Combination of trehalose and low boron in presence of decreased glycerol improves post-thawed ram sperm parameters: A model study in boron research.

BACKGROUND: Sperm cryopreservation has been widely used in the field of reproductive biotechnology. It applies to certain males of economic and scientific values, including livestock breeds or endangered animal species. The development of a semen extender with a low cryoprotectant concentration and an appropriate amount of trehalose and boron can prevent the deterioration of sperm parameters. OBJECTIVE: The main goal of this study is to establish a suitable ram extender model, by examining different combinations of high (5%) and low (3%) glycerol concentrations (to reduce its toxic effects on sperm freezing), a fixed amount of trehalose and an increased dose of boron to prevent the deterioration of sperm parameters, and investigate the levels of gene expressions. MATERIALS AND METHODS: The Merino ram ejaculates were collected. The collected ejaculates providing the defined criteria were pooled. The pooled ejaculates were divided into eight aliquots and diluted with the Tris extender including different combinations of glycerol (5% and 3%) and boron (0.25, 0.5, and 1 mm) concentrations and a fixed amount of trehalose, then frozen. After freeze-thawing process, sperm motility, mitochondrial membrane activity, plasma membrane integrity, acrosomal membrane integrity, DNA damage (single cell gel electrophoresis (COMET) and TUNEL assays) as well as NAD(P)H quinone oxyreductase (NQO1), glutamate-cycteine ligase (GCLC), and glutathione S-transferase (GSTP1) for molecular mechanisms of sperm cell response to oxidative stress were assessed for different extender groups following freeze-thawing process: 5% glycerol + 0 mm boron (G5B0.00), 5% glycerol + 0.25 mm boron (G5B0.25), 5% glycerol + 0.5 mm boron (G5B0.50), 5% glycerol + 1 mm boron (G5B1.00), 3% glycerol + 0 mm boron (G3B.00), 3% glycerol + 0.25 mm boron (G3B0.25), 3% glycerol + 0.5 mm boron (G3B0.50), and 3% glycerol + 1 mm boron (G3B1.00). RESULTS: G3B0.25 presented higher percentages of subjective motility, mitochondrial activity, and viability of spermatozoa comparing with G5B0.00 and groups with boron. Supplementation of 0.25 mm boron with and without trehalose (G3B0.25 and G5B0.25) showed higher acrosome integrity, compared with G5B0.00, G5B1.00, G3B0.50, and G3B1.00. For TUNEL analysis, G3B1.00 showed the highest DNA integrity among the experimental groups which was statistically significant only with G5B0.50 (p < 0.05). The mRNA levels of NQO1 were significantly decreased in G5B1.00, G3B0.50, and G3B1.00, when compared to G5B0.00. In comparison with G5B0.00, supplementation of 1 mm boron with and without trehalose had significantly lower expression of GCLC. The level of GSTP1 gene was significantly lower (approximately threefold) in G3B1.00, compared to G5B0.00 (p < 0.05). DISCUSSION AND CONCLUSION: It can be assumed that the increase of the boron concentration in the extender may have important adverse effects on sperm parameters and antioxidant gene expression after thawing. The results obtained from this study will help to understand the toxicity limits of boron and eliminate the toxicity of glycerol in studies of gametes and tissue freezing. Therefore, it can be concluded that the use of sufficient boron can decrease cryodamages of cryopreservation of mammalian spermatozoa as well tissue engineering.

Expression patterns of major genes in fatty acid synthesis, inflammation, oxidative stress pathways from colostrum to milk in Damascus goats.

The molecular regulation of milk secretion and quality in the transition period from colostrum to milk in goats is largely unknown. In the present study, mammary gland secretion of goats was collected in 0th, 4th, 7th, 14th and 28th days after parturition. In addition to composition and fatty acid profile of colostrum or milk, FASN, SCD, ACACA, COX-2, NRF2, TLR2, NF-kB, LTF and PTX3 genes expression patterns were determined from milk somatic cells. While somatic cell count (SCC), malondialdehyde (MDA), fat, fat-free dry matter, protein and lactose were highest as expression levels of the oxidative and inflammatory genes, freezing point and electrical conductivity were lowest in colostrum. With the continuation of lactation, most of the fatty acids, n3 ratio, and odour index increased but C14:0 and C16:0 decreased. While FASN was upregulated almost threefolds in 14th day, ACACA was upregulated more than fivefolds in 7th and 14th days. Separately, the major genes in fatty acid synthesis, inflammation and oxidative stress were significantly associated with each other due to being positively correlated. MDA was positively correlated with SCC and some of the genes related inflammation and oxidative stress. Furthermore, significant negative correlations were determined between SCC and fatty acid synthesis related genes. With this study, transition period of mammary secretion was particularly clarified at the molecular levels in Damascus goats.

Combination of fetuin and trehalose in presence of low glycerol has beneficial effects on freeze-thawed ram spermatozoa.

BACKGROUND: Freeze-thawing process negatively affects ram spermatozoa in terms of sperm quality, DNA integrity and antioxidant defence system. Thus, antioxidant supplementation of spermatozoa during freeze-thawing is suggested to improve sperm parameters. OBJECTIVES: The aim of this study was to determine the effects of fetuin and trehalose added into ram semen extender on sperm parameters, antioxidant parameters, antioxidant-related gene expressions and DNA integrity during the freeze-thawing process, in low glycerol concentration. METHODS: Semen samples collected from six mature rams were pooled and splitted into equal aliquots and diluted with a tris-based extender containing different concentrations of glycerol (G5; %5 and G3; %3), fetuin (F; 2.5, 5 and 15 mg/mL) and trehalose (60 mm) as eight groups (G5F0, G5F2.5, G5F5, G5F15, G3F0, G3F2.5, G3F5 and G3F15). RESULTS: G3F5 group resulted in the highest motility, mitochondrial activity and viability and the lowest DNA fragmentation and DNA damage (p < 0.05). Also, G3F0 displayed considerably more cryoprotective effect compared with G5F0 group (p < 0.05) in terms of motility, mitochondrial activity and viability rates. Lipid peroxidation levels decreased in G5F5 group compared with G5F0 group (p < 0.05). The levels of total glutathione increased in G3F2.5 group (p < 0.05) in comparison with the G5F0 group. NQO1 gene levels were upregulated approximately twofold in G5F5, G5F15, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). The levels of GCLC gene were approximately twofold higher in G3F0, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). GSTP1 gene levels were significantly higher with different levels in all treatment groups except for G5F2.5 and G3F0 groups in comparison with G5F0 group (p < 0.05). CONCLUSIONS: Co-supplementation of tris-based extender having low glycerol (3%) with trehalose and fetuin to enhance the quality of ram spermatozoa after freeze-thawing process is recommended.

Effect of essential oil supplementation to diet on meat quality, fatty acid composition, performance parameters and intestinal microbiota of Japanese quails.

The effect of essential oil (EO) supplementation on carcass characteristics of Japanese quails and interactions between ingredients and intestinal morphology were investigated in this study. A total of 250 quails were fed different diet: D1, basal diet (BD); D2, BD plus palmarosa oil (PO; 100 µg/kg diet); D3, BD plus lemon myrtle oil (LMO; 100 µg/kg diet); D4, BD plus α-Tops (mixture of α-terpineol, cineole and terpinene-4-ol; 100 µg/kg diet); and D5, BD plus cyclodextrin. Overall growth performance was determined at multiple time points during 35 days of experiment. Carcass characteristics (fatty acid, pH and colour), intestinal morphology and the expression levels of meat quality-related genes including the insulin-like growth factor (IGF-1), myogenin and avian uncoupling protein (avUCP) were examined at the end of the trial. Additionally, intestinal microbiome of quails was studied by next-generation sequencing-based culture-independent analysis. Although the inclusion of EOs into the diet had no effect on the growth performance of quails and the microbial profile, the significant changes in pH24 and colour (a*) of the quail's breast muscle (p < .05) in the group receiving PO were observed. Additionally, oleic acid content in the breast muscle was significantly higher in the EOs supplemented groups (p < .01). Quails fed the PO supplemented diet had higher villus and relatively rich in oleic acid. The expression levels of IGF-1 and myogenin genes in quail's muscle were not affected, but the expression of avUCP gene was significantly lower in quails fed with LMO and α-Tops (p < .05). The results demonstrated variable effects of these treatments on intestinal morphology. Taken together, dietary inclusion of EOs is found to be beneficial and hence can be recommended for improving the quality of poultry meat.

Decreasing glycerol content by co-supplementation of trehalose and taxifolin hydrate in ram semen extender: Microscopic, oxidative stress, and gene expression analyses.

This study aimed to evaluate the comparative effects of taxifolin hydrate and trehalose on the quality of frozen-thawed ram spermatozoa for the first time. Ejaculates collected from six mature rams were pooled, and divided to eight equal aliquots to extend them with different concentrations of glycerol (%5 and %3), taxifolin hydrate (10, 100, and 500 µM), and trehalose (60 mM) as eight groups (G5T0, G5T10, G5T100, G5T500, G3T0, G3T10, G3T100, and G3T500). After freeze-thawing process of cryopreservation, microscopic and oxidative stress parameters, and gene expression levels were investigated for understanding of possible impacts of taxifolin hydrate and trehalose. The study showed that G3T10 resulted in the highest post-thawed viability and mitochondrial activity. Moreover, all extenders with taxifolin hydrate reduced DNA fragmentation in comparison to G5T0, but DNA damage was prevented at the highest rate in presence of G5T10. The level of LPO significantly decreased in the groups G5T500 and G3T100, and the expression levels of NQO1, GCLC, and GSTP1 genes significantly increased in the groups G5T100, G5T500, G3T10, and G3T100 compared to the group G5T0. Finally, co-supplementation of tris-based extender having 3% glycerol with 60 mM trehalose and 10 µM taxifolin hydrate in cryopreservation extender may be recommended to improve the quality of post-thawed ram spermatozoa. However, further in vivo and in vitro studies are suggested to evaluate fertility rates of frozen-thawed ram spermatozoa co-supplemented with trehalose and taxifolin hydrate.

Dietary high calories from sunflower oil, sucrose and fructose sources alters lipogenic genes expression levels in liver and skeletal muscle in rats.

INTRODUCTION AND OBJECTIVES: The objectives of this study were to investigate the underlying mechanism of PPARα, LXRα, ChREBP, and SREBP-1c at the level of gene and protein expression with high-energy diets in liver and skeletal muscle. MATERIALS AND METHODS: Metabolic changes with consumption of high fat (Hfat), high sucrose (Hsuc) and high fructose (Hfru) diets were assessed. Levels of mRNA and protein of PPARα, LXRα, ChREBP, and SREBP-1c were investigated. Body weight changes, histological structure of liver and plasma levels of some parameters were also examined. RESULTS: In Hfru group, body weights were higher than other groups (P<0.05). In liver, LXRα levels of Hsuc and Hfru groups were upregulated as 1.87±0.30 (P<0.05) and 2.01±0.29 (P<0.01). SREBP-1c levels were upregulated as 4.52±1.25 (P<0.05); 4.05±1.11 (P<0.05) and 3.85±1.04 (P<0.05) in Hfat, Hsuc, and Hfru groups, respectively. In skeletal muscle, LXRα and SREBP-1c were upregulated as 1.77±0.30 (P<0.05) and 2.71±0.56 (P<0.05), in the Hfru group. Protein levels of ChREBP (33.92±8.84ng/mg protein (P<0.05)) and SREBP-1c (135.16±15.57ng/mg protein (P<0.001)) in liver were higher in Hfru group. In skeletal muscle, LXRα, ChREBP and SREBP-1c in Hfru group were 6.67±0.60, 7.11±1.29 and 43.17±6.37ng/mg, respectively (P<0.05; P<0.01; P<0.05). The rats in Hfru group had the most damaged livers. CONCLUSION: Besides liver, fructose consumption significantly effects skeletal muscle and leads to weight gain, triggers lipogenesis and metabolic disorders.

A randomized-controlled trial of prolotherapy injections in the treatment of plantar fasciitis.

OBJECTIVES: This study aims to evaluate the efficacy of prolotherapy injections for the treatment of plantar fasciitis. MATERIAL AND METHODS: Between October 2014 and October 2015, 60 patients with symptomatic chronic plantar fasciitis were randomly divided into two groups, as control (n=31) and prolotherapy (n=29) groups. In the prolotherapy group, ultrasound-guided prolotherapy injections into the plantar fascia up to five different points were performed three times every 21 days. In the control group, the patients received instructions for plantar fascia and Achilles tendon stretching exercises three times a week for three months. Additionally, all patients were given heel lifts and instructed to refrain from heavy loading activity. The patients were evaluated via the Visual Analog Scale (VAS), Food and Ankle Outcome Score (FAOS), and Foot Function Index (FFI) at baseline and at 21, 42, 90, and 360 days during follow-up. RESULTS: A total of 50 patients completed follow-up (26 patients in the prolotherapy group and 24 patients in the control group). The VAS, FAOS, and FFI scores were significantly improved in both groups (p<0.001). There were no statistically significant differences in the pain and functional scores at 21 days of treatment between the groups. The VAS and FAOS scores were higher in the prolotherapy group than the control group at 42, 90, and 360 days of treatment. The FFI scores were also higher in the prolotherapy group than the controls at 42 and 90 days of treatment; however, both groups had similar scores at 360 days. CONCLUSION: Our study results suggest that prolotherapy is an effective auxiliary method for treating chronic plantar fasciitis.

Evaluation and comparison of the effect of hypothermia and ozone on ischemia-reperfusion injury of skeletal muscle in rats.

BACKGROUND: Tourniquet-induced ischemia-reperfusion, which affects local and distant organs, is very common in orthopedic surgery. Hypothermia is used in traumatic tissue during ischemic period commonly. Ozone (O3) has been recommended as a novel therapeutic agent in various medical conditions. The objective of the study was to evaluate and compare the effect of hypothermia (H) and O3 on ischemia-reperfusion injury of skeletal muscle in rats by measuring oxidative parameters and inducible nitric oxide synthase (iNOS) levels. MATERIALS AND METHODS: Eighteen rats (Wistar albino) were separated into five groups randomly (sham, IR, IR + H, IR + O3, IR + H + O3; n = 6). The lower right extremity of all rats was subjected to 2 h of ischemia and 22 h of reperfusion clamping the common iliac artery and using the rubber-band technique at the level of the lesser trochanter under general anesthesia. Two hours of hypothermia were applied during the first 2 h of reperfusion in two groups. O3 was applied in two groups. All rats were sacrificed after the IR period with high dose of anesthesia. The tibialis anterior muscle and blood were saved. Levels of superoxide dismutase, glutathione peroxidase, MDA, NOx, and interleukin-1ß were measured in the muscle. Creatinine kinase, lactate dehydrogenase, aspartate aminotransferase, urea, creatinine, and electrolytes were measured in serum. Immunohistochemical iNOS staining was performed on muscle samples. RESULTS: The levels of MDA, NOx, and interleukin-1ß in muscle were raised in the IR group compared with those in the sham group. The same parameters were lower in the groups of IR + H, IR + O3, and IR + H + O3 compared with those in the IR group. Superoxide dismutase and glutathione peroxidase activities in muscle were lower in the IR group compared with those in the sham group; however, same parameters were higher in the groups of IR + H, IR + O3, and IR + H + O3 compared with those in the IR group. Score and intensity of iNOS staining in skeletal muscle in the IR group was increased compared with that in the sham group and decreased in the groups of IR + H, IR + O3, and IR + H + O3 compared with that in the IR group. Levels of creatinine kinase, aspartate aminotransferase, and K in the three treatment groups decreased compared with those in the IR group. CONCLUSIONS: These findings showed that hypothermia, which has more affect, and O3 decreased the tourniquet-induced IR injury in the rat's muscle-skeletal system by reducing the levels of oxidative and nitrosative stress parameters and enhancing antioxidant enzymes. Hypothermia and O3 had no synergistic effect. Hypothermic reperfusion and O3 preconditioning might be beneficial in skeletal muscle IR injury-associated tourniquet.

Evaluation and comparison of the effects of hyperbaric oxygen and ozonized oxygen as adjuvant treatments in an experimental osteomyelitis model.

BACKGROUND: We evaluated and compared the efficacy of ozone (O(3)) and hyperbaric oxygen (HBO) therapies in an experimental rat model of osteomyelitis. MATERIALS AND METHODS: Forty-eight male Sprague-Dawley rats were divided into sham, osteomyelitis (control), vancomycin (V), vancomycin + HBO (VHB), vancomycin + O(3) (VO), and vancomycin + HBO + O(3) (VOHB) groups. Osteomyelitis was induced by a bone injection of 10(8) CFU/mL methicillin-resistant Staphylococcus aureus. HBO was administered daily at 2.8-atm pressure for 90 min; O(3) therapy was provided as intraperitoneal injections of 0.7 mg/kg O(3)/O(2) gas mixture once daily. Treatments were continued from d 7 to 21 after induction of osteomyelitis. Bone tissues and blood samples were harvested for biochemical, histopathologic, and microbiologic analyses. RESULTS: Rats in the sham, VO, and VOHB groups gained weight but those in the control, V, and VHB groups did not. Levels of malondialdehyde, superoxide dismutase, and glutathione peroxidase were lower in the VHB, VO, and VOHB groups than in V and control groups. Levels of interleukin-10 and -1ß and tumor necrosis factor-α were decreased in the VHB, VO, and VOHB groups; transforming growth factor-ß was increased in these groups compared with V and control groups (P ≤ 0.001). Bacteria counts in VOHB were significantly lower than those in group of V (P = 0.012). Histopathologic scores in group VO were significantly lower than those in group V (P = 0.046). CONCLUSIONS: O(3) was as effective as HBO in decreasing oxidative parameters and inflammatory cytokines. Rats in the VO and VOHB groups gained more weight than did the other groups. Bacteria counts were significantly decreased in group VOHB compared with the other groups. Histopathologic scores in group VO were significantly decreased compared with the other groups.

Effect of preconditioned hyperbaric oxygen and ozone on ischemia-reperfusion induced tourniquet in skeletal bone of rats.

BACKGROUND: The aim of the study was to investigate effect of I/R injury on bone tissue and protective role of hyperbaric oxygen precondition (HBO-PC) and ozone precondition (O(3)-PC) on I/R injury by using biochemical analysis. MATERIALS AND METHODS: Thirty-two rats were included in study. The animals were divided into four equal groups: sham operation, I/R, I/R+HBO and I/R+O(3). One session of 60 min, 3 ATA, 3-4 L/min, 100% oxygenation was defined as one dose of HBO. First dose of HBO was administrated 72 h before ischemia. Subsequent, one-dose of HBO administrated per 12 hours until ischemia time (total seven doses); 0.7 mg/kg ozone/oxygen mixture intraperitoneally was defined as one dose of ozone. First dose of O(3) was administered 72 h before ischemia (total four doses). I/R model was induced in anesthetized rats by unilateral (right) femoral artery clipping for 2 h followed by 22 h of reperfusion. The right tibia and were harvested. Tissue was assayed for levels of malondialdehyde (MDA) and protein carbonyl (PCO), activities of superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). RESULTS: MDA and PCO levels were increased in I/R group. SOD activity was increased; GSH-Px activity was decreased in I/R group. MDA and PCO levels were decreased, SOD and GSH-Px activities were increased in both HBO+I/R and O(3)+I/R groups. CONCLUSION: It has been shown that levels of MDA and PCO in bone were increased followed by 2 h of ischemia and 22 h of reperfusion period. Ozone-PC and HBO-PC has protective effect against skeletal bone I/R injury by decreasing levels of MDA and PCO, increasing activities of SOD and GSH-Px in rats.