Vibration Emissions Reduce Boar Sperm Quality via Disrupting Its Metabolism.

Artificial insemination (AI) with liquid-preserved semen has recently become common in pig breeding. The semen doses are produced in a centralized manner at the boar stud and then subsequently distributed and transported to sow farms. However, vibration emissions during transportation by logistic vehicles may adversely affect the quality of boar sperm. Therefore, this study aimed to explore the impact of vibration-induced emissions on sperm quality and function under simulated transportation conditions. Each time, ejaculates from all 15 boars were collected and then pooled together to minimize individual variations, and the sample was split using an extender for dilution. Different rotational speeds (0 rpm, 80 rpm, 140 rpm, 200 rpm) were utilized to simulate varying intensities of vibration exposure using an orbital shaker, considering different transportation times (0 h, 3 h, and 6 h). Subsequently, evaluations were conducted regarding sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial function, adenosine triphosphate (ATP) levels, mitochondrial reactive oxygen species (ROS) levels, pH, glycolytic pathway enzyme activities, and capacitation following exposure to vibration emissions. Both vibration time and intensity impact sperm motility, plasma membrane integrity, and acrosomal integrity. Vibration exposure significantly reduced sperm ATP levels, mitochondrial membrane potential, and the levels of mitochondria-encoded proteins (MT-ND1, MT-ND6) (p < 0.05). After vibration emission treatment, the pH value and mitochondrial ROS levels significantly increased (p < 0.05). Inhibition of sperm glycolysis was observed, with reduced activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), along with decreased lactate levels (p < 0.05). Additionally, sperm tyrosine phosphorylation levels were significantly reduced by vibration emissions compared to the control group (p < 0.05). After the vibration emission treatment, the number of sperm bound to each square millimeter of oviduct explants decreased significantly compared to the control group (p < 0.05). Similarly, compared to the control group, using semen subjected to vibration stress for AI results in significantly reduced pregnancy rates, total born litter size, live-born litter size, and healthy born litter size (p < 0.05).

Pyrroloquinoline Quinone Improves Ram Sperm Quality through Its Antioxidative Ability during Storage at 4 °C.

Sperm motility is an important factor in the migration of sperm from the uterus to the oviduct. During sperm preservation in vitro, sperm generates excessive ROS that damages its function. This study aims to investigate whether the addition of pyrroloquinoline quinone (PQQ) to the diluted medium could improve chilled ram sperm quality, and then elucidates the mechanism. Ram semen was diluted with Tris-citric acid-glucose (TCG) medium containing different doses of PQQ (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM), and stored at 4 °C. Sperm motility patterns, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) levels, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and ATP levels were measured after preservation. Furthermore, the expressions of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) in sperm were also detected by western blotting. In addition, sperm capacitation and the ability of sperm to bind to the zona pellucina were also evaluated. It was observed that the addition of PQQ significantly (p < 0.05) improved ram sperm motility, membrane integrity, and acrosome integrity during preservation. The percentage of sperm with high mitochondrial membrane potential in the PQQ treatment group was much higher than that in the control. In addition, supplementation of PQQ also decreased the sperm MDA and ROS levels, while increasing ATP levels. Interestingly, the levels of MT-ND1 and MT-ND6 protein in sperm treated with PQQ were also higher than that of the control. Furthermore, the addition of 100 nM PQQ to the medium decreased ROS damage in MT-ND1 and MT-ND6 proteins. The addition of 100 nM PQQ significantly (p < 0.05) increased protein tyrosine phosphorylation in ram sperm after induced capacitation. Furthermore, the value of the sperm-zona pellucida binding capacity in the 100 nM PQQ treatment group was also much higher than that of the control. Overall, during chilled ram- sperm preservation, PQQ protected ram sperm quality by quenching the ROS levels to reduce ROS damage and maintain sperm mitochondrial function, and preserved the sperm's high ability of fertilization.

Carboxylated ε-Poly-l-lysine Improves Post-Thaw Quality, Mitochondrial Functions and Antioxidant Defense of Goat Cryopreserved Sperm.

Carboxylated ε-poly-l-lysine (CPLL), a novel cryoprotectant, can protect the sperm membranes by inhibiting ice crystal formation during the cryopreservation process. The present study was conducted to investigate the consequence of CPLL supplementation on the post-thaw quality of cryopreserved goat sperm. For this, different doses (0, 0.5%, 1%, 1.5%, and 2%; v/v) of CPLL were added to the cryopreservation medium, and the motility, membrane and acrosome integrity, mitochondrial membrane potential (MMP), ATP level, ROS production, anti-oxidant defense system, malondialdehyde (MDA) level, and apoptosis in post-thaw sperm were evaluated. It was observed that the addition of 1% CPLL significantly (p < 0.05) increased the total motility, membrane integrity, acrosome integrity, and catalase (CAT) activity of post-thaw sperm compared to those of control and other CPLL doses. The ATP content was observed significantly (p < 0.05) higher in 0.5% and 1% CPLL, however, the SOD activity and progressive motility were significantly (p < 0.05) increased by adding CPLL at 1% and 1.5% level. Moreover, the addition of CPLL at 1% dose not only showed a lower percentage of apoptosis, but also significantly (p < 0.05) increased the MMP while reducing ROS production and MDA levels compared to those of other CPLL doses and/or control. Therefore, it is clear that the supplementation of 1% CPLL can remarkably improve the post-thaw goat sperm motility, membrane and acrosome integrity, antioxidant abundance, mitochondrial potentials, and ATP supply by protecting the sperm from cryodamage and undergoing apoptosis. These findings will provide novel insights into sperm cryobiology.

Retardation of oxidative rancidity in ghee adding orange peel powder at different storage temperature.

This study is aimed to determine and compare the antioxidant activity of Orange Peel Powder (OPP) in ghee at different temperatures (4 °C, 25 °C and 60 °C) for divergent storage periods (0, 7, 14 and 21 days). To compare the antioxidant potentiality, synthetic antioxidant BHA (Butylated Hydroxy Anisole) is used. Twelve ghee samples were prepared where one was control, another one was BHA treated and the rest ten were admixing OPP in ghee at different ratios. After sensory evaluation three highest scored ghee samples (0.5%. 1.0% and 1.5%) were selected. Samples were analyzed for peroxide (PV), thiobarbituric acid (TBA), free fatty acids (FFA) value and radical scavenging activity. Though storage temperature and storage period were increased OPP treated ghee samples peroxide, TBA and FFA values were lowered significantly compared to control samples. Moreover, 1.0% and 1.5% OPP treated ghee samples such values were lowered than BHA treated ghee samples and all these are on the favor of ghee quality. OPP treated ghee samples' DPPH quench potentiality is also stronger than BHA treated ghee samples. Therefore, OPP is a great source of antioxidants and this can be used in ghee as a natural source of antioxidants.

Beneficial Effect of Proline Supplementation on Goat Spermatozoa Quality during Cryopreservation.

Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.

Adverse effect of superoxide-induced mitochondrial damage in granulosa cells on follicular development in mouse ovaries.

High mitochondrial oxidative phosphorylation (mt-OXPHOS) levels are required to supply the ATP necessary for follicle-stimulating hormone (FSH)-induced granulosa cell proliferation during the follicular development process. Consequently, excessive reactive oxygen species (ROS) might be generated and have an adverse effect on follicular health. This study aimed to elucidate the negative effects of ROS on mitochondrial functions in FSH-stimulated granulosa cells during the follicular development process and to investigate whether pyrroloquinoline quinone (PQQ) treatment could accelerate this process by ameliorating the adverse effects. To do this, both in vitro and in vivo experiments were performed with granulosa cells from superovulated immature (3-week-old) mice that were pretreated with or without PQQ, and a natural mating study was also performed. The ROS level in FSH-/eCG-stimulated granulosa cells was significantly increased. Moreover, high oxidative stress and mtDNA damage levels were evident in the granulosa cells. PQQ treatment not only reduced the ROS and oxidative stress levels but also ameliorated mtDNA damage, accelerated FSH-/eCG-induced ATP production, and increased the mitochondrial membrane potential and the expression levels of mitochondrial genes (Nd1, Cytb, Cox1, ATPase6) and the mt-ND1 protein. Accordingly, the proliferation and viability of granulosa cells, numbers of healthy preovulatory follicles and ovulated oocytes and serum estrogen level were significantly improved, while the apoptosis of granulosa cells was reduced. However, PQQ treatment did not change the fertility parameters in mature mice with natural cycles but did significantly increased the number of offspring born per delivery. These results revealed that ROS-associated damage in FSH-stimulated granulosa cells adversely affects their physiology and follicular health during the follicular development process. Treatment with PQQ is a beneficial tool to increase both the number of ovulated oocytes and pups per delivery.

Exogenous Oleic Acid and Palmitic Acid Improve Boar Sperm Motility via Enhancing Mitochondrial Β-Oxidation for ATP Generation.

: It takes several hours for mammalian sperm to migrate from the ejaculation or insemination site to the fertilization site in the female reproductive tract in which glucose, amino acids, and fatty acids are regarded as the primary substrates for ATP generation. The present study was designed to investigate whether oleic acid and palmitic acid were beneficial to boar sperm in vitro; and if yes, to elucidate the mechanism that regulates sperm motility. Therefore, the levels of oleic acid and palmitic acid, motility, membrane integrity, acrosome integrity, and apoptosis of sperm were evaluated. Moreover, the enzymes involved in mitochondrial ß-oxidation (CPT1: carnitine palmitoyltransferase 1; ACADVL: long-chain acyl-coenzyme A dehydrogenase) were detected with immunofluorescence and Western blotting. Consequently, the ATP content and the activities of CPT1, ACADVL, malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) were also measured. We observed that CPT1 and ACADVL were expressed in boar sperm and localized in the midpiece. The levels of oleic acid and palmitic acid were decreased during storage at 17 °C. The addition of oleic acid and palmitic acid significantly increased sperm motility, progressive motility, straight-line velocity (VSL), membrane integrity, and acrosome integrity with a simultaneous decrease in sperm apoptosis after seven days during storage. When sperm were incubated with oleic acid and palmitic acid at 37 °C for 3 h, the activities of CPT1 and ACADVL, the ATP level, the mitochondrial membrane potential, the activities of MDH and SDH, as well as sperm motility patterns were significantly increased compared to the control (p﹤0.05). Moreover, the addition of etomoxir to the diluted medium in the presence of either oleic acid or palmitic acid and the positive effects of oleic acid and palmitic acid were counteracted. Together, these data suggest that boar sperm might utilize oleic acid and palmitic acid as energy substrates for ATP production via ß-oxidation. The addition of these acids could improve sperm quality.

Resveratrol Improves Boar Sperm Quality via 5'AMP-Activated Protein Kinase Activation during Cryopreservation.

Mammalian sperm is highly susceptible to the reactive oxygen species (ROS) stress caused by biochemical and physical modifications during the cryopreservation process. 5'AMP-activated protein kinase (AMPK) is involved in regulating both cell metabolism and cellular redox status. The aim of the present study was to investigate whether the resveratrol protects boar sperm against ROS stress via activation of AMPK during cryopreservation. Boar sperm was diluted with the freezing medium supplemented with resveratrol at different concentrations (0, 25, 50, 75, 100, and 125 µM). It was observed that the addition of 50 µM resveratrol significantly improved the postthaw sperm progressive motility, membrane integrity, acrosome integrity, mitochondrial activity, glutathione (GSH) level, activities of enzymatic antioxidants (glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase), and the phosphorylation of AMPK. Meanwhile, the lipid peroxidation, ROS levels, and apoptosis of postthaw sperm were reduced in the presence of 50 µM resveratrol. Furthermore, when fresh boar sperm was incubated with the medium in the presence of 50 µM resveratrol and 30 µM Compound C (an AMPK inhibitor), the effects of the resveratrol were partly counteracted by the Compound C. These observations suggest that the resveratrol protects boar sperm via promoting AMPK phosphorylation. In conclusion, the addition of resveratrol to the freezing extenders protects boar sperm against ROS damage via promoting AMPK phosphorylation for decreasing the ROS production and improving the antioxidative defense system of postthaw sperm. These findings provide novel insights into understanding the mechanisms of resveratrol on how to protect boar sperm quality contrary to the ROS production during cryopreservation.

Glycogen Synthase Kinase-3 Regulates Sperm Motility and Acrosome Reaction via Affecting Energy Metabolism in Goats.

Hyperactivation and acrosome reaction of sperm are pre-requisite steps for fertilization. However, the hyperactivation and acrosome reaction are critically controlled through the phosphorylation of specific proteins. Glycogen synthase kinase-3 (GSK3), a serine/threonine kinase with two different isoforms (α and ß), is involved in biochemical signaling pathways. This study was aimed to investigate whether the GSK3α/ß is present in goat sperm and its regulatory role in sperm motility and acrosome reaction. GSK3α/ß was detected with immunofluorescence and Western blotting. Sperm motility, membrane integrity, acrosome reaction, mitochondrial membrane potential, phospho-Ser21-GSK3α and phospho-Ser9-GSK3ß were analyzed. The ATP production and activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) were measured. It was observed that the GSK3α/ß was expressed in goat sperm, especially in the peri-acrosomal, mid-piece and principal piece of the tail. The abundance of GSK3α/ß in sperm was increased during transit along the epididymis. Addition of either 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or CHIR99021 significantly increased the sperm motility patterns and GSK3α/ß phosphorylation. Interestingly, the adenosine triphosphate (ATP) production, activities of LDH, MDH and SDH were observed to be increased in the CHIR99021 treatment. The results suggested that GSK3α/ß regulates sperm motility and acrosome reaction via phospho-ser21-GSK3α and phospho-ser9-GSK3ß that involved in the regulation of sperm energy metabolism.

Negative effects of ROS generated during linear sperm motility on gene expression and ATP generation in boar sperm mitochondria.

Mitochondrial oxidative phosphorylation (OXPHOS) is essential for ATP production to maintain sperm linear motility during migration from the uterus to the oviduct. However, ROS are generated as by-products of OXPHOS, causing stress and damaging the sperm quality. This study aimed to clarify the ROS targets in sperm mitochondria that decrease linear motility and to investigate whether mitochondria-target antioxidants (PQQ and CoQ10) affect mitochondrial activity and sperm motility. Sperm linear motility pattern, ATP production, and mitochondrial activity were decreased with increasing ROS levels during incubation in the low-glucose medium. However, sperm motility patterns and ROS levels were not significantly changed in the high-glucose medium. Moreover, the gene expression system (mt-DNA, mitochondrial transcription factor-A (TFAM) and RNA polymerase (POLRMT)) in sperm mitochondria was damaged during incubation in the low-glucose medium. Interestingly, PQQ treatment increased the mt-DNA stability and decreased the damage to TFAM and POLRMT, which resulted in high expression of mitochondrial genes. Furthermore, the antioxidants increased mitochondrial activity and maintained sperm linear motility under the low glucose condition. These results revealed that both ATP production and the mitochondrial transcription system are damaged with increasing ROS levels in sperm that show a linear motility pattern. Treatment with antioxidants, such as PQQ and CoQ10, is beneficial tool to maintain sperm linear motility.

Gene Expression and Protein Synthesis in Mitochondria Enhance the Duration of High-Speed Linear Motility in Boar Sperm.

Sperm motility patterns are continuously changed after ejaculation to fertilization in the female tract. Hyperactivated motility is induced with high glucose medium in vitro or the oviduct fluids in vivo, whereas sperm maintain linear motility in the seminal plasma or the uterine fluids containing low glucose. Therefore, it is estimated that sperm motility patterns are dependent on the energy sources, and the mitochondrial oxidative phosphorylation is activated to produce ATP in low glucose condition. To elucidate these hypotheses, boar sperm was incubated in different energy conditions with the transcription and translation inhibitors in vitro. Sperm motility parameters, mitochondrial activity, ATP level, gene expression and protein synthesis were analyzed. Sperm progressive motility and straight-line velocity were significantly increased with decreasing glucose level in the incubation medium. Moreover, the mitochondrial protein turnover meaning transcription and translation from mitochondrial genome in sperm is activated during incubation. Incubation of sperm with mitochondrial translation inhibitor (D-chloramphenicol) suppressed mitochondrial protein synthesis, mitochondrial activity and ATP level in sperm and consequently reduced the linear motility speed, but not the motility. Thus, it is revealed that the mitochondrial central dogma is active in sperm, and the high-speed linear motility is induced in low glucose condition via activating the mitochondrial activity for ATP generation.

Mitochondrial Protein Turnover Is Critical for Granulosa Cell Proliferation and Differentiation in Antral Follicles.

Granulosa cell (GC) proliferation is essential for follicular development. FSH is a key factor in GC proliferation, and a continuous supply of high levels of ATP is necessary for cell proliferation. However, genes encoding proteins of the glycolytic pathways are poorly expressed in GCs. Therefore, we hypothesized that mitochondrial gene expression and protein synthesis play a primary role in ATP production during GC proliferation. To test this hypothesis, we performed an in vivo study of GCs collected from 23-day-old mice ovaries with or without equine chorionic gonadotropin (eCG) priming. It was observed that mitochondrial activity with membrane potential, expression of protein-coding genes (Nd1-6, Cytb, Atpase6,8) and transcription-related genes (Polrmt, Tfam, Tfb2m), copy number of mitochondrial (mt-)DNA, and protein synthesis were increased in GCs after 24 hours of eCG injection and mostly maintained elevated up to 48 hours. Therefore, we performed in vitro culture of GCs in DMEM medium supplemented with FSH, testosterone, and serum and containing different glucose concentrations with or without d-chloramphenicol (CRP) for 24 hours. GC proliferation and ATP production were observed to be independent of glucose concentration. Furthermore, FSH-induced mitochondrial activity with membrane potential, ATP content, BrdU-incorporated cell proliferation, intensity of mt-ND1 and mt-ND6 proteins, and expressions of marker genes for proliferation and differentiation were significantly decreased by CRP treatment. These results revealed the crucial role of mitochondria in the supply of ATP and the necessity of mitochondrial gene expression and protein synthesis in not only the proliferation but also the differentiation of GCs during follicular development.

Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice.

It has been known that EGF-like factor secreted from LH-stimulated granuloma cells acts on granulosa cells and cumulus cells to induce ovulation process. Granulosa cells are changed the morphology with differentiating cell functions to produce progesterone. Cumulus cells are detached to make a space between the cells to accumulate hyaluronan rich matrix. LH also changes extracellular matrix (ECM) components including fibronectin in the follicular walls and granulosa cell layers. EGF like factor and fibronectin synergistically play important roles in numerous cell functions, especially cancer cell migration, estimating that fibronectin would impact on granulosa cells and cumulus cells. To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. The functions were monitored by the detection of downstream signaling pathway, focal adhesion kinase (FAK). The pharmacological approach in both in vivo and in vitro were used for analyzing the physiological roles of FAK during ovulation process. The immunofluorescence staining revealed that fibronectin and integrin were observed in granulosa cells, cumulus cells and the space between cumulus cells and oocyte at 4 and 8 h after hCG injection. Concomitantly with the changes of fibronectin-integrin localization, FAK was phosphorylated in periovulatory follicles. The injection of FAK inhibitor suppressed not only ovulation but also luteinization of granulosa cells and cumulus expansion. In cultured-granulosa cells, fibronectin-integrin synergistically activated FAK with amphiregulin (AREG). Such cooperative stimulations induced a morphological change in granulosa cells, which resulted in the maximum level of progesterone production via the induction of Hsd3b. When cumulus-oocyte complexes (COCs) were cultured with AREG in the presence of serum, the maximum level of cumulus expansion was observed. The AREG-induced cumulus expansion was also suppressed by FAK inhibitor. Thus, it is concluded that fibronectin and AREG synergistically activate FAK not only in granulosa cells and cumulus cells to induce successful ovulation process.