Synthesis of green zinc-oxide nanoparticles and its dose-dependent beneficial effect on spermatozoa during preservation: sperm functional integrity, fertility and antimicrobial activity.

Introduction: The development of an effective extender is important for semen preservation and the artificial insemination (AI) industry. This study demonstrates the beneficial effect of zinc oxide nanoparticles (ZnO-NPs) as an additive to semen extenders to improve semen quality, fertility, and antibacterial activity during liquid preservation in a boar model. Methods: Initially, to find out the safe concentration of ZnO-NPs in sperm cells, a wide range of ZnO-NP concentrations (0, 5, 10, 50, 100, 500, and 1,000 µM) were co-incubated with sperm at 37°C for a cytotoxic study. These NP concentrations were compared to their salt control zinc acetate (ZA) at the same concentrations and to a control group. The effect of the different concentrations of ZnO-NPs on sperm motility, membrane integrity, mitochondrial membrane potential (MMP), and apoptosis was assessed. Accordingly, the non-toxic dose was selected and supplemented in MODENA extender to determine its beneficial effect on the boar semen parameters mentioned and the lipid peroxidation (LPO) levels during liquid preservation at 16°C for 6 days. The non-cytotoxic dosage was subsequently chosen for AI, fertility investigations, and the evaluation of the antibacterial efficacy of ZnO-NPs during preservation hours. An antibacterial study of ZnO-NPs and its salt control at doses of 10 µM and 50 µM was carried out by the colony forming unit (CFU) method. Results and discussion: The cytotoxic study revealed that 5, 10, and 50 µM of ZnO-NPs are safe. Consequently, semen preserved in the MODENA extender, incorporating the non-toxic dose, exhibited 10 and 50 µM ZnO-NPs as the optimal concentrations for beneficial outcomes during liquid preservation at 16°C. ZnO-NPs of 10 µM concentration resulted in a significantly (p < 0.05) improved conception rate of 86.95% compared to the control of 73.13%. ZnO-NPs of 10 and 50 µM concentrations exhibit potent antimicrobial action by reducing the number of colonies formed with days of preservation in comparison to the negative control. The investigation concluded that the incorporation of 10 µM ZnO-NPs led to enhancements in sperm motility, membrane integrity, and MMP, attributed to a reduction in the malondialdehyde (MDA) levels. This improvement was accompanied by a concurrent increase in fertility rates, including farrowing rate and litter size, during the liquid preservation process. Furthermore, ZnO-NPs exhibited an antimicrobial effect, resulting in decreased bacterial growth while preserving boar semen at 16°C for 6 days. These findings suggest that ZnO-NPs could serve as a viable alternative to antibiotics, potentially mitigating antibiotic resistance concerns within the food chain.

Effect of membrane stabilizers on semen quality and sperm membrane protein expression during cryopreservation of goat semen.

BACKGROUND: Semen cryopreservation is a complex process during which there is alteration in the expression of sperm and seminal plasma proteins, molecular weight of protein or loss of membrane proteins during the process. In order to compensate for these changes, different membrane stabilizers are used in freezing semen extenders. However, there is scarcity of such studies during cryopreservation of goat semen. OBJECTIVE: To investigate the effect of membrane stabilizers on sperm membrane protein expression during cryopreservation of goat semen. MATERIALS AND METHODS: A total of 36 semen ejaculates from nine Assam Hill Goat bucks aged 2 to 2.5 years was collected by artificial vagina method. Three membrane stabilizers, each at two different concentrations viz. 50 and 80 mM sucrose, 50 and 100 mM trehalose, and 100 and 150 ng per mL IGF-1 (insulin-like growth factor 1 protein) were added to Tris-citric acid fructose egg yolk glycerol (TCFEYG) extender and semen samples were cryopreserved. The sperm membrane protein profile was studied in fresh and cryopreserved semen by SDS-PAGE. RESULTS: SDS- PAGE of sperm membrane extract of fresh semen revealed the presence of 24 protein bands with molecular weights ranging from 10 kDa to 240 kDa. Samples supplemented with 50 mM sucrose and 80 mM sucrose revealed 21 protein bands with molecular weights ranging from 10 kDa to 240 kDa. All the 21 protein bands were same as those observed in the sperm membrane of fresh spermatozoa, except that the 23 kDa, 29 kDa and 42 kDa bands were absent in frozen semen. Similarly, frozen semen extended with 50 mM trehalose and 100 mM trehalose revealed 22 protein bands with molecular weights ranging from 10 kDa to 240 kDa, but lacking the 29 kDa and 42 kDa bands. Proteins with molecular weights of 29 kDa, 130 kDa and 240 kDa were absent in frozen semen supplemented with 100 ng per mL IGF-1 and 150 ng per mL IGF-1. CONCLUSION: The present study revealed that supplementation of tris basic extender with trehalose at 100 mM and or IGF-1 at 100 ng/mL or 150 ng per mL improves the post-thaw semen characteristics and protects certain fertility related sperm membrane proteins. Doi.org/10.54680/fr23510110612.

Productive and reproductive performances of two-breed and three-breed pig crosses with Niang Megha, Hampshire and Duroc inheritance reared under subtropical Eastern Himalayan hilly climate.

The objective of the study was to evaluate the performance of different crossbreeds, viz., two-breed crosses including HN-50 (50% Hampshire × 50% Niang Megha) and HN-75 (75% Hampshire × 25% Niang Megha) and three-breed cross, HND (25% Hampshire × 25% Niang Megha × 50% Duroc) for suggesting suitable crossbred pigs with appropriate inheritance for subtropical Eastern Himalayan hilly climate. These crossbreed pigs were reared in standard management conditions in Nucleus Pig Breeding Farm of ICAR RC for NEH region. A total of 1995 records were collected which included data on production performance (n = 1466), reproductive performance (n = 428) and carcass traits (n = 101) from farm record book maintained over a period of 7 years. Productive performance included body weight, ADG and FCR at different stages of growth. The study revealed productive performance was highest in two-breed cross of pigs with 75% H inheritance (HN-75) and three-breed cross (HND) pigs. Reproductive performance included ages at puberty, first conception and first farrowing, along with inter-farrowing interval, pregnancy and farrowing rate as well as litter performance. The HN-75 was found to be having shorter inter-farrowing interval and higher pregnancy rate than other genetic groups. Regarding carcass traits, three-breed cross had a higher dressing percentage and less back-fat thickness than other crossbred pigs. Two breed crosses of pigs were found to be having a higher back-fat thickness than three-breed cross pig, and HN-75 had a better dressing percentage than HN-50. Thus, it was concluded that three-breed cross was recommended for lean meat production, and two-breed cross HN-75 was recommended for both breeding and fattening purposes for subtropical Eastern Himalayan hilly climate.

Dietary flaxseed oil improve boar semen quality, antioxidant status and in-vivo fertility in humid sub-tropical region of North East India.

Environmental stress in the form of high temperature humidity index (THI) in tropical and sub-tropical region negatively affects semen quality and fertility of boar. Therefore, the present study was done to evaluate the effect of supplementing flaxseed oil (FLO) to boar's diet on its semen quality, antioxidant status, fatty acid composition of seminal plasma and fertility under sub-tropical climate. For this purpose, six Hampshire crossbreed (50% Hampshire and 50% Gunghroo) boars were divided into two groups i.e control (CON) and treatment (FLO). In FLO and CON group, flaxseed and vegetable oil, respectively, was top dressed at the rate of 3% in basal diets for each boar on daily basis for 16 weeks during monsoon season. A total of 60 ejaculates, comprising 30 ejaculates from each group (ten ejaculates from each boar) were collected. Semen samples were evaluated for sperm quality parameters (SQPs: motility, viability, abnormality, acrosomal integrity and Hypo-osmotic swelling test) and velocity attributes by computer assisted semen analysis (CASA) at fresh and after 72 h of preservation at 17 °C. Antioxidant (glutathione peroxidase; GPx, catalase; CAT, total antioxidant capacity; TAC and malondialdehyde; MDA) were analyzed in seminal plasma and serum. Fatty acid compositions of seminal plasma were analyzed by gas chromatography-mass spectrometry (GC-MS). In-vivo fertility study was also conducted. Reaction time and false mounts were significantly (p < 0.05) reduced in FLO group as compared to CON group. Semen quality parameters were significantly (p < 0.05) improved at fresh stage and after 72 h of liquid storage in FLO group as compared to CON group. Velocity attributes (VAP, VSL, VCL, ALH, BCF and LIN) were significantly (p < 0.05) higher in FLO group. Flaxseed oil supplementation significantly (p < 0.01) enhanced serum GPx and CAT concentration. Serum and seminal plasma MDA concentration decreased significantly (p < 0.01) in FLO group. Similarly, GPx, TAC and CAT were significantly (p < 0.01) elevated in seminal plasma of FLO group. The study revealed that feeding of flaxseed oil altered the fatty acid composition of seminal plasma and significantly (p < 0.05) improved the farrowing rate. In summary, flaxseed oil supplementation improved the semen quality parameters and fertility of boars in sub-tropical climate by improving the antioxidant capacity and altering the fatty acid composition of seminal plasma.

Market potential and opportunities for commercialization of traditional meat products in North East Hill Region of India.

The North Eastern (NE) India is renowned for its preference for animal-based food. This region is known for its traditional meat products. However, the popularity of these products remains confined to the specific community/location. The knowledge on the traditional preparation methods is generally passed across generations through practice and word of mouth. The traditional style of preparation and the specific ingredients added to each product makes them unique. In this review, an attempt has been made to identify the initiatives, opportunities, and market potential for commercialization of the traditional meat products. These unique features and properties of the traditional meat products have been highlighted. The commercialization of these products will enhance entrepreneurship development and ensure quality ethnic products to the consumer in the NE hill region of India.

Multiple proteins from ejaculated and epididymal porcine spermatozoa bind glycan motifs found in the oviduct.

Previously, we demonstrated that porcine spermatozoa bind selectively to Lewis(x) (Le(X) ) and bi-antennary α2-6 sialylated N-acetyllactosamine (bi-SiaLN) glycan motifs found in the oviduct. Recognition of these motifs mediates sperm binding to oviduct epithelial cells to form a reservoir. Here, we characterize proteins that bind to Le(X) and bi-SiaLN in ejaculated and cauda epididymal spermatozoa. Fluoresceinated 3'-O-sulphated-Le(X) (suLe(X) ) and bi-SiaLN were used to locate the binding proteins on spermatozoa. Binding to N-acetyllactosamine (LN), a glycan that spermatozoa bind less, was assessed as a control. Over 60% of ejaculated and epididymal spermatozoa bound to suLe(X) and bi-SiaLN, but only 25% bound to LN. Specific binding to suLe(X) and bi-SiaLN required divalent cations. Fluoresceinated suLe(X) bound mostly to the plasma membrane at the apex of the head, whereas bi-SiaLN bound to a broader area of the head. Further characterization of receptors was accomplished by probing, with specific glycans, sperm proteins that had been separated by SDS-PAGE and transferred to blots. Glycan blot assays identified several suLe(X) -binding bands that migrated as 12-250 kDa. Migration of some of the bands that bound bi-SiaLN was similar to those that bound suLe(X) . A 12-kDa band protein was detected in seminal fluid and ejaculated spermatozoa, but absent in epididymal spermatozoa. In summary, we determined that: (i) multiple glycan-binding proteins recognize suLe(X) and bi-SiaLN motifs, (ii) specific binding required divalent cations and (iii) glycan-binding proteins were identified before and after sperm contact with accessory gland fluids. While the identity of these molecules is still unknown, this information predicts characteristics of the authentic oviduct suLe(X) and bi-SiaLN receptors on spermatozoa.

Effect of conventional and controlled freezing method on the post thaw characteristics of boar spermatozoa.

The objective of the present study was to evaluate the effectiveness of conventional, and controlled freezing method adopting three freezing rates 20°C, 40°C and 60°C/min for cryopreservation of boar semen. Sixty sperm-rich fractions of ejaculates from six boars were utilized for freezing of semen with different freezing methods in lactose-egg yolk glycerol extender using 0.5 ml straws. Semen samples were evaluated for sperm motility, live sperm, acrosome integrity, plasma membrane integrity (PMI) by carboxyfluorescein diacetate plus propidium iodide (PI) staining, mitochondrial membrane potential (MMP) by combined JC-1 plus PI staining and lipid peroxidation (LPO) by BODIPY (581/591)-C11 probe after equilibration and after freezing. The results revealed that the post thaw sperm motility, live sperm, live intact acrosome and plasma membrane integrity were significantly (p<0.05) higher in all the three controlled freezing methods (20°C, 40°C and 60°C/min) as compared to that in conventional method. In addition, the controlled freezing methods yielded higher (p>0.05) mean values of live sperm with high MMP as compared to conventional freezing. However, the post thaw sperm LPO did not influence by difference in freezing methods. No significant difference on the post thaw sperm qualities was recorded among the three controlled freezing rates. All the sperm parameters assessed declined significantly (p<0.05) after freezing as compared to that after equilibration irrespective of freezing method employed. In conclusion, cryopreservation of boar semen with controlled freezing methods conferred better post thaw sperm quality as compared to conventional method, and the freezing rates of either 20, 40 or 60°C/min could provide better freezability of boar semen.

Effect of cryopreservation on apoptotic-like events and its relationship with cryocapacitation of buffalo (Bubalus bubalis) sperm.

This study investigated the apoptosis-like events associated with cryopreservation process and their relationship with cryocapacitation in buffalo (Bubalus bubalis) sperm. A total of 49 semen ejaculates from seven bulls were studied for structural changes in sperm following cryopreservation. Apoptotic changes were detected by assays specific for translocation of phosphatidylserine (PS) to the cell surface, alterations in membrane permeability and mitochondrial membrane potential (MMP), and DNA integrity. A significant (p < 0.01) percentage of cryopreserved sperm showed externalization of PS and early apoptotic changes and lowered MMP when compared with the fresh sperm. Freezing and thawing of sperm increased permeability to YOPRO-1, an impermeant fluorescent dye. However, on TUNEL staining, cryopreserved sperm showed no breach in DNA integrity. The sperm capacitation status was evaluated by chlortetracycline (CTC) fluorescence pattern, in which a significant (p < 0.01) percentage of cryopreserved sperm were found to be capacitated. The capacitated sperm (Pattern B) was positively correlated with the aforementioned apoptotic events. In conclusion, cryopreservation process induced early apoptosis-like changes in buffalo sperm, and a close link exists between cryocapacitation and apoptosis during cryopreservation of sperm.

Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa.

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 µg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.

Objective sperm motion analysis to assess dairy bull fertility using computer-aided system--a review.

Motility is one of the most important characteristics associated with the fertilizing ability of spermatozoa and is an expression of their viability and structural integrity. Computer-assisted semen analyser (CASA) provides precise and accurate information on different sperm motion characteristics. This article reviews various aspects of computer-aided motility analysis of bull sperm like sample preparation, standardization of instrument settings, importance of various motility parameters evaluated by the system and its impact on basic functional studies of spermatozoa. It gives special emphasis to various aspects of bull sperm motion analysis especially sub-populations of spermatozoa, hyper-activation, motion characteristic in different genetic and age groups, etc. and their utility in predicting the fertility of dairy bulls. The need to fill the gap in research and the necessity of universal standardization of the equipment has been discussed.

Early sexual maturity in local boars of Northeastern India: age-related changes in testicular growth, epididymal sperm characteristics and peripheral testosterone levels.

The present study reports the age related changes in the peripheral testosterone levels, testicular and epididymal growth and development and cauda epididymal spermiogram in local pigs of Northeastern India, which attain sexual maturity around 3 months of age. Local boars (n = 20) were castrated at monthly intervals from 2 to 6 months of age (4 boars per month) to study the testicular growth and development and the epididymal spermiogram. Blood samples, collected from local boars (n = 6) at monthly intervals from 2 to 6 months of age, were analyzed for testosterone levels by radioimmunoassay. Compared to Hampshire boars, significantly (P < 0.05) high testosterone levels were observed in the local boars as early as 2 months of age. The mean (± SEM) level of testosterone in the local boars at 2, 3 and 4 months of age was 11.89 ± 1.52, 20.45 ± 1.33 and 20.38 ± 2.0 ngml(-1), respectively. Though there was consistently significant (P < 0.05) difference in the body weight between Hampshire and local pigs, the same was not observed in case of testicular weight except at 3 and 6 months of age. In line with the above observation, the testis:body weight ratio (gram testis per kg body weight) was significantly (P < 0.05) higher in the local boars compared to the Hampshire boars at any time of observation, which ranged from 0.8 to 1.0 in case of Hampshire and from 2.3 to 3.0 in local boars. The sperm concentration in the cauda epididymal fluid of local boars at 2, 3 and 6 months of age was 2255 ± 186.6, 3685 ± 103.8 and 4325 ± 146.2 million/ml, respectively and the sperm motility, viability and total abnormality was 73.3, 75.2 and 6.2%, respectively at 3 months of age. Taken together, the testosterone level, testicular growth and development and epididymal spermiogram indicate the trait of early sexual maturity in the local pigs as compared to Hampshire.

Comparative studies on egg, meat, and semen qualities of native and improved chicken varieties developed for backyard poultry production.

The objective of the present study was to evaluate/compare the sensory attributes of eggs and meat, egg qualities, proximate composition of eggs, and semen qualities of slow growing native (Miri and Mizo-local) and fast growing improved chicken varieties (Gramapriya and Vanaraja) under hill ecosystem of northeastern India. Significantly higher egg weight, egg volume, and albumen volume were observed in Gramapriya followed by Vanaraja, Mizo-local, and Miri chickens. However, yolk volume was significantly higher in Vanaraja and Gramapriya varieties as compared to native chickens. Yolk to albumen ratio was significantly lower in Gramapriya as compared to Vanaraja and Miri chicken. Consumer liking of eggs for aroma, flavor, and overall acceptability of Miri, Mizo-local, and Vanaraja were significantly higher than that of Gramapriya. Genetic groups did not differ significantly in appearance and proximate composition of eggs. No significant differences were observed between various genetic groups for sensory attributes of meat samples. Semen volume was significantly (p < or = 0.01) lower while sperm concentration was significantly (p < or = 0.01) higher in native chicken as compared to the improved chicken varieties. However, pH, mass activity, sperm motility, and livability did not differ significantly among genetic groups although Mizo-local had significantly higher abnormal sperm count. The study concluded that the genetic groups with different growth rate differed significantly for various egg quality parameters and semen characteristics but not for sensory attributes of meat and proximate composition of eggs.

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium.

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated (4 degrees C for 4h) and frozen at -196 degrees C in LN(2). The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P<0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P<0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P<0.01) reduced after freezing-thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r=0.81) and high membrane fluidity (r=0.65), and negatively correlated with cholesterol level (r=-0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.

Preservation of boar semen at 18 degrees C induces lipid peroxidation and apoptosis like changes in spermatozoa.

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (DeltaPsi(m)) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 degrees C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, DeltaPsi(m), and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of DeltaPsi(m) was done using 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly (p<0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10(9) sperm) were low in sperm (99.83+/-2.69) and seminal plasma (191.98+/-11.58), which gradually increased up to the 96 h of storage. Highest negative correlation (r value) was observed between MDA levels and sperm motility (-0.97), live percent (-0.97), acrosomal integrity (-0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (-0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage (r=0.98). There was a significant (p<0.05) increase in the sperm cells with low DeltaPsi(m) from 0 to 96 h of preservation. Before preservation, 14.85+/-4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00+/-6.25%. The apoptotic sperm population was 8.33+/-2.31% in fresh semen, while this population was 25.19+/-4.25% at 96 h of preservation and the difference was significant (p<0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 degrees C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.

Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen.

Several factors affect sperm motility and functional integrity during preservation; the damage caused by reactive oxygen species (ROS) being an important factor. The present study investigated intracellular ROS generation and its relationship with sperm motility, lipid peroxidation (LPO), mitochondrial membrane potential (MMP) and DNA integrity during preservation (liquid preservation at 4 degrees C and cryopreservation) of buffalo semen. Fifty-six ejaculates, eight each from seven buffalo bulls (Bubalus bubalis) were utilized for the study. Intracellular ROS level was detected using 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and propodium iodide (PI) by flow cytometry. 3,3'-Dihexyloxacarbocyanine iodide [DiOC(6) (3)]/PI and acridine orange were used for detection of MMP and DNA integrity of spermatozoa, respectively. Results revealed that ROS and LPO level in sperm increased linearly between 0 h and 72 h of liquid preservation at 4 degrees C, with significantly (P<0.01) higher levels at 48 h and 72 h of storage compared to fresh semen. The ROS level in viable sperm in frozen-thawed semen did not differ significantly from fresh semen, but the LPO was significantly (P<0.05) higher in frozen-thawed sperm compared to fresh sperm. There was a linear reduction in the sperm with high MMP and DNA integrity in liquid semen, which was significantly (P<0.01) higher at 48 h and 72 h of storage compared to fresh semen. The intracellular ROS was strongly associated to sperm motility, LPO, MMP and DNA integrity during liquid preservation, while this association did not exist in frozen-thawed sperm. The study concluded that ROS generation and its associated effects are likely to be an important contributor to the reduced sperm motility and functional integrity during liquid preservation of buffalo semen at 4 degrees C, but ROS generation and its damage had only minor effects during freezing and thawing process.