Antioxidant Effects of Marigold (Calendula officinalis) Flower Extract on the Oxidative Balance of Bovine Spermatozoa

Summary The aim of our study was evaluation of potential antioxidant effects of marigold (Calendula officinalis) extract and assessment of its in vitro impact on the selected quality parameters of bovine spermatozoa. Marigold is medicinal herb from the family Asteraceae native to southern Europe, and it commonly used in pharmacology and medicine. Its well-known positive properties include antioxidant, antibacterial, antiflammatory, antiviral, antifungal, antihelmintic and wound-healing activities. In this study, the flower extracts were subjected to high performance liquid chromatography (HPLC), which identified especially phenolic acids (rosmarinic and chlorogenic acids) and polyphenols (rutin, kaempferol, resveratrol, quercetin and apigenin). These substances are known for their antioxidant activity and protective effects against oxidative stress. For our experiments, 10 samples of semen from sexually mature Holstein bulls were collected on a single day by using an artificial vagina, diluted in physiological saline solution and exposed to solutions with different concentration of marigold flower extract (75, 150 and 300 µg/mL). Selected quality parameters (motility, mitochondrial activity, production of reactive oxygen species – ROS, protein oxidation and lipid peroxidation – LPO) were analyzed after 0, 2 and 24 hours of in vitro culture. The motility evaluation was performed by using the computer-assisted sperm analysis (CASA) method. This method revealed that 75 and 150 µg/mL extract had positive effects and increased the motility (P<0.01) and mitochondrial activity (P<0.0001) of bovine spermatozoa compared to the control group following 2 and 24 hours. This phenomenon was observed also in case of ROS production, protein oxidation and LPO. Marigold extract concentrations of 75 and 150 µg/mL decreased the levels of ROS, protein oxidation and damage to the membranes caused by LPO compared to the control group (P<0.05; P<0.01) at time 2 and 24 hours. At the same time, 300 µg/mL extract exhibited positive, although less significant, effects compared to 75 and 150 µg/mL extracts. The data acquired from our study confirm that 75 and 150 µg/mL of marigold flower extract have positive effects on the motility and mitochondrial activity of bovine spermatozoa, and decrease ROS generation, LPO and protein oxidation in spermatozoa. Based on our results, the flower extract from marigold could be used for protection against oxidative stress in in vitro cultures of male gametes.

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Biological characterization of implant surfaces - in vitro study

Revista de Odontologia da UNESP ◽

10.1590/1807-2577.1087 ◽

2015 ◽

Vol 44 (4) ◽

pp. 195-199 ◽

Cited By ~ 1

Author(s):

Priscilla Barbosa Ferreira Soares ◽

Camilla Christian Gomes Moura ◽

Huberth Alexandre da Rocha Júnior ◽

Paula Dechichi ◽

Darceny Zanetta-Barbosa

Keyword(s):

Alkaline Phosphatase ◽

Cell Viability ◽

Total Protein ◽

Cell Attachment ◽

Control Group ◽

Mitochondrial Activity ◽

Serum Total Protein ◽

Trypan Blue Exclusion ◽

Experimental Surface

<title>Abstract</title><sec><title>Objective</title>Evaluate the biological performance of titanium alloys grade IV under different surface treatments: sandblasting and double etching (Experimental surface 1; Exp1, NEODENT); surface with wettability increase (Experimental surface 2; Exp2, NEODENT) on response of preliminary differentiation and cell maturation.</sec><sec><title>Material and method</title>Immortalized osteoblast cells were plated on Exp1 and Exp2 titanium discs. The polystyrene plate surface without disc was used as control group (C). Cell viability was assessed by measuring mitochondrial activity (MTT) at 4 and 24 h (n = 5), cell attachment was performed using trypan blue exclusion within 4 hours (n = 5), serum total protein and alkaline phosphatase normalization was performed at 4, 7 and 14 days (n = 5). Data were analyzed using one-way ANOVA and Tukey test.</sec><sec><title>Result</title>The values of cell viability were: 4h: C– 0.32±0.01A; Exp1– 0.34±0.08A; Exp2– 0.29±0.03A. 24h: C– 0.43±0.02A; Exp1– 0.39±0.01A; Exp2– 0.37±0.03A. The cell adhesion counting was: C– 85±10A; Exp1- 35±5B; Exp2– 20±2B. The amounts of serum total protein were 4d: C– 40±2B; Exp1– 120±10A; Exp2– 130±20A. 7d: C– 38±2B; Exp1– 75±4A; Exp2– 70±6A. 14 d: C– 100±3A; Exp1– 130±5A; Exp2– 137±9A. The values of alkaline phosphatase normalization were: 4d: C– 2.0±0.1C; Exp1– 5.1±0.8B; Exp2– 9.8±2.0A. 7d: C– 1.0±0.01C; Exp1– 5.3±0.5A; Exp2– 3.0±0.3B. 14 d: C– 4.1±0.3A; Exp1– 4.4±0.8A; Exp2– 2.2±0.2B. Different letters related to statistical differences.</sec><sec><title>Conclusion</title>The surfaces tested exhibit different behavior at dosage of alkaline phosphatase normalization showing that the Exp2 is more associated with induction of cell differentiation process and that Exp1 is more related to the mineralization process.</sec>

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In Vitro Assessment of the Antioxidant Properties of Aqueous Byproduct Extracts of Vitis vinifera

Food Technology and Biotechnology ◽

10.17113/ftb.57.01.19.5879 ◽

2019 ◽

Vol 57 (1) ◽

pp. 29-38 ◽

Cited By ~ 1

Author(s):

Roberto Puglisi

Keyword(s):

Plasma Membranes ◽

Antioxidant Properties ◽

Dermal Fibroblasts ◽

Total Phenolic ◽

Mitochondrial Activity ◽

Cabernet Sauvignon ◽

Leaf Extracts ◽

Volume Fractions ◽

Positive Effects

Aqueous extracts were obtained at low temperature with the Naviglio technology from grapevine stalks (Merlot), marc (Merlot and Cabernet Sauvignon) and leaves (Merlot) as typical byproducts of winemaking industry, and their properties were evaluated cytofluorometrically on human dermal fibroblasts. Leaf extracts had the greatest total phenolic ((47.6±3.5) mg/g) and proanthocyanidin ((24.2±0.1) mg/g) contents compared to the others. The preliminary colorimetric MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay individuated two consecutive non-toxic volume fractions of each extract (from 0.8 to 12.8 %) that were adopted for three cytofluorometric tests. The first cell membrane test did not evidence any harmful effects against plasma membranes at the two non-toxic volume fractions. The second mitochondrial membrane test showed a decreased (p<0.01) percentage of cells ((15.7±8.3) vs (32.5±1.3) %) with active polarized mitochondrial membranes at the higher non-cytotoxic volume fractions of extracts from Cabernet Sauvignon marc in response to 4.5 mM H2O2, and from Merlot stalks (p<0.05) at 1.5 mM H2O2 ((49.3±6.1) vs (64.6±2.4) %) and without H2O2 ((89.7±2.4) vs (96.9±1.8) %), compared to the controls submitted to the same H2O2 concentration. Conversely, mitochondrial activity of leaf extracts significantly (p<0.05) increased ((96.3±1.8) and (96.4±1.4) %) after treatment with 0.5 mM H2O2 at both non-cytotoxic volume fractions compared to control ((88.2±1.1) %). Finally, as evidenced by the third oxidative status test, stalk extracts did not evidence relevant effects on the cellular oxidative state, while the extracts of marc and leaves demonstrated significantly medium (p<0.05) to highly (p<0.001) positive effects following exposure to H2O2 ranging from 0.5 to 4.5 mM, compared to controls.

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Transcriptional, Mitochondrial Activity, and Viability of Egyptian Buffalo’s Granulosa Cells In Vitro Cultured under Heat Elevation

World s Veterinary Journal ◽

10.54203/scil.2021.wvj25 ◽

2021 ◽

Vol 11 (2) ◽

pp. 193-201

Author(s):

Nasser Ghanem ◽

Marwa Said Faheem ◽

Romysa Samy ◽

Ashraf Hesham Barkawi

Keyword(s):

Heat Stress ◽

Granulosa Cells ◽

Transcriptional Activity ◽

The Other ◽

Control Group ◽

Mitochondrial Activity ◽

Fluorescent Intensity ◽

Stressed Group ◽

Other Hand

It is documented that heat stress caused impairment on the reproductive performance of dairy animals. However, there are few reports that have focused on the molecular and intracellular responses of in vitro cultured buffalo granulosa cells during heat elevation. The present study was conducted to investigate the effect of heat elevation during in vitro culture of buffalo granulosa cells on their viability, quality, mitochondrial activity, and transcriptional activity. Granulosa cells were harvested after aspiration of cumulus-oocytes complexes that were collected from abattoir ovaries. The granulosa cells were cultured in vitro either at a normal physiological temperature suitable for oocyte maturation and embryo development (38.5°C) or exposed to the elevated temperature of 40.5°C on day 3 of culture (the first two days were for confluence) for two hours of culture then continued at 38.5°C up to day 7 of culture. The viability of granulosa cells was measured using trypan blue and quality was estimated by measuring the level of intracellular reactive oxygen species (ROS) on day 7. Moreover, metabolic activity was performed by measuring the fluorescent intensity of mitochondria. Moreover, transcriptional activity was done by profiling four selected candidate genes using quantitative real-time PCR. The results indicated that the granulosa cells viability rate significantly decreased in the heat stress group (25.1 ± 3.7), compared to the control group (36.6 ± 5.3) on confluence day (day 3). In addition, the viability rate on the last day of culture (day 7) decreased in heat stress, compared to control (83.7 ± 4.5 and 97.4 ± 0.4, respectively). On the other hand, there was a nonsignificant difference in ROS profile between the control (21.7*104 ± 1.3) and the heat-stressed group (15.7 ± 0.7) on day 7 of culture. However, the mitochondrial fluorescent intensity was higher in the control (21.9 ± 1.9) than in the heat-stressed group (15.4 ± 0.8) on day 7 of culture. The expression of cellular defense (HSF1) and apoptosis-inducing gene (P53) were significantly up-regulated in granulosa cells exposed to heat elevation, compared to the control group. On the other hand, the steroidogenesis-regulating gene (StAR) was down-regulated in granulosa cells cultured under heat shock, compared to the control group. In conclusion, heat stress reduced the viability of granulosa cells by inducing the expression of an apoptosis-related gene (P53) and compromised expression of genes regulating the steroid biosynthesis, which resulted in up-regulation of cell defense gene (HSF1) in an attempt to ameliorate the deleterious effect of heat stress on the biological activity of the granulosa cells.

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Investigation of the Synergistic Effect of Brown Sugar, Longan, Ginger, and Jujube (Brown Sugar Longan Ginger Tea) on Antioxidation and Anti-Inflammation in In Vitro Models

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/3596085 ◽

2020 ◽

Vol 2020 ◽

pp. 1-6

Author(s):

Ping Lin ◽

Kai-Wen Kan ◽

Jia-Haur Chen ◽

Yung-Kai Lin ◽

Yung-Hao Lin ◽

...

Keyword(s):

Synergistic Effect ◽

Umbilical Vein ◽

In Vitro Models ◽

Mitochondrial Activity ◽

Brown Sugar ◽

Nonalcoholic Fatty Liver ◽

Beneficial Effects ◽

Positive Effects ◽

Anti Inflammation

This research unveils the synergistic effect of brown sugar, longan, ginger, and jujube on the beneficial effects of antioxidation and anti-inflammation. Longan, ginger, and jujube are ubiquitous herbs in traditional Chinese medicine (TCM) and are frequently used in folk remedies. Longan and ginger have been reported to be beneficial for antioxidation, anti-inflammation, ant-obesity, and nonalcoholic fatty liver disease (NAFLD) improvements. However, the potential scientific and medical benefits of their combination Brown Sugar Longan Ginger Tea (BSLGT), a popular drink in Chinese cultures, are elusive. Through the in vitro methodologies, we discovered that BSLGT could significantly improve the mitochondrial activity, antioxidant capacity, lipid content, and inflammatory response in human hepatocytes. In addition, BSLGT also exerted positive effects on the downregulation of atherosclerosis-associated, vasoconstrictor, and thrombosis-related gene expression in human umbilical vein endothelial cells. In short, our experimental results successfully revealed that the antioxidative and anti-inflammatory effects of BSLGT may have the potential to improve liver metabolism and cardiovascular inflammation although solid evidence requires further investigation.

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Baicalin improves the in vitro developmental capacity of pig embryos by inhibiting apoptosis, regulating mitochondrial activity and activating sonic hedgehog signaling

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz036 ◽

2019 ◽

Vol 25 (9) ◽

pp. 538-549 ◽

Cited By ~ 3

Author(s):

Qing Guo ◽

Mei-Fu Xuan ◽

Zhao-Bo Luo ◽

Jun-Xia Wang ◽

Sheng-Zhong Han ◽

...

Keyword(s):

Embryonic Development ◽

Signaling Pathway ◽

Sonic Hedgehog ◽

Hedgehog Signaling ◽

Control Group ◽

Mitochondrial Activity ◽

Cell Stage ◽

Shh Signaling ◽

Developmental Capacity

Abstract Baicalin, a traditional Chinese medicinal monomer whose chemical structure is known, can be used to treat female infertility. However, the effect of baicalin on embryonic development is unknown. This study investigated the effects of baicalin on in vitro development of parthenogenetically activated (PA) and in vitro fertilized (IVF) pig embryos and the underlying mechanisms involved. Treatment with 0.1 μg/ml baicalin significantly improved (P < 0.05) the in vitro developmental capacity of PA pig embryos by reducing the reactive oxygen species (ROS) levels and apoptosis and increasing the mitochondrial membrane potential (ΔΨm) and ATP level. mRNA and protein expression of sonic hedgehog (SHH) and GLI1, which are related to the SHH signaling pathway, in PA pig embryos at the 2-cell stage, were significantly higher in the baicalin-treated group than in the control group. To confirm that the SHH signaling pathway is involved in the mechanism by which baicalin improves embryonic development, we treated embryos with baicalin in the absence or presence of cyclopamine (Cy), an inhibitor of this pathway. Cy abolished the effects of baicalin on in vitro embryonic development. In conclusion, baicalin improves the in vitro developmental capacity of PA and IVF pig embryos by inhibiting ROS production and apoptosis, regulating mitochondrial activity and activating SHH signaling.

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Evaluation of theIn VitroandIn VivoAntioxidant Potentials ofSudarshanaPowder

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/6743862 ◽

2018 ◽

Vol 2018 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

Weerakoon Achchige Selvi Saroja Weerakoon ◽

Pathirage Kamal Perera ◽

Dulani Gunasekera ◽

Thusharie Sugandhika Suresh

Keyword(s):

Antioxidant Activity ◽

Antioxidant Capacity ◽

Thiobarbituric Acid ◽

Trolox Equivalent Antioxidant Capacity ◽

Control Group ◽

Thiobarbituric Acid Reactive Substance ◽

Trolox Equivalent ◽

Antioxidant Effects

Sudarshanapowder (SP) is one of the most effective Ayurveda powder preparations for paediatric febrile conditions. The objective of the present study was to evaluate thein vitroandin vivoantioxidant potentials of SP. Thein vitroantioxidant effects were evaluated using ABTS radical cation decolourization assay where the TROLOX equivalent antioxidant capacity (TEAC) was determined. Thein vivoantioxidant activity of SP was determined in Wistar rats using the Lipid Peroxidation (LPO) assay in serum. Thein vitroassay was referred to as the TROLOX equivalent antioxidant capacity (TEAC) assay. For thein vivoassay, animals were dosed for 21 consecutive days and blood was drawn to evaluate the MDA level. Thein vitroantioxidant activity of 0.5 μg of SP was equivalent to 14.45 μg of standard TROLOX. The percentage inhibition against the radical formation was50.93±0.53%. The SP showed a statistically significant (p<0.01) decrease in the serum level of thiobarbituric acid-reactive substance in the test rats when compared with the control group. These findings suggest that the SP possesses potent antioxidant activity which may be responsible for some of its reported bioactivities.

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83 VITAMIN K2 SUPPLEMENTATION IMPROVES BLASTOCYST RATE BY RECOVERY OF MITOCHONDRIA IN IN VITRO-CULTURED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab83 ◽

2014 ◽

Vol 26 (1) ◽

pp. 155

Author(s):

L. Baldoceda ◽

C. Vigneault ◽

P. Blondin ◽

C. Robert

Keyword(s):

In Vitro Culture ◽

Fluorescent Microscopy ◽

Vitamin K2 ◽

Control Group ◽

Mitochondrial Activity ◽

Bovine Embryo ◽

Bovine Embryos ◽

Blastocyst Rate ◽

Mitotracker Red

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.

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Antioxidant Effects of Ocimum Gratissimum Leaf Essential Oils as a Supplement to Extender on Chilled Canine Sperm Quality

10.21203/rs.3.rs-26157/v1 ◽

2020 ◽

Author(s):

Vui Van Nguyen ◽

Samorn Ponchunchoovong ◽

Sajeera Kupittayanant ◽

Pakanit Kupittayanant

Keyword(s):

Essential Oils ◽

Sperm Quality ◽

Antioxidant Properties ◽

Quality Parameters ◽

Control Group ◽

Dependent Manner ◽

Ocimum Gratissimum ◽

Significant Difference ◽

Antioxidant Effects

Abstract Background:Oxidative stress during chilled storage is a major problem withcanine sperm. To improve the quality of chilled canine sperm during storage, many synthetic antioxidants have been examined, but different outcomes were investigated depending on antioxidant properties. The bioactive compounds of essential oils fromOcimum gratissimumleaves are known as a natural antioxidant source. This study aimed to evaluate the antioxidant effects of essential oils from Ocimum gratissimumleavesas a supplement in extender on chilled canine sperm during 12 days of storage. Results:The results showed thatlow concentrations of Ocimum gratissimum essential oils (25, 50, and 100µg/mL) have beneficial effectson sperm quality, whereasOcimum gratissimumessential oils athigh levels (above 200µg/mL) have harmful effects. Specifically, the addition of 100µg/mL ofOcimum gratissimum essential oilsto the extender had the greatestbeneficial effect in improving the quality of chilled canine sperm, and had a significant difference in all sperm quality parameters except motility when compared to the control group (p<0.05). Conclusions:Ocimum gratissimum essential oilshave an impact on chilled canine sperm quality in a dose-dependent manner, and the best results areachieved with a maximum dose ofOcimum gratissimum essential oils of 100µg/mL.

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Effects of Taurine on Sperm Quality during Room Temperature Storage in Hu Sheep

Animals ◽

10.3390/ani11092725 ◽

2021 ◽

Vol 11 (9) ◽

pp. 2725

Author(s):

Liuming Zhang ◽

Yanhu Wang ◽

Tariq Sohail ◽

Yan Kang ◽

Haoyuan Niu ◽

...

Keyword(s):

Room Temperature ◽

Sperm Quality ◽

Membrane Integrity ◽

Quality Parameters ◽

Control Group ◽

Negative Effects ◽

Positive Effects ◽

Antioxidant Parameters ◽

Acrosome Integrity ◽

Hu Sheep

The present study aimed to investigate whether the presence of Tau protected Hu sheep sperm from ROS stress during storage at room temperature. The semen was diluted with extender (Tris-based) at room temperature, supplemented with different concentrations of Tau (0, 10, 20, 40, 80, or 100 mM), and stored at 15 °C. Sperm quality parameters (sperm progressive motility, kinetic parameters, plasma membrane integrity rate, acrosome integrity rate, and MMP) and antioxidant parameters (ROS, MDA, SOD, CAT, and T-AOC) were evaluated during the preservation of semen. The addition of Tau, especially at a concentration of 20 mM, exerted positive effects on sperm quality parameters and antioxidant parameters compared to the sperm without Tau treatment (control group). The addition of Tau, especially at a concentration of 100 mM, exerted negative effects on sperm quality parameters and antioxidant parameters compared to the control group. Interestingly, the results indicated that the sperm acrosome integrity rate did not change during storage time. In conclusion, the addition of Tau to sperm preserved at room temperature can enhance the antioxidant ability of sperm, reduce the LPO on the 5th day, and improve the quality of semen preserved at room temperature. These results implied that Tau had potential to enhance Hu sheep sperm reproductive performance.

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Positive effects of trehalose and cysteine on ram sperm parameters

Veterinární Medicína ◽

10.17221/131/2016-vetmed ◽

2017 ◽

Vol 62 (No. 5) ◽

pp. 245-252 ◽

Cited By ~ 2

Author(s):

S. Gungor ◽

C. Ozturk ◽

AD Omur

Keyword(s):

Sperm Motility ◽

Sperm Parameters ◽

Control Group ◽

Mitochondrial Activity ◽

Sperm Viability ◽

Significant Protection ◽

Positive Effects ◽

Liquid Storage ◽

Acrosome Integrity ◽

Ram Sperm

The aim of this study was to determine the effects of trehalose and cysteine on sperm motility, viability, mitochondrial activity and acrosome integrity during liquid storage of Merino ram semen. Ejaculates were collected using artificial vaginas from five Merino rams, microscopically evaluated and pooled at 37 °C. The pooled semen samples were diluted in a Tris-based extender, including cysteine (2 mM and 4 mM), trehalose (10 mM and 25 mM) and no antioxidant (control). Diluted semen samples were kept in tubes and cooled from 37 to 5 °C in a cold cabinet, and maintained at 5 °C. Cooled samples were evaluated for sperm motility, viability, mitochondrial activity and acrosome integrity at 0, 24, 48, 72 and 96 h. Extender supplemented with trehalose (10 and 25 mM) and cysteine (2 and 4 mM) led to higher motility in comparison to the control at 24, 48, 72 and 96 h of liquid storage (P < 0.05). Trehalose at the doses of 10 mM, 25 mM and 2 mM cysteine led to higher viability between 24–48–72 h and at 96 h of liquid storage (P < 0.05). Further, 4 mM of cysteine improved sperm viability rates at 24 and 48 h of storage compared to the control group (P < 0.05), and resulted in improved acrosome integrity rates compared to the control group at 72 and 96 h of storage (P < 0.05). Extender supplemented with 10 and 25 mM trehalose at 24 and 72 h and 4 mM cysteine at 24 and 96 h of storage led to higher sperm mitochondrial activity rates when compared to the control group (P < 0.05). In conclusion, the findings of this study show that trehalose and cysteine provided significant protection to ram sperm parameters during liquid storage.

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