Comparative evidence support better antioxidant efficacy of mitochondrial-targeted (Mitoquinone) than cytosolic (Resveratrol) antioxidant in improving in-vitro sperm functions of cryopreserved buffalo (Bubalus bubalis) semen.

The present study compared the effect of mitochondria-targeted (Mitoquinone, MitoQ) and untargeted cytosolic antioxidant (Resveratrol, RESV) supplementation on lipid peroxidation (LPO) and in-vitro sperm functions of cryopreserved buffalo bull semen. To optimize additive's concentration, sperm pellet obtained from twenty-four ejaculates was supplemented with different concentrations of MitoQ (20 nM, 100 nM, 200 nM); and RESV (10 μM, 25 μM, 50 μM) against control in the extender. The post-thaw sperm motility, livability, and membrane integrity were higher (P < 0.05) in 200 nM MitoQ and 50 μM RESV than other concentrations used. In another experiment, sperm pellet from thirty-two ejaculates was supplemented with 200 nM MitoQ and 50 μM RESV in the extender. Pre-freeze and post-thaw progressive motility and livability were higher (P < 0.05) in MitoQ (200 nM) than RESV (50 μM) treatment. MitoQ supplementation improved post-thaw membrane integrity (CFDA-PI) higher (P < 0.05) than RESV, however, hypo-osmotic swelling response observed no improvement with RESV treatment. Post-thaw LPO rate was lower (P < 0.05) and Bovine cervical mucus penetration was higher (P < 0.05) in MitoQ than RESV treatment. In post-thaw semen, MitoQ showed higher (P < 0.05) proportion of acrosome intact (FITC-PNA), live non-apoptotic (P < 0.01) sperm with a higher reduction (P < 0.05) in membrane scrambling. MitoQ improved (P < 0.01) proportion of sperm with high Mitochondrial Membrane Potential and low LPO (P < 0.01) than RESV treatment. In conclusion, improvement in post-thaw in-vitro sperm functions and cryo-tolerance was more evident in MitoQ than RESV supplemented buffalo bull semen. Our study provides a better strategy to mitigate oxidative stress by enhancing mitochondrial antioxidant system with targeted antioxidants than cytosolic antioxidant supplementation.

Tiwari S ，Mohanty TK ，Bhakat M ，Kumar N ，Baithalu RK ，Nath S ，Yadav HP ，Dewry RK ... -  《-》

The addition of resveratrol in tris citric acid extender ameliorates post-thaw quality parameters, antioxidant enzymes levels, and fertilizing capability of buffalo (Bubalus bubalis) bull spermatozoa.

Resveratrol is a natural grape-derived polyphenol with potent antioxidant properties that protect spermatozoa against lipid peroxidation (LPO) by eradicating free radicals. The objectives of this study were to 1) appraise the effects of resveratrol in extender on post-thaw quality parameters, antioxidant enzymes, adenosine triphosphate (ATP), DNA fragmentation, LPO and 2) fertilizing capability of buffalo bull spermatozoa. Semen was collected from four fertility proven bulls with artificial vagina thrice, evaluated initially, and diluted in five different extenders containing resveratrol (T4 = 100 μM, T3 = 50 μM, T2 = 20 μM, T1 = 10 μM), and control (no resveratrol), and evaluated after post-dilution and post-thawing stage of cryopreservation. Analysis of variance revealed that, there was no difference (P > 0.05) in any parameters due to treatments at post-dilution. However, at post-thawing, sperm progressive motility (%), plasma membrane integrity (%), mitochondrial membrane potential (%) and ATP levels (nmol/106) were found higher in semen samples cryopreserved in T3 and 4 than control. Sperm supravital plasma membrane integrity (%) and viable/acrosome integrity were found higher in semen samples cryopreserved in T4 than control and T1. Furthermore, sperm catalase (U/mL), glutathione peroxidase (μM) and superoxide dismutase (U/mL) concentrations were found significantly higher in resveratrol treated groups as compared to the control. Conversely, DNA fragmentation (%) and LPO (μM/mL) were significantly (P > 0.05) decreased in semen samples cryopreserved in T4 in comparison to the control. Fertilizing capability was found higher in semen samples cryopreserved in T4 as compared to the control (%, 77.33 vs. 57.41, P < 0.05). It is concluded that the addition of resveratrol in extender ameliorates quality parameters, antioxidant enzymes levels and fertilizing capability, and alleviate DNA fragmentation and LPO in buffalo spermatozoa during cryopreservation.

Ahmed H ，Jahan S ，Ullah H ，Ullah F ，Salman MM ... -  《-》

Effect of naringenin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Our objectives were to explore the effect of naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 μM naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 μM naringenin compared to 200 μM naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150 μM naringenin compared to all groups except 100 μM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 μM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 μM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 μM naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03 % (114/200) vs. 46.50 ± 0.04 % (93/200), respectively]. Taken together, it is concluded that naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 μM concentration.

Khan GS ，Tahir MZ ，Zahoor MY ，Hifz-Ul-Rahman ，Riaz A ... -  《-》

Effect of graphene oxide as cryoprotectant on post-thaw sperm functional and kinetic parameters of cross bred (HF X Sahiwal) and Murrah buffalo (Bubalus bubalis) bulls.

Graphene oxide (GO) greatly suppresses the growth and recrystallization by curving the hexagonal shape of ice crystals. Study was conducted to evaluate effect of GO as cryoprotectant in semen extender for augmenting sperm viability in dairy (cattle and buffalo) animals. In experiment one, semen was extended with TRIS Egg Yolk Glycerol (TEYG) extender supplemented with different concentrations of GO: 0.0125, 0.25, 0.5, 0.1 and 0.2 mg ml-1. Freezing of semen samples was conducted at 30 °C min-1 from temperature drop from 4 °C to -15 °C and -15 °C to - 60 °C followed by 50 °C min-1 from - 60 °C to -140 °C, and the semen straws were plunged in liquid nitrogen. Second experiment evaluated the performance of TEYG extender supplemented with combinations of GO (G05 as 0.05 and G10 as 0.1 mg ml-1) and glycerol (T48 as 4.8 and T64 as 6.4%) in four groups as G05T48, G05T64, G10T48 and G10T64. Freezing rates of 30 °C min-1[Protocol (PRT) I], 40 °C min-1 (PRT II) and 50 °C min-1 (PRT III) in the critical temperature fall zone of -15 °C to -60 °C were evaluated for semen extender supplemented with glycerol 6.4% and GO 0.05 mg ml-1 in the third experiment. Cattle (n = 3) and buffalo (n = 3) bulls were chosen for the study taking six ejaculates per bull per treatment. Post-thaw sperm motility, membrane integrity, viability and abnormalities were observed by means of CASA, Hypo-osmotic swelling test (HOST), Eosin-Nigrosin stain and Rose Bengal stain procedures, respectively. Post-thaw total motility (TM), progressive motility (PM), VCL, VSL, VAP, HOST response and viability increased significantly in extender with GO concentrations of 0.1 and 0.05 mg ml-1 as compared to control. Per cent abnormalities were significantly (p < .05) lower in group with GO 0.025 and 0.0125 mg ml-1 as compared to control. Results from the second experiment showed higher post-thaw TM, PM, VCL, VAP, VSL, HOST response, viability increased significantly (p < .05) in G05T64 and G05T48 as compared to G10T64. Sperm abnormalities did not vary among the groups as compared to control for cattle spermatozoa. In the third experiment post-thaw TM, PM, VCL, VSL, VAP, HOS response and sperm viability increased significantly (p < .05) in PRT III as compared to PRT I for buffalo and cattle spermatozoa. Sperm abnormalities were significantly (p < .05) lower in PRT II and PRT III as compared to PRT I for buffalo, whereas, lower in PRT II as compared to PRTI for cattle spermatozoa. GO as cryoprotectant when added to semen extender at the rate of 0.05 and 0.1 mg ml-1, resulted in better plasma membrane function and viability. Glycerol concentration below 6.4% in buffalo semen extender reduced post-thaw quality of sperm even when GO was added to the extender. Higher freezing rate of 50 °C min-1 in the critical temperature fall zone of -15 to -60 °C perform better than the freezing rate of 30 °C min-1. It is concluded that TEYG extender having glycerol 6.4% and GO 0.05 mg ml-1 improved post-thaw semen quality of cattle and buffalo.

Singh P ，Bedi MK ，Singhal S ，Singh AK ，Kumar A ，Honparkhe M ... -  《-》

Effect of Milk Type Subjected to Different Heat Treatments on Cryo-Survivability and In Vivo Fertility of Buffalo (Bubalus bubalis) Spermatozoa in a Milk-Based Extender.

Tariq HA ，Tariq A ，Ahmad N ，Nadeem M ，Riaz A ... -  《-》