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 ... -  《-》

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

Buffalo bull sperm suffer more cryoinjuries due to lipid peroxidation of high structural polyunsaturated fatty acid contents than cattle sperm. Consequently, the post-thaw fertilization potential of buffalo bull sperm is compromised. Crocin is a carotenoid known for its antioxidant potential through scavenging reactive oxygen species. Objectives of the current study were to investigate the effect of crocin addition in the semen extender on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm. Semen samples (n = 32) from four Nili-Ravi buffalo bulls were extended with tris-citric acid extender containing different concentrations of crocin (0 mM; control, 0.5, 1, 1.5 and 2 mM). The extended semen was packed in 0.5 mL French straws (25 × 106 sperm/straw) and cryopreserved in liquid nitrogen. 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 integrity, 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. The fertility of semen doses containing the most potent concentration of crocin (based on optimum post-thaw semen quality) was compared with control during the breeding season. Buffaloes (n = 400; 200/group) were inseminated 24 h after the onset of oestrus and transrectally palpated for pregnancy at least 60 days post-insemination. Results revealed that 0.5 and 1 mM crocin improved sperm post-thaw total motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential and viability, and 1 and 1.5 mM crocin enhanced catalase activity and reduced lipid peroxidation compared to control (p < .05). Moreover, 1 mM crocin improved sperm post-thaw progressive motility, kinematics, and DNA integrity, and 1.5 mM crocin enhanced plasma membrane integrity than control (p < .05). Expression levels of SPACA3, PRM1 and BCL2 genes were higher (p < .05) with 1 mM crocin compared to other groups. In contrast, no difference (p > .05) was noticed in expressions of BAX and ROMO1 genes among all groups. The fertility rate of semen doses containing the most potent concentration (1 mM) of crocin was higher (p = .0465) compared to control (56 ± 0.03% vs. 46 ± 0.04%, respectively). In conclusion, 1 mM crocin in the semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm.

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

Effect of carboxylated poly l-Lysine as a cryoprotectant on post-thaw quality and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull semen.

Buffalo bull sperm are more prone to cryo-injuries. Glycerol being the most common permeable cryoprotectant exerts cytotoxic effects on sperm which cause a reduction in fertility. Thus, the exploration of new cryoprotectant is needed. For this purpose, we investigated the effect of carboxylated poly l-Lysine (CPLL) as cryoprotectant used with different concentrations of glycerol on post-thaw sperm motility, kinematics, plasma membrane integrity, mitochondrial membrane potential (MMP), lipid peroxidation (LPO), catalase concentration and in vivo fertility of Nili Ravi buffalo bull semen. In experiment 1, semen samples (n = 15, bulls = 3) were diluted with Tris-citrate-egg yolk extender containing different concentration of CPLL [0% (C0), 0.25% (C0.25), 0.5% (C0.5), 0.75% (C0.75), 1% (C1)]. Each concentration of CPLL was added in extender containing either 7% (G7) or 5% (G5) glycerol. Diluted semen samples were cooled and cryopreserved using standard procedures. Post-thaw total and progressive motility, plasma membrane integrity, acrosome integrity, and MMP were found higher (P < 0.05) in group (G5C0.75) containing 0.75% CPLL and 5% glycerol as compared to the control group (G7C0) and other groups while LPO was recorded lower (P < 0.05) in the same group (G5C0.75). In experiment 2, in vivo fertility was compared between G5C0.75 (5% Glycerol+ 0.75% CPLL; depicted better post-thaw quality) and control group G7C0. Buffaloes were inseminated after 24 h of onset of estrus. Pregnancy diagnosis was performed per rectum at least 60 days post insemination. The fertility rates [56% (58/102) vs. 36% (37/103)] were higher (P < 0.05) in G5C0.75 as compared to the control group G7C0. Based upon these results, this study concludes that the addition of 0.75% CPLL in combination with 5% glycerol in freezing extender improves the post-thaw structure, function and in vivo fertility of Nili Ravi buffalo bull semen.

Tariq A ，Ahmad M ，Iqbal S ，Riaz MI ，Tahir MZ ，Ghafoor A ，Riaz A ... -  《-》

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.

Iqbal S ，Andrabi SMH ，Riaz A ，Durrani AZ ，Ahmad N ... -  《-》

Ostrich egg yolk improves post thaw quality and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa.

Egg yolk containing a higher ratio of phospholipids and cholesterol may have better cryoprotective effect to buffalo spermatozoa during cryopreservation. Our objectives were to ascertain the comparison of Ostrich and chicken egg yolk in semen extender on post thaw quality, motion dynamics and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 45) from five bulls were collected once a week for a period of nine weeks and diluted in Triladyl® extender having different concentrations of Ostrich egg yolk (10%, 15%, 20%) and 20% chicken egg yolk as control at 37 °C. Diluted semen samples were frozen in 0.54 mL French straws with programmable freezer. Post thaw sperm progressive motility (%), morphology (%), average path velocity (μm/s), straight line velocity (μm/s), Linearity (%), straightness (%), length of straight line path (μm), plasma membrane integrity (%), acrosome membrane integrity (%), DNA integrity (%) and mitochondrial activity were higher (P < 0.05) in spermatozoa cryopreserved in extender containing 20% Ostrich egg yolk as compared to 20% chicken egg yolk and other groups. The fertility rates (67.61% vs 54.2%) were higher (P < 0.05) in buffaloes inseminated with semen doses frozen in extender containing 20% Ostrich egg yolk than the 20% chicken egg yolk. It is concluded that 20% Ostrich egg yolk in extender improves post thaw semen quality, motion dynamics and in vivo fertility in Nili Ravi buffaloes.

Naz S ，Umair M ，Iqbal S 《-》