Molecular Characterization of Lineage-IV Peste Des Petits Ruminants Virus and the Development of In-House Indirect Enzyme-Linked Immunosorbent Assay (IELISA) for its Rapid Detection".

Peste des petits ruminants (PPRV), a highly contagious viral disease, causes significant economic losses concerning sheep and goats. Recently, PPR viruses (PPRVs), have adopted new hosts and lineage IV of PPRVs represents genetic diversity within the same lineage. 350 samples, including blood, swabs, and tissues from sheep/goats, were collected during the 2020-2021 disease outbreaks in Pakistan. These samples were analysed through RT-PCR and three isolates of PPRV with accession numbers, MW600920, MW600921, and MW600922, were submitted to GenBank, based on the partial N-gene sequencing. This analysis provides a better understanding of genetic characterizations and a targeted RT-PCR approach for rapid PPRV diagnosis. An IELISA test was developed using the semi-purified antigen MW600922 isolate grown in Vero cells. The PPRV isolates currently present high divergence with the Turkish strain; conversely, similarities equivalent to 99.73% were observed for isolates collected from Pakistan. The developed indirect ELISA (IELISA) test demonstrated antibody detection rates at dilutions of 1:200 for antibodies (serum) and 1:32 for antigens. In comparison to cELISA, high specificity (85.23%) and sensitivity (90.60%) rates were observed. In contrast to the virus neutralization test (VNT), IELISA was observed to be 100% specific and 82.14% sensitive in its results. Based on these results, serological surveys conducted for PPR antibodies using IELISA can be a more effective strategy on a larger scale. Furthermore, our results demonstrate a significant breakthrough in the research in terms of cost-effectiveness and storage efficiency, and the developed IELISA test is highly recommended for use in developing countries.

Peste des petits ruminants (PPRV) is a transboundary, highly contagious, and economically significant viral disease affecting small ruminants and wildlife. PPRV, a disease that only targets animals, is the focus of the Global Eradication Programme (PPRV GEP), which aims to eradicate the disease by 2030. Following the completion of the first phase of the GEP (2017­2021), Pakistan has initiated the second phase: PPRV presence and the implementation of a control strategy. Rapid and accurate laboratory diagnosis is vital to the disease's effective control and eradication. In the present study, we have improved diagnosis by reverse transcriptase polymerase chain reaction (RT-PCR), which not only can detect low viral concentrations but also contributes to the genetic analysis of lineage-IV viruses. However, the development of cost-effective indirect ELISA (iELISA) may allow for the analysis of serum samples obtained from larger populations of small ruminants.

Keratin as an effective coating material for in vitro stem cell culture, induced differentiation and wound healing assays.

The utilization of stem cells in tissue engineering holds great promise as efficient tools for tissue regeneration and in treating numerous musculoskeletal diseases. However, several limiting factors, such as precise delivery and control of differentiation of these stem cells as well as mimicking the microenvironment required to modulate stem cell behaviour in-vivo, have given rise to an urgent need for the development of new biomaterials which could be tailored to enhance cell renewal and/or direct cell fates. Keratin-rich biological materials offer several advantages, such as biocompatibility, tailorable mechanical properties, huge bioavailability, non-toxicity, non-immunogenic, and intrinsic tissue repair and/or regeneration capabilities, which makes them highly valued. In the present work, we report the preparation of keratin-based bio-materials from goat hair waste and its effectiveness as a coating material for in vitro culture and induced differentiation of mesenchymal stem cells (MSC's) and primary goat fibroblast cells. Since no known keratinase enzymes are expressed as such in human and/or animal systems, these keratin biomaterials could be used to slow the rate of degradation and deliver keratin-loaded stem cell scaffolds to induce their directed differentiation in vivo. The generated keratin materials have been characterized for surface morphology, protein structures, size and other properties using SDS-PAGE, LC/MS-MS, SEM, FTIR etc. Also, in vitro cell culture assays such as cell adhesion, viability using MTT, live dead assays, differentiation assays and in vitro scratch/wound healing assays were performed. Our results provide important data supporting tissue engineering applications of these keratinous biomaterials by combining the unique biological characteristics of goat hair-derived keratin material with the regenerative power of stem cells and their combinatorial use in applications such as disease treatment and injury repair as well as their use in the preparation of wound healing products, such as dressings and bandages, for management of clinical care in animals.

Non-surgical and guided-bone-regeneration surgical management of Type-3b dens invaginatus with an apico-marginal defect: A case report.

BACKGROUND: Dens invaginatus (DI), an unusual developmental anomaly is a challenge for the operating dentist with regard to its diagnosis and treatment. This case report presents the successful management of a Type-3b DI in a permanent maxillary lateral incisor associated with a large radicular cyst and communicating apico-marginal defect (Von Arx type IIb). METHODS AND RESULTS: A 19-year-old female patient reported pain and palatal swelling. During the clinical examination, tooth #12 exhibited tenderness to percussion, and presented a deep periodontal pocket depth (PPD) of 12 mm, along with grade I mobility. Radiographic examination revealed a large peri-radicular radiolucency with atypical tooth morphology. Cone beam computed tomography clarified the complicated root canal anatomy to be Type-3b DI associated with an apico-marginal defect. The case was managed successfully by non-surgical endodontic therapy followed by surgical intervention utilizing a guided bone regenerative (GBR) approach. Eighteen-month follow-up showed an asymptomatic and functional tooth with a significant reduction in pocket depth. The periapical radiographs showed continued healing of the osseous defect. CONCLUSIONS: The successful healing outcome of a challenging case, characterized by a complex DI morphology, a large peri-radicular lesion, a through-and-through defect, and a combined endodontic-periodontal apico-marginal defect was achieved through accurate diagnosis, treatment planning, and execution using contemporary endodontic and periodontal treatment techniques. The application of GBR techniques during the surgical phase of treatment may have contributed to the improved regenerative healing outcome in this case, which was initially considered prognostically questionable. KEY POINTS: Why is this case new information? Type-3b DI exhibits a complex root canal structure, each case displaying unique characteristics, necessitating a case-specific treatment plan. In this case report the Type-3b DI morphology was associated with a large peri-radicular, through and through defect and combined endodontic periodontal apico-marginal defect. The treatment approach involved incorporating guided bone regenerative (GBR) principles during the surgical phase. This case report contributes to the existing evidence on the diagnosis and successful management of Type-3b DI with a concurrent apico-marginal defect. What are the keys to successful management of this case? The successful management of a prognostically challenging case was achieved through a closely integrated multidisciplinary coordination between the endodontist and periodontist. Utilization of contemporary techniques and tools contributed to the successful management The use of three-dimensional radiological examination through cone beam computed tomography enabled a precise preoperative assessment, facilitating the formulation of a treatment plan for managing both the Type-3b DI morphology and the associated peri-radicular lesion. Employing GBR techniques in peri-radicular surgery may have assisted in the healing of through-and-through periapical defects with concurrent apico-marginal defects (Von Arx type IIb). What are the primary limitations to the success of this case? A complex root canal anatomy associated with Type-3b DI morphology A large peri-radicular through and through defect with concurrent apico-marginal defect. Difficulty in weekly and long-term follow-up of the patient.

Alpha-tocopheryl Succinate in Extender Improves the Post Thaw Quality of Water Buffalo Spermatozoa.

BACKGROUND: Alpha-tocopheryl succinate, a major chain-splitting antioxidant, is the most effective form of vitamin E and may be used in the semen extender for cryopreservation of buffalo spermatozoa. OBJECTIVE: To use different concentrations of alpha-tocopheryl succinate (T1, 0.3 mM, T2, 0.6 mM, and T3, 0.9 mM) and control (0.0 mM) in extender for dose optimization and hence improve the frozen-thawed quality of water buffalo spermatozoa. MATERIALS AND METHODS: Semen samples were collected from three mature buffalo bulls with artificial vagina (42°C) and this study was replicated for five times. Semen was cryopreserved by conventional method which included filling of semen per experimental treatment into 0.5 mL French straws, sealing with polyvinyl alcohol powder and keeping them 5 cm above the liquid nitrogen vapors for 12 min and storing in liquid nitrogen tank. Frozen-thawed semen was also processed for total antioxidant capacity content (TAC) and lipid peroxidation (LPO) level by thiobarbituric acid (TBA). Computer-assisted semen analysis (CASA) and other assays were also performed. RESULTS: TAC levels were higher (P<0.05) with T2 and T3 as compared to T1 and control. LPO levels were lower (P<0.05) with T2 and T3 as compared to T1 and control. Sperm progressive motility (%) and rapid velocity (%) were higher (P<0.05) with T2 and T3 as compared to control. The extender containing T3 had higher (P<0.05) sperm average path velocity (µm/s) and straight line velocity (µm/s) as compared to control. At 1 and 2 h incubation period (37 °C) T2 and T3 in extenders had higher (P<0.05) progressive motility and rapid velocity compared to control. Sperm supra vital plasma membrane integrity (%), mitochondrial transmembrane potential (%), viable and intact acrosome (%) and DNA integrity (%) were higher (P<0.05) with T2 and T3 as compared to T1 and control, respectively. CONCLUSION: The supplementation of alpha-tocopheryl succinate in extender, either at 0.6 (T2) or 0.9 (T3) mM concentrations improves the post thaw quality of water buffalo spermatozoa by sustaining the TAC levels and keeping the LPO levels lower as compared to the control. It is suggested that future study should be aimed to explore the influence of these optimal concentrations of alpha-tocopheryl succinate on in vivo fertility of buffalo bull spermatozoa.

Superoxide Dismutase in Extender Improves the in vitro Quality and in vivo Fertility of Cryopreserved Water Buffalo (Bubalus bubalis) Spermatozoa.

BACKGROUND: Superoxide dismutase (SOD) as an antioxidant in semen extender may be used for the cryopreservation of buffalo spermatozoa and in vivo fertility. OBJECTIVE: This study was aimed to evaluate the effects of SOD (SOD1, 100 IU/mL; SOD2, 200 IU/mL; SOD3, 300 IU/mL) and control (0.0) in Tris citric acid extender on in vitro quality and in vivo fertility of cryopreserved water buffalo bull spermatozoa. MATERIALS AND METHODS: Semen collection was carried out on a weekly basis (four bulls, three replicates, and n = 24 ejaculates). The conventional freezing of semen loaded straws (0.5 mL) was undertaken by placing them horizontally on a steel rack inside a Styrofoam box for 10 min containing liquid nitrogen (LN2) vapours, and plunging into a liquid nitrogen tank (-196 °C) for storage, followed by thawing at 37 °C for 30 s and analysis by computer-assisted sperm analyzer (CASA) and other assays. RESULTS: At post-dilution, the acrosome integrity (ACR-I, %) was significantly improved (P < 0.05) in extender supplemented with SOD3 as compared to other experimental groups. In addition, DNA integrity (DNA-I, %) was significantly higher (P < 0.05) in SOD1 and SOD3 compared to SOD2 and control. At post-thawing, the mean values of sperm progressive motility (PM, %), average path velocity (VAP, µm/s) and straight line velocity (VSL, µm/s) were significantly higher (P < 0.05) in extender supplemented with SOD3 compared to the control. At post-thawing, mean values of subjective motility (SM, %), plasma membrane integrity (PMI, %) and ACR-I were significantly higher (P < 0.05) in extender supplemented with SOD3 compared to the control. At post-thawing, sperm DNA-I was significantly higher (P < 0.05) in extender supplemented with all SOD doses compared to the control in a dose-dependent manner. The in vivo fertility rate (%) was significantly higher with SOD3 compared to the control (68.2 % vs. 49.5 %). CONCLUSION: The supplementation of SOD3 (300 IU/mL) in Tris citric acid extender improves both in vitro quality and in vivo fertility of buffalo bull spermatozoa.

Effect of EDTA as Chelating Agent in Extender on the Post Thaw Quality of Buffalo Bull Spermatozoa.

BACKGROUND: Spermatozoa are prone to mechanical and biochemical stresses upon freezing. The influx of Ca++ causes early capacitation and production of reactive oxygen species. OBJECTIVE: To evaluate the effect of ethylenediaminetetraacetic acid (EDTA) as a calcium chelator in a semen extender. MATERIALS AND METHODS: The effect of EDTA concentrations (0%, 0.1%, 0.2% and 0.3%) on the post thaw quality of buffalo bull spermatozoa were studied. RESULTS: The extender with 0.2% EDTA improved significantly visual motility, progressive motility and plasma membrane integrity. Sperm kinematics, such as beat cross frequency (BCF), curved line velocity (VCL), straight line velocity (VSL) and average path velocity (VAP), were higher in the extender with 0.2% EDTA. EDTA at 0.2% improves semen parameters (visual motility, supra vital plasma membrane integrity, chromatin integrity and percentage viable spermatozoa with intact acrosome. CONCLUSION: The EDTA supplementation in a semen extender improves the post-thaw quality of Nili-Ravi buffalo bull semen.

Effect of Cryopreservation on Casa Characteristics, Mitochondrial Transmembrane Potential, Plasma and Acrosome Integrities, Morphology and in vivo Fertility of Buffalo Bull Spermatozoa.

BACKGROUND: Application of frozen-thawed semen is an important tool for improving the vivo fertility, but the process of freezing and thawing causes significant damage to spermatozoa. OBJECTIVE: The aim of this study was to evaluate the effect of cryopreservation on CASA characteristics, mitochondrial transmembrane potential, plasma, and acrosome integrities, morphology and in vivo fertility of buffalo bull spermatozoa. MATERIALS AND METHODS: Semen was collected from four mature buffalo bulls with artificial vagina at 42 °C. Ejaculates having > 1 mL volume, > 60 % sperm visual motility and > 0.5 x 109 sperm/mL concentration from each bull were diluted in Tris-citric acid egg yolk glycerol extender (TCA) making two aliquots per bull for analysis at post dilution and cryopreserved respectively. RESULTS: Analysis of variance (ANOVA) showed that the process of freezing and thawing significantly reduced (P < 0.05) CASA characteristics including total motility (TM, %), progressive motility (PM, %), rapid velocity (RV, %), average path velocity (VAP, µm/sec), straight line velocity (VSL, µm/sec), curvilinear velocity (VCL, µm/sec), beat cross frequency (BCF, Hz), straightness (STR, %) and linearity (LIN, %). Furthermore, the process of freezing and thawing significantly reduced (P < 0.05) subjective motility (SM, %), Supra-vital plasma membrane integrity (SVPMI, %), high mitochondrial membrane potential (HMMP, %), viable spermatozoa with intact acrosome (V/IACR, %). Moreover, it was observed that the freezing thawing process significantly decreased the in vivo fertility (%, 50.35 % vs. 61.39 %; P < 0.05) as compared to post diluted semen. CONCLUSION: It is concluded that the process of freezing and thawing significantly reduced semen quality and in vivo fertility of buffalo bull in terms of various functional parameters.

Impact of weight loss on sperm DNA integrity in obese men.

The objective of the study was to determine whether weight loss in obese men improves their fertility with respect to DNA fragmentation index and morphology. Collected fertility parameters included DFI and morphology. Body mass index (BMI) was calculated for all patients with comparisons to their fertility parameters before and after weight loss using paired t test and chi-square tests. The mean BMI was significantly higher in group 1, before weight loss (33.18 kg/m2 ), than in group 2, after weight loss (30.43 kg/m2 ). Overall, 53.3% of men had DFI <20% while 43.8% had a DFI between 20% and 40%, and 2.9% of men had DFI >40%. The mean DFI of participants was higher before weight loss (20.2%) and had improved significantly after weight loss (17.5%) (p = <.001). The weight loss had significant positive correlation with percentage of DFI. There was a significant improvement in morphology after weight loss (p = <.05). In one of the largest cohorts of male fertility and obesity, DFI and morphology demonstrated significant relationship with adiposity, possibly contributing to subfertility in this population.

Cryoprotectant effect of trehalose in extender on post-thaw quality and in vivo fertility of water buffalo (Bubalus bubalis) bull spermatozoa.

This study was designed to ascertain the cryoprotectant effects of different concentrations of trehalose [0 (T0), 25 (T25), 35 (T35), 45 (T45) mm], egg yolk [20% (E20), 15% (E15) v/v] and glycerol [7% (G7), 5% (G5) v/v] in Tris-citric acid-based extender on post-thaw quality and in vivo fertility of buffalo bull spermatozoa. Twenty-five ejaculates were collected from five bulls and split into four parts. After that, the split ejaculates from each of the bull were diluted either in T0E20G7 (control) or T25E20G5 or T35E15G5 or T45E15G5 extender. Finally, the sperm suspension was frozen in 0.54-ml French straws. Post-thaw sperm total motility (%), progressive motility (%), rapid velocity (%), average path velocity (µm/s), straightline velocity (µm/s), curvilinear velocity (µm/s), linearity (%), plasma membrane and acrosome integrities (%) were higher (p < .05) in T45E15G5 extender as compared to other treatment groups and control. The fertility rate (56.8% versus 41.3%) was higher (p < .05) in buffaloes inseminated with semen doses cryopreserved in extender containing T45E15G5 combination of cryoprotectants than the control. In conclusion, addition of 45 mm trehalose along with 15% egg yolk and 5% glycerol in extender improves the post-thaw quality and in vivo fertility of buffalo bull spermatozoa.

Determination of an Optimal Membrane-permeable Cryoprotectant Addition and Dilution Protocol for Water Buffalo Spermatozoa.

BACKGROUND: There is a possibility to reduce the toxicity of glycerol and osmotic stress of DMSO by lowering their concentrations in freezing extenders. OBJECTIVE: To investigate the effect of glycerol and DMSO in tris-citric acid based extender on post- thaw quality of buffalo (Bubalus bubalis) bull spermatozoa. MATERIALS AND METHODS: Semen was collected from five adult buffalo bulls with artificial vagina. Five aliquots of semen per bull were separated for dilution with the treatment extenders. The first aliquot was diluted at 37C with 6 percent glycerol (T1). The second aliquot was diluted at 37C with extenders containing 4.5 percent glycerol and 1.5 percent DMSO (T2). The third aliquot was diluted with extenders containing 4.5 percent glycerol at 37C and 1.5 percent DMSO at 4С (T3). The fourth aliquot was diluted with extenders containing 1.5 percent DMSO at 37C and 4.5 percent glycerol at 4С (T4). The fifth aliquot was diluted with extender containing 2.5percent DMSO at 37 as well as at 4C (T5). The final concentration of spermatozoa was 50×106/ml in all the treatment groups. Semen was cooled from 37 to 4C in 2 h and equilibration was done at 4 C for 4 h. Later on, packing of cooled semen was undertaken in 0.54 ml French straws and frozen in a programmable cell freezer. RESULTS: At post thawing, treatment groups T1 and T2 yielded significant (P < 0.05) outcome for CASA parameters, longevity, acrosomal integrity, plasma membrane integrity, mitochondrial transmembrane potential and DNA integrity. CONCLUSION: We concluded that by decreasing glycerol concentration (4.5 percent) and combining it with DMSO (1.5 percent) at 37C (T2) in tris-citric acid based extender provided similar results to those observed when glycerol (6 percent) alone is used at 37C (T1) for improving the post-thaw quality of buffalo bull spermatozoa.

Freezability of water buffalo spermatozoa is improved with the addition of catalase in cryodiluent.

BACKGROUND: Catalase enzyme is usually distributed in mammalian seminal plasma, where it decomposes hydrogen peroxide into water and oxygen and enhances sperm survivability. OBJECTIVE: To evaluate the effect of catalase (0, 100, 200 or 300 IU/ml) added in tris-citric acid (TCA) based extender on motion characteristics, viability and DNA integrity of bubaline spermatozoa at post dilution (PD) and post thawing (PT) stages of cryopreservation. MATERIALS AND METHODS: Collection of semen was done in four Nili-Ravi bulls with an artificial vagina (42 degree C). Qualified semen samples from each bull were further subdivided into four aliquots for dilution with the experimental TCA extender containing either 0.0 (T1), 100 IU (T2), 200 IU (T3) or 300 IU (T4) catalase (activity12660 U/mg). RESULTS: At PT, mean computer progressive motility, average path velocity, straight line velocity, curvilinear velocity, visual motility and DNA integrity were higher (P < 0.05) in catalase fortified treatment groups as compared with control. Regarding plasma membrane integrity and supra-vital plasma membrane integrity, at PT the mean values were higher (P < 0.05) in T4 as compared with control. At PD and PT, mean acrosomal integrity of buffalo bull spermatozoa was higher (P < 0.05) in T4 group as compared with control. CONCLUSION: Addition of catalase at a concentration of 300IU/ml in TCA cryodiluent improved the freezability of water buffalo spermatozoa.

Chicken egg yolk plasma in tris-citric acid extender improves the quality and fertility of cryopreserved water buffalo (Bubalus bubalis) spermatozoa.

This study was primarily designed to evaluate the effect of different concentrations of ultraviolet (UV)-C-irradiated chicken egg yolk plasma (EYP; v:v; 10%, P1; 15%, P2; 20%, P3) or 20% (v:v) of whole chicken egg yolk (WCEY) in tris-citric acid (TCA) extender on water buffalo sperm quality during cryopreservation (postdilution, PD; postequilibration, PE; post-thawing, PT). Also the effect of best evolved concentration of UV-C-irradiated EYP in extender on in vivo fertility of buffalo spermatozoa was evaluated. At PE and PT, computer-assisted sperm analysis progressive motility (PM, %) was significantly higher in P3 compared with P1 and WCEY. Rapid velocity (RV, %) was higher (P < 0.05) in P3 compared with P1 and WCEY during cryopreservation (PD, PE, and PT). Average path velocity (µm/s) and straight line velocity (µm/s) were higher (P < 0.05) in P2 and P3 than WCEY at PE and PT. The decline percentage (%, longevity) in PM and RV was lower (P < 0.05) in P3 compared with WCEY during 2 hours incubation under in vitro condition at PT. Supravital plasma membrane integrity (%) was higher (P < 0.05) in P2 and P3 compared with control at different stages (PE and PT). Mitochondrial transmembrane potential (%) was higher (P < 0.05) in P2 and P3 compared with P1 and WCEY at different stages (PD and PT). Percentage of viable sperm with intact acrosome, and sperm DNA integrity (%) were higher (P < 0.05) in P2 and P3 compared with WCEY at PT. The in vivo fertility rate (%) was significantly higher with P3 compared with WCEY (76.61 vs. 64.49). In conclusion, WCEY (20%) can be replaced with UV-C-irradiated chicken EYP (20%) in TCA extender for cryopreservation of water buffalo spermatozoa.

Use of post-thaw semen quality parameters to predict fertility of water buffalo (Bubalus bubalis) bull during peak breeding season.

This study was designed to predict the fertility of water buffalo bull using post-thaw semen quality parameters during peak breeding season. Thirty ejaculates were collected from five bulls with artificial vagina and cryopreserved. At post-thaw, semen was analysed for motility parameters, velocity distribution, kinematics, DNA integrity/fragmentation, viability, mitochondrial transmembrane potential, morphology, plasma membrane and acrosome integrity. Data of 514 inseminations were collected for estimation of in vivo fertility. Pearson's correlation coefficients showed that progressive motility (PM), rapid velocity, average path velocity, straight line velocity, straightness, supravital plasma membrane integrity, viable spermatozoon with intact acrosome or with high mitochondrial activity were correlated with in vivo fertility (r = .81, p < .01; r = .85, p < .01; r = .64, p < .05; r = .73, p < .05; r = .57, p < .05; r = .88, p < .01; r = .84, p < .01 and r = .81, p < .01 respectively). Step forward multiple regression analysis showed that the best single predictor of fertility was PM. However, combinations of semen quality parameters to predict fertility were better as compared to single parameter. In conclusion, fertility of buffalo bull can be predicted through some of the post-thaw in vitro semen quality tests during peak breeding season.

Freezability of water buffalo bull (Bubalus bubalis) spermatozoa is improved with the addition of curcumin (diferuoyl methane) in semen extender.

Effects of curcumin as antioxidant in extender were evaluated on freezability of buffalo spermatozoa. Semen from each of the five bulls (n = 3 replicates, six ejaculates/bull, a total of 30 ejaculates) was diluted in Tris-citric acid extender containing curcumin (0.5, 1.0, 1.5 or 2.0 mM) or control. At pre-freezing and post-thawing, total antioxidant contents (µM/L) and lipid peroxidation levels (µM/ml) were higher (p < .05) and lower (p < .05) respectively, with 1.5 and 2.0 mM compared to 0.5 and 1.0 mM curcumin and control. At post-thawing, progressive motility (PM, %) and rapid velocity (RV, %) were higher (p < .05) with 1.5 mM compared to other doses of curcumin and control (except in case of RV, 1.5 was similar with 1.0 mM). Kinematics (average path velocity, µm/s; straight-line velocity, µm/s; curved-line velocity, µm/s; straightness, %; linearity, %), in vitro longevity (%, PM and RV) and DNA integrity (%) at post-thawing were higher (p < .05) with 1.5 mM compared to control. At post-thawing, supravital plasma membrane integrity (%) and viable spermatozoa with intact acrosome (%) were higher with 1.5 compared to 2.0 mM curcumin and control. We concluded that freezability of water buffalo spermatozoa is improved with the addition of 1.5 mM curcumin in extender.

Programmable fast-freezing method improves the post-thaw motion dynamics, integrities of plasmalemma, mitochondrial transmembrane, DNA and, acrosome, and in vivo fertility of water buffalo (Bubalus bubalis) spermatozoa.

The effects of freezing methods (FR1, nonprogrammable/static, 5 cm above liquid nitrogen [LN2 ] for 10 min, plunging in LN2 ; FR2, programmable medium, +4°C to -15°C at 3°C min-1 , from -15 to -80°C at 10°C min-1 and final holding for 1 min at -80°C, plunging in LN2 ; FR3, programmable fast, from initial holding at +4°C for 2 min, from +4°C to -20°C at 10°C min-1 , from -20°C to -100°C at 30°C min-1 , final holding for 1 min at -100°C and plunging in LN2 ) were assessed on post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. Mean sperm progressive motility (%), rapid velocity (%), average path velocity (µm s-1 ), straight line velocity (µm s-1 ), curved line velocity (µm s-1 ), integrities (%) of plasmalemma, mitochondrial transmembrane, DNA and acrosome were higher (p < .05) in samples cryopreserved with FR3 compared to FR1 and FR2. Similarly, in vivo fertility (%) of buffalo spermatozoa was higher (p < .05) with FR3 than FR1 (%; 68.0 versus 50.0). We concluded that programmable fast-freezing method (FR3) improves the post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa.

Semen quality parameters as fertility predictors of water buffalo bull spermatozoa during low-breeding season.

The present study was carried out to assess various postthaw semen quality parameters for the prediction of fertility in buffalo bull during low-breeding season. Semen (30 ejaculates) was collected from five adult buffalo bulls with artificial vagina (42 °C). Sperm motility parameters, velocity distribution, motion kinematics, and subpopulations were analyzed by computer-aided sperm motion analyzer (CASA). Moreover, sperm visual motility, supravital plasma membrane integrity, viability/acrosome integrity, viability/mitochondrial transmembrane potential, DNA fragmentation/integrity, and morphology were analyzed by phase-contrast microscope, supravital hypoosmotic swelling test, Trypan blue/Giemsa staining, propidium iodide/"5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide" (JC-1) fluorochromes, neutral comet assay/acridine orange assay and wet mount technique, respectively. Outcome of 528 inseminations was analyzed for in vivo fertility. Pearson's correlation coefficients revealed that sperm progressive motility (%), rapid velocity (%), average path velocity (µm/s), straight line velocity (µm/s), subpopulation one (most rapid, and progressive) of motile spermatozoa (%), supravital plasma membrane integrity (%), and viable spermatozoa with intact acrosome (%) were significantly correlated with in vivo fertility (r = 0.64, P < 0.01; r = 0.57, P < 0.01; r = 0.52, P < 0.01; r = 0.56, P < 0.01; r = 0.73, P < 0.001; r = 0.74, P < 0.001; r = 0.88, P < 0.001); whereas nonviable spermatozoa with damaged acrosome or low-mitochondrial transmembrane potential and comet length (µm) of neutral comet assay were negatively associated with in vivo fertility (r = -0.79, r = -0.75, P < 0.001, and r = -0.60, P < 0.05, respectively). Multiple regression analysis reported that combination of semen quality parameters as predictor of fertility were better (R(2) adjusted = 81.30%, P < 0.001) as compared with single parameter (R(2) adjusted = 50.20%, P < 0.007). It is concluded that assessment of CASA parameters and some other sperm structural and functional parameters, that is, integrity of plasma membrane and acrosome, and transmembrane potential of mitochondria were able to predict the in vivo fertility of water buffalo bull during low-breeding season.

Effect of equilibration times, freezing, and thawing rates on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa.

The effects of equilibration times (E1, 2 h; E2, 4 h; E3, 6 h), freezing rates (FR1, manual, 5 cm above liquid nitrogen (LN2 ) for 10 min, plunging in LN2 ; FR2, programmable ultra-fast, holding at +4 °C for 2 min, from 4 to -10 °C at -10 °C/min, from -10 to -20 °C at -15 °C/min, from -20 to -120 °C at -60 °C/min, holding at -120 °C for 30 sec, plunging in LN2 ), and thawing rates (T1, 37 °C for 30 sec; T2, 50 °C for 15 sec; T3, 70 °C for 7 sec) were evaluated on quality of buffalo bull spermatozoa. Progressive motility (%), rapid velocity (%), average path velocity (VAP, µm/s), straight line velocity (VSL, µm/s), and mitochondrial transmembrane potential (%) were higher (p < 0.05) with E2, FR2, and T3 compared to other groups. Sperm curved line velocity (VCL, µm/s) was higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm straightness (%) and linearity (LIN, %) were higher (p < 0.05) with E2 compared to other groups. Sperm LIN was affected (p < 0.05) with T3 compared to other groups. Supravital-plasma membrane integrity (%), viability and acrosome integrity (%) of spermatozoa were higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm DNA integrity (%) was higher (p < 0.05) with FR2 and T1 compared to other groups. We concluded that inclusion of 4 h-equilibration time, programmable ultra-fast freezing rate, and rapid thawing at 70 °C for 7 sec in cryopreservation protocol improves the post-thaw quality of buffalo bull spermatozoa.

Isolation of bacteria in semen and evaluation of antibiotics in extender for cryopreservation of buffalo (Bubalus bubalis) bull spermatozoa.

This study was designed to investigate the occurrence of bacterial species in water buffalo semen at the time of collection/processing and to assess the efficacy of some selected antibiotics (GTLS; gentamycin, tylosin and linco-spectin or SP; streptomycin and penicillin) in cryodiluent on bacterial control and quality including in vivo fertility of buffalo spermatozoa. For this purpose, four experiments were conducted. In experiment 1, a total of 11 bacterial species were isolated from buffalo ejaculates. In experiment 2, total aerobic bacterial counts at post dilution and thawing were lower (P < 0.05) in GTLS than in SP or control. The majority of the bacterial isolates from ejaculates were more susceptible to GTLS than SP. In experiment 3, sperm acrosome integrity was higher (P < 0.05) in GTLS and SP compared to control. In experiment 4, the in vivo fertility results for GTLS were higher (P < 0.05) than that for SP. In conclusion, a number of bacterial species were isolated from the bubaline semen, which requires an efficient control before its use in artificial insemination program. The GTLS combination of antibiotics may be incorporated into a freezing extender/protocol without compromising the post-thaw quality and in vivo fertility of buffalo bull spermatozoa.

l-Cysteine improves antioxidant enzyme activity, post-thaw quality and fertility of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa.

The effects of l-cysteine in extender on antioxidant enzymes profile during cryopreservation, post-thaw quality parameters and in vivo fertility of Nili-Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of l-cysteine (0.0, 0.5, 1.0, 2.0 and 3.0 mm) and frozen in 0.5-ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P < 0.05) at pre-freezing and post-thawing in extender containing 2.0 mm l-cysteine as compared to other groups. Post-thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (µm s-1 ), straight line velocity (µm s-1 ), curvilinear velocity (µm s-1 ), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0 mm l-cysteine as compared to other groups (P < 0.05). The fertility rates (59 versus 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mm of l-cysteine than in the control. In conclusion, the addition of 2.0 mm l-cysteine in extender improved the antioxidant enzymes profile, post-thaw quality and in vivo fertility of Nili-Ravi buffalo bull spermatozoa.

Polyoma small T antigen triggers cell death via mitotic catastrophe.

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint. Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.