug against trypanosomiasis under field conditions was studied. For this purpose a total of 300 free range camels were selected at different grazing and watering point in Cholistan desert. The study population of camels included 150 clinically suspected camels for trypanosomiasis and 150 healthy camels with normal temperature, pulse and respiration. For therapeutic trials 36 positively diagnosed animals were randomly divided into three experimental groups for therapeutic trials. Group A was treated with Imidocarb dipropionate (ID) @ 1.2 mg kg-1 body weight; Group B was treated with Diaminazine aceturate (DA) @ 3.5 mg kg-1 body weight and Group C was treated with Isometamidium chloride hypochloride (IC) @ 0.75 mg kg-1 body weight of camels. Data on risk factors of age,sex, ectoparasites, housing was also collected. Results revealed an overall 15% point prevalence of trypanosomiasis. There was significant (P < 0.05) decline in the values of physiological biomarkers of total erythrocyte counts, hemoglobin concentration, packed cell volume, serum total proteins and albumin while erythrocyte sedimentation rate was increased in infected camels as compared to healthy ones. Different hepatic enzymes including aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and alkaline phosphatase were also significantly increased in the infected animals. Therapeutic trials indicated that Isometamidium chloride hypochloride (IC) was more effective than Imidocarb dipropionate (ID) and Diaminazine aceturate (DA). It is concluded that haemato-biochemical parameters were important physiological biomarkers and IC was the most responsive therapeutic agent against trypanosomiasis in camels in field conditions. The risk factors analysis showed older camels (>5 years) showed highest infection while infection was found to be lowest in less than 1 year age group.

Association between bacterial strain type and host biomarkers in Clostridium perfringens infected goats.

The study project was designed to determine the effects of Clostridium perfringens type D infection on hematological and biochemical parameters in goats. Purposive blood samples were collected from 6 healthy and 12 diseased goats positive for C. perfringens infection. Neither the animals nor their mother were vaccinated against Clostridium perfringens from whom samples were obtained. Study was carried out in two different topographic areas; hilly (district Swat) and plain (district Mardan) of Khyber Pakhtunkhwa, Pakistan but nonsignificant (P > 0.05) statistical difference was recorded between the prevalence of Clostridium perfringens infected goats. Mean erythrocytes count (RBC) and hemoglobin level decreased significantly (P < 0.05) while the white blood cells (WBC) increased significantly (P < 0.05) in diseased animals compared to the healthy animals. However non-significant differences (P > 0.05) were observed in packet cell volume (PCV) and platelets count in healthy and diseased animals. According to biochemical analysis, a significant increase (P < 0.05) in liver enzymes, total bilirubin, serum creatinine, blood urea and glucose was recorded in diseased goats. . The results demonstrated that fluctuation in most of the mean hematological values remained within the normal range however the mean liver enzymes, total bilirubin, serum creatinine, blood urea and glucose levels gone beyond the normal levels which demonstrated severe damages to liver and kidneys.

A review on epidemiology, global prevalence and economical losses of fasciolosis in ruminants.

Fasciolosis is an important plant borne trematode zoonosis in ruminants caused by the Fasciola hepatica and Fasciola gigentica, It is classified as a neglected tropical disease and found in more than 50 countries especially where sheep and cattle are reared. Fasciolosis is a serious animal health problems in many rural and urban areas of world, causing significant financial losses due to decrease in production and viscera condemnation in animals. Accurate diagnosis of fasciolosis is always remained a challenging task for the field practitioners. There is no comprehensive summary on the occurrence and distribution of the infection at international level. Therefore, we intended to provide a complete overview on the prevalence and epidemiology of fasciolosis in farm animals from a global prospective. It includes to map the global distribution of fasciolosis in different areas of the world to identify the endemic regions which may be a source of potential disease outbreak. The financial liability related to fasciolosis on the livestock production has also been addressed. For this purpose, the published data during 2000-2015 (15 years) on fasciolosis was reviewed and collected by electronic literature search of four databases including Google, PubMed, Science Direct, and Web of Science. Data presented are contemplated to enhance our current understanding of the parasite's geographical distribution, host range, and economic losses. Information provided would be useful for the application of more effective control strategies against fasciolosis in different geo-economics regions of the world.

Spatiotemporal analysis and clinico-epidemiological study for seroprevalence of canine leptospirosis.

Leptospirosis is a worldwide re-emerging zoonotic disease. The study was conducted to estimate the Seroprevalence of canine leptospirosis in a total of 450 dogs, from a total of 97 puppies and 353 adult dogs selected for examination Sampling, started from January to December 2023 in District Kasur in the province Punjab of the country Pakistan. Leptospira IgG ELISA kit manufactured by DRG Instruments GmbH, Germany was used for the screening of canine Leptospira antibodies. Out of 450 tested dogs, 183 dogs (40.67%) were tested positive for Leptospira antibody for the screening of Leptospira antibodies. The estimated Seroprevalence of leptospirosis in various age groups of dogs, were 23.7% (23/97) and 45.3% (160/353), in puppies and adults, respectively (P < 0.05). It was found that out of the sampled dogs, a total of 35/127 (27.6%), 29/100 (29%), 73/130 (56.2%), and 46/93 (49.5%) dogs were tested seropositive for Leptospira antibodies in winter, spring, summer and fall, respectively (P < 0.05).

Impact of Farm Management Practices on Tick Infestation in Punjab's Livestock: A Comprehensive Epidemiological Study.

Tick infestation poses an important challenge to livestock in Pakistan. Farm management practices and environmental variables can influence tick infestation prevalence in animals. To this end, a cross-sectional survey of 96 farms in four different geographical districts (24 farms from each district) was conducted in Punjab, Pakistan, between October 2021 and January 2022. An epidemiological questionnaire was designed focusing on farm management practices and their impact on tick infestations at these farms. Data were collected via in-person interviews. Regional and farm-specific variables' associations were evaluated using Pearson's chi-square test and Fischer's exact test, respectively. A multivariable logistic regression model was used to identify significant risk factors. This study identified that using soft bedding materials, e.g., wheat straw, leaf litter or plain soil posed a significant risk of tick infestation. Additionally, the absence of quarantine measures, open sheds and inadequate drainage were found as contributing factors in univariable analysis. Higher tick prevalence in the hotter seasons highlighted the influence of Punjab's extreme weather on tick infestation. Despite regular veterinary visits and the use of acaricidal drugs, the prevalence of tick infestation at these farms suggested potential drug resistance in the ticks. The study recommended establishing quarantine practices, improving farms' drainage systems and bedding and using a combination of chemical and traditional remedies to tackle drug resistance in ticks. Education and awareness programs on tick-borne diseases and control measures are advocated to reduce the tick infestation burden on animals. Further research on longitudinal studies to better understand tick population dynamics and develop effective acaricides is encouraged. This called for collaborative control efforts among farmers, veterinarians and research institutions.

Humoral and Cell-Mediated Immune Response Validation in Calves after a Live Attenuated Vaccine of Babesia bigemina.

The current vaccines to control bovine Babesia bigemina (B. bigemina) infection are not fully protective and vaccination failures incur heavy losses to the cattle industry around the world. Using modified micro-aerophilous stationary phase, we developed a culture-derived attenuated live vaccine against B. bigemina and tested a single subcutaneous inoculation of 2 × 108 infected erythrocytes in calves. The protection was measured after a lethal intravenous challenge with 5 × 108 virulent calf-derived B. bigemina. Our results demonstrated that a single shot of attenuated vaccine was capable of inducing robust humoral and cell-mediated immune responses in calves. We found a significant increase in the IgG antibody titers post-challenge and a strong proliferation of both CD4+ and CD8+ T cells contributing towards the protection. Our vaccine provided complete protection and parasitic clearance, which was followed for more than 100 days post-challenge. This immunity against babesiosis was directly linked to strong humoral responses; however, the parasitic clearance was attributed to significant T cells effector responses in vaccinated calves as compared to the infected control calves. We anticipate that these results will be helpful in the development of more efficient culture-derived vaccines against Babesia infections, thus reducing significant global economic losses to farmers and the cattle industry.

Tick-Borne Pathogens Shape the Native Microbiome Within Tick Vectors.

Ticks are blood-feeding arthropods and transmit a variety of medically important viral, bacterial, protozoan pathogens to animals and humans. Ticks also harbor a diverse community of microbes linked to their biological processes, such as hematophagy, and hence affect vector competence. The interactions between bacterial and/or protozoan pathogens and the tick microbiome is a black-box, and therefore we tested the hypothesis that the presence of a protozoan or bacterial pathogen will alter the microbial composition within a tick. Hence, this study was designed to define the microbial composition of two tick species, Hyalomma (H.) anatolicum and Rhipicephalus (R.) microplus. We used a combination of PCR based pathogen (Anaplasma marginale and Theileria species) and symbiont (Wolbachia species) identification followed by metagenomic sequencing and comparison of the microbial communities in PCR positive and negative ticks. A total of 1786 operational taxonomic units was identified representing 25 phyla, 50 classes, and 342 genera. The phylum Proteobacteria, Firmicutes, Actinobacteriota, and Bacteroidota were the most represented bacteria group. Alpha and beta diversity were not significantly affected in the presence or absence of Theileria sp. and A. marginale as see with H. anatolicum ticks. Interestingly, bacterial communities were significantly reduced in Theileria sp. infected R. microplus ticks, while also exhibiting a significant reduction in microbial richness and evenness. Putting these observations together, we referred to the effect the presence of Theileria sp. has on R. microplus a "pathogen-induced dysbiosis". We also identify the presence of Plasmodium falciparum, the causative agent of human malaria from the microbiome of both H. anatolicum and R. microplus ticks. These findings support the presence of a "pathogen-induced dysbiosis" within the tick and further validation experiments are required to investigate how they are important in the vector competence of ticks. Understanding the mechanism of "pathogen-induced dysbiosis" on tick microbial composition may aid the discovery of intervention strategies for the control of emerging tick-borne infections.

Study of antimicrobial resistance and physiological biomarkers with special reference to Salmonellosis in diarrheic foals in Punjab, Pakistan.

Antimicrobial resistance results in selective colonization in animals. In the present study, 447 diarrheic foals (235 horse foals, 165 donkey foals and 47 mule foal) were selected from Lahore and Sahiwal districts of Punjab, Pakistan. Fresh fecal and blood samples from diarrheic foals were collected for isolation and confirmation of Salmonella Polymerase chain reaction. Results revealed that 50 (11.25%) foals (horse n=29, donkey n=12 and mule n=9) were positive. Fifty Salmonella enterica isolates belonging to 7 serovars S. Paratyphi B (15), S. Saintpaul (7), S. Newport (6), S. Typhimu-rium (11), S. Kottbus (4), S. Lagos (2), and S. enterica ssp salamae (5). Salmonella was common in foals that visited veterinary hospital, as compared to those in stud farms and individual foals reared in low income household. Out of the total 50 samples, 92% of isolates were resistant to three or more than three antimicrobials. The highest resistance (86%) was against Sulphamethoxazole (23.75mg) and lowest (4%) against trimethoprime (5mg). The isolates also showed resistance against Doxycycline (30mg), Oxytetracycline (30mg), Streptomycin (10mg), Neomycin (30mg), Amikacin (30mg), chloramphenicol (30mg), Ampicillin (10mg), Amoxicillin (10mg), kanamycin (30mg), Norfloxacin (10mg), Gentamicin 10mg, Cefotaxime (30mg), Ciprofloxacin (5mg) and Ceftriaxone (30mg). Blood analysis of salmonella infected foals showed That Hemoglobin, PCV and TEC were significantly higher and (while) TLC, PCV, Monocytes, Lymphocytes, Basophils, Eosinophil and Neutrophils were significantly lower than normal. Albumin were lower and BNU, Biluribin, ALT and creatinine were higher than normal values.

Trehalose improves semen antioxidant enzymes activity, post-thaw quality, and fertility in Nili Ravi buffaloes (Bubalus bubalis).

Our objectives were to study the effect of trehalose in extender on (1) antioxidant enzymes profile during cryopreservation (after dilution, before freezing, and after thawing), (2) in vitro quality (after thawing), and (3) in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 20) from four buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of trehalose (0.0, 15, 30, 45, and 60 mM) and frozen in French straws. At post dilution, profile of sperm catalase (U/mL) was higher (P < 0.05) in extenders containing 15, 30, and 45 mM of trehalose as compared to control. Although profiles of superoxide dismutase (U/mL) and total glutathione (µM) were higher (P < 0.05) in extenders containing 15 and 30 mM of trehalose as compared to control. At prefreezing, sperm catalase, superoxide dismutase, and total glutathione profiles were higher (P < 0.05) in all the treatment groups as compared to control. At post thawing, the profiles of catalase and total glutathione were higher (P < 0.05) in extender containing 30-mM trehalose as compared to other treatment groups and control. Whereas, profile of superoxide dismutase was higher (P < 0.05) in extenders containing 30, 45, and 60 mM of trehalose as compared to control and 15mM group. Post thaw total sperm motility (%) was higher (P < 0.05) in extender containing 30-mM trehalose as compared to control and 15 and 60-mM groups. Although sperm progressive motility (%), rapid velocity (%), average path velocity (µm/s), straight line velocity (µm/s), curvilinear velocity (µm/s), plasma membrane (structural and functional, %), acrosome (%), and DNA (%) integrity were higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment groups and control. The fertility rates (61% vs. 43%) were higher (P < 0.05) in buffaloes inseminated with semen doses cryopreserved in extender containing 30 mM of trehalose than the control. It is concluded that addition of 30-mM trehalose in extender improves the semen antioxidant enzymes activity, post thaw quality, and fertility in Nili Ravi buffaloes.