Ram Spermatozoa Research Articles

Effects of extender type and storage time on sperm quality parameters of Kail ram semen stored at 5 °C

The study was designed to evaluate the effect of different extenders and storage times on sperm quality parameters of extended Kail ram semen. Semen was collected from five adult Kail rams using an artificial vagina. Semen samples with >70% total sperm motility were pooled, diluted with Tris (TR), sodium citrate (SC), and skim milk (SM)-based extenders, and stored at 5 °C. Sperm motility and kinematics, viability, and plasma and acrosomal membrane integrity were assessed every 24 hrs for 120 hrs. Sperm longevity was estimated by placing semen in a water bath at 37°C to assess sperm kinematics. Storage time as the main effect had a negative (P < 0.05) impact on sperm quality parameters. The percentages of total motile (TM), progressive motile (PM), rapid, and medium progressive (MP) motile sperm were similar at 24 hrs of storage for the three extenders. However, semen extended in TR had a higher (P < 0.05) percentage of TM, PM, rapid, and MP sperm compared to SM and SC extenders at 48 hrs of storage. The sperm kinematics (VCL, VSL, VAP, and ALH) of progressive sperm were similar for all three extenders up to 72 hrs of storage. In addition, semen extended in TR had a higher (P < 0.05) percentage of sperm with intact plasma and acrosomal membranes at a storage time of 48 hrs. At 37 °C, the percentage of TM and PM sperm was higher (P < 0.05) in the TR extender than in the SM and SC extenders at 60 minutes and beyond. In conclusion, the Kail ram sperm extended in TR and stored at 5 °C maintained better quality after 48 hrs storage than sperm extended in SM and SC extenders. At 37 °C, sperm extended in TR also retained better quality parameters at 60 min and afterward than sperm extended in SM and SC.

Association between different soy lecithin-based extenders and freezing rates in ram semen cryopreservation

The aim of the study was to evaluate the effect of the association between glycine-milk (GM) based extenders made with different concentrations of soy lecithin (SL) and freezing rates (FR) on semen quality after thawing. Pooled semen from rams (n = 12) were diluted in GM extenders with 20% egg yolk (EY–20%) or with different concentrations of SL: 0.5% (SL–0.5%), 1.0% (SL–1.0%), and 2.0% (SL–2.0%). The diluted semen (150 ×106 spermatozoa/0.25 mL) was frozen at three FR of − 10, − 20, and − 60 °C/min, and subsequently thawed and analyzed. Results revealed that EY–20% and SL–2.0% had better kinetic parameters, and showed higher proportions of viable, non-apoptotic, plasma-membrane-intact spermatozoa (A-/PI-) and non-capacitated spermatozoa (F), and had lower acrosome-reacted spermatozoa (AR) in the EY–20% and satisfactory values for SL-2.0% compared to SL–0.5% and SL–1.0% (P < 0.05). The FR at − 20 and − 60 °C/min maintained higher A-/PI- and viable spermatozoa compared to − 10 °C/min. The combination EY–20% and − 60 °C/min showed the highest A-/PI- and F (P < 0.05) and the lowest AR, and it did not differ from the combinations EY–20% at − 20 °C/min and SL–2.0% at − 20 °C/min (P > 0.05). In conclusion, the combination EY–20% and − 60 °C/min, showed the best cryoprotective effects on ram spermatozoa. Changes in spermatozoa after thawing were related to the use of the type of extender, the amounts of the same compound in the extender, and the freezing rates to which they were subjected.

Proteomic analysis reveals the potential positive effects of Mito-TEMPO on ram sperm motility and fertility during cryopreservation

The aim of this study was to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on the protein profile of ram sperm during cryopreservation and evaluate the cryoprotective roles of Mito-TEMPO on ram sperm quality and fertilization capacity. Semen collected from 8 Dorper rams was cryopreserved in TCG-egg yolk extender supplemented with various concentrations of Mito-TEMPO (0, 20, 40 and 60 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The cervical artificial insemination (AI) was performed to evaluate the fertilization ability of cryopreserved ram sperm. The alterations of sperm proteomic profile between the control and MT40 groups were determined using iTRAQ-coupled LC-MS. Supplementation with 40 μM of Mito-TEMPO resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed ram sperm were elevated in the MT40 group. The inclusion of 40 μM Mito-TEMPO in freezing extender also resulted in the higher pregnancy rate of ewes. A total of 457 proteins including 179 upregulated proteins and 278 downregulated proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05. Sixty-one DEPs with (FC > 1.5) were dramatically regulated by Mito-TEMPO. These DEPs are mainly involved in sperm motility, energy metabolism and capacitation. Our data suggest that the beneficial effects of Mito-TEMPO on sperm motility and fertility potential of cryopreserved ram semen are achieved by regulating sperm antioxidant capacity and sperm proteins related to energy metabolism and fertility.

Ram sperm cryopreservation disrupts metabolism of unsaturated fatty acids

In ram sperm, metabolites are important components of the plasma membrane, energy metabolism cycle, and precursors for other membrane lipids, and they may have important roles in maintaining plasma membrane integrity, energy metabolism, and regulation of cryotolerance. In this study, the ejaculates from 6 Dorper rams were pooled and sperm were systematically investigated by metabolomics at various steps of cryopreservation (37 °C, fresh [F]; from 37 to 4 °C, cooling [C]; and from 4 to −196 to 37 °C, frozen-thawed [FT]) to identify differential metabolites (DM). There were 310 metabolites identified, of which 86 were considered DMs. Regarding the DMs, there were 23 (0 up and 23 down), 25 (12 up and 13 down), and 38 (7 up and 31 down) identified during cooling (C vs F), freezing (FT vs C), and cryopreservation (FT vs F), respectively. Furthermore, some key polyunsaturated fatty acids (FAs), particularly, linoleic acid (LA), docosahexaenoic acid (DHA), and arachidonic acid (AA) were down-regulated during cooling and cryopreservation. Significant DMs were enriched in several metabolic pathways including biosynthesis of unsaturated FAs, LA metabolism, mammalian target of rapamycin (mTOR), forkhead box transcription factors (FoxO), adenosine monophosphate-activated protein kinase (AMPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathways, regulation of lipolysis in adipocytes, and FA biosynthesis. This was apparently the first report to compare metabolomics profiles of ram sperm during cryopreservation and provided new knowledge to improve this process.

Glycerol-Free Equilibration with the Addition of Glycerol Shortly before the Freezing Procedure: A Perspective Strategy for Cryopreservation of Wallachian Ram Sperm.

This study investigated the effect of glycerol added in different phases of sperm equilibration on CASA and flow cytometry parameters of thawed ram spermatozoa. Sperm was collected from adult Wallachian rams. The freezing extender was glycerol-free ANDROMED® (Minitub GmbH, Tiefenbach, Germany) supplied by 6% exogenous glycerol at different stages of the cryopreservation process. The purpose of this study was to compare two strategies of glycerol addition for sperm cryopreservation. The first strategy included the use of a glycerol-free extender for the procedure of glycerol-free equilibration and chilling, with the glycerolation of the extender by 6% glycerol shortly before sperm slow freezing (GFA). The second strategy included the use of a freezing extender already glycerolated by 6% glycerol before the equilibration and chilling of sperm and following slow freezing (GA). Sperm samples were analyzed after equilibration (but before freezing) and after thawing (at T0, T1 h, and T2 h time points). iSperm® mCASA (Aidmics Biotechnology Co., LTD., Taipei, Taiwan) was used for the evaluation of sperm kinematics. Flow cytometry was used to measure sperm viability (plasma membrane/acrosome intactness) and mitochondrial membrane potential. The obtained results significantly demonstrated that the glycerol-free equilibration with the addition of glycerol shortly before freezing is a perspective strategy for cryopreservation of Wallachian ram sperm.

Variation of existence and location of aquaporin 3 in relation to cryoresistance of ram spermatozoa.

Osmotic changes during the process of freeze-thawing involve changes in the location of aquaporins (AQPs) in membrane domains of spermatozoa. Some AQPs, like aquaporin 3 (AQP3), are linked to sperm cryotolerance in the porcine species. Conspicuous individual variability exists between rams and their ejaculates, which may be classified as displaying good freezability (GFE) or poor freezability (PFE), depending on several endogenous and environmental factors. The present work aimed to examine whether differences in freezability could even involve changes in location and expression of AQP3 in ram spermatozoa. Thirty ejaculates from 10 rams (three of each) were evaluated and subsequently classified as GFE (n = 13) or PFE (n = 17) through a principal component analysis (PCA) and k-means cluster analysis. Spermatozoa were examined for the presence, abundance and distribution of AQP3 by western blot and immunocytochemistry, employing a commercial rabbit polyclonal antibody (AQP3 - ab125219). Although AQP3 was found in the sperm acrosome, midpiece, principal and end piece of the tail in both fresh and after frozen-thawed samples, its highest immunolabeling was found in the mid- and principal piece. In the GFE group, the expression of AQP3 in the mid- and principal piece was greater (P < 0.05) in frozen-thawed samples than in fresh specimens while such differences were not detected in the PFE group. Sperm cryotolerance relates to changes in AQP3 expression and thus AQP3 could be used as a biomarker for cryotolerance. A greater capacity of AQP3 localization in mid- and principal piece of the spermatozoa could be linked to an increase the osmo-adaptative capacity of ejaculates with better capacity to withstand freeze-thawing processes.

X-Y Chromosom bearing Sperm Proportion of Local Ram after Sexing with Different Combination of Bovine Serum Albumin (BSA) Concentration

The research aims to evaluate proportion of X-Y chromosom bearing sperm after sexing of local ram semen with different combination of BSA concentration. The research object was ten ejaculated semen of local ram, three years old. The research design used CRD (completely randomize design) with four treatments of BSA concentration combination on upper and bottom layer (T1: 3% &amp; 6%, T2: 4% &amp; 6%, T3: 5% &amp; 10%, and T4: 6% &amp; 12%), and 10 repetitions. Data analysis using analysis variance and Duncan’s Multiple Range Test. The result showed that combination of BSA concentration was significantly effect on proportion of X-Y chromosome bearing sperm of local ram sperm. The higher average proportion of X- and Y- chromosome bearing sperm was obtained at combination 5% (75.55±1.09% for X) at upper layer and 10% BSA (76.45±1.12% for Y) of bottom layer. The conclusion is that combination of BSA concentration significantly effect on proportion of X-Y chromosome bearing sperm, and concentration of 5% and 10% BSA at upper layer and bottom layer gave the higher proportion of X-Y chromosome bearing sperm of local ram sperm.

Influence of trans-ferulic acid on the quality of ram semen upon cold preservation.

Due to lower antioxidant capacity and higher amounts of polyunsaturated fatty acids, ram spermatozoa are very susceptible during cooling process. The objective was to examine the effect of the trans-ferulic acid (t-FA) on the ram semen during liquid preservation. Semen samples were collected from the Qezel rams, pooled, and extended with the Tris-based diluent. Pooled samples enriched with different amounts of the t-FA (0, 2.5, 5, 10, and 25mM) and preserved at 4°C for 72h. Spermatozoa's kinematics, membrane functionality, and viability were assessed by CASA system, hypoosmotic swelling test, and eosin-nigrosin staining, respectively. Moreover, biochemical parameters were measured at 0, 24, 48, and 72h. Results showed that 5 and 10mM t-FA improved forward progressive motility (FPM) and curvilinear velocity compared to the other groups at 72h (p < 0.05). Samples treated with 25mM t-FA showed the lowest total motility, FPM, and viability at 24, 48, and 72h of storage (p < 0.05). Higher total antioxidant activity levels were observed in the 10mM t-FA-treated group compared to the negative control at 72h (p < 0.05). Treatment with 25mM t-FA increased malondialdehyde amounts and decreased superoxide dismutase activity compared to other groups at the final time assessment (p < 0.05). Nitrate-nitrite and lipid hydroperoxides values were not affected by treatment. The current study indicates the positive and negative influences of different concentrations of t-FA on the ram semen upon cold storage.

Inclusion of Spirulina platensis and Salvia verbenaca extracts to boost semen quality and fertilization ability in sheep.

The aim of this study was to investigate the effect of Spirulina platensis (SP) and Salvia verbenaca (SV) extracts added to skimmed milk (SM) extender on ram sperm quality and fertility. Semen was collected using an artificial vagina, extended in SM to reach a final concentration of 0.8 × 109 spermatozoa/mL, stored at 4°C and evaluated at 0, 5H and 24 H. The experiment has been performed in three steps. Firstly, from four extracts (methanol: MeOH, acetone: Ac, ethyl acetate: EtOAc and hexane: Hex) of SP and SV, only acetonic and hexanoic extracts of SP and acetonic and methanolic extracts of SV showed the highest in vitro antioxidant activities and were then selected for the following step. Thereafter, the effects of four concentrations (1.25, 3.75, 6.25, and 8.75 μg/mL) of each selected extract on stored sperm motility were evaluated. The output of this trial led to select the best concentrations having beneficial effects on sperm quality parameters (viability, abnormalities, membrane integrity, and lipid peroxidation) and fertility after insemination. The results showed that the same concentration (1.25 μg/mL) of both Ac-SP and Hex-SP, as well as 3.75 μg/mL of Ac-SV and 6.25 μg/mL of MeOH-SV, maintain all sperm quality parameters at 4°C during 24 H of storage. Besides, no difference was found in fertility between the selected extracts and the control. In conclusion, SP and SV extracts were shown to improve the quality of ram sperm and to maintain fertility rate after insemination as similar or competitive to many previous studies published in the field.

Improvement of the Seminal Characteristics in Rams Using Agri-Food By-Products Rich in Phytomelatonin

The aim of this study was to evaluate the effect of a phytomelatonin-rich diet, including by-products from the food industry, on ram sperm quality and seminal plasma composition. Melatonin content in several by-products before and after in vitro ruminal and abomasal digestion was determined by HPLC-ESI-MS/MS. Finally, 20% of a mix of grape pulp with pomegranate and tomato pomaces was included in the rams' diet, constituting the phytomelatonin-rich diet. Feeding the rams with this diet resulted in an increase in seminal plasma melatonin levels compared with the control group (commercial diet) in the third month of the study. In addition, percentages higher than those in the control group of morphologically normal viable spermatozoa with a low content of reactive oxygen species were observed from the second month onwards. However, the antioxidant effect does not seem to be exerted through the modulation of the antioxidant enzymes since the analysis of the activities of catalase, glutathione reductase and glutathione peroxidase in seminal plasma revealed no significant differences between the two experimental groups. In conclusion, this study reveals, for the first time, that a phytomelatonin-rich diet can improve seminal characteristics in rams.

Inhibition of extracellular ice crystals growth for testing the cryodamaging effect of intracellular ice in a model of ram sperm ultra-rapid freezing

ABSTRACT We aimed to test the common belief, that as the sperm cell is the smallest cell in an organism and, therefore, it contains a small amount of intracellular water, the intracellular ice should not strongly affect the sperm cell during the cryopreservation. To investigate the effect of intracellular ice, we developed a novel approach of ram spermatozoa ultra-rapid freezing with the inhibition of the growth of extracellular ice crystals during freezing. In our approach, ram sperm was supplemented with cell-impermeable synthetic ice blocker 1,4-Cyclohexanediol, leaving a sample unsupplemented as a control. Sperm in cryostraws were ultra-rapidly frozen, and then thawed. We hypothesized, that only in the case when the intracellular ice plays the role in the cryodamage, the post-freezing intactness of spermatozoa frozen in the presence of extracellular ice inhibitors should be equal (or even less) to the intactness of spermatozoa frozen without extracellular inhibitors. No statistically significant difference (p = 0.98) was observed between the post-freezing intactness of sperm ultra-rapidly frozen in the presence of 1,4-Cyclohexanediol and the intactness of sperm frozen without 1,4-Cyclohexanediol. We concluded that the intracellular ice plays the role in sperm cryodamage, at least in the model of ram sperm ultra-rapid freezing. Highlights Here, we investigated the cryodamaging factors during ram sperm ultra-rapid freezing; Optimized ultra-rapid freezing will provide positive economic implications for the sheep industry worldwide.

Post-thaw quality of ram sperm frozen with different concentrations of low-density lipoproteins associated with non-enzymatic antioxidants.

The cryopreservation reduces ram sperm quality, decreasing the pregnancy rate of ewes inseminated with thawed sperm. Hence, we aimed to improve the post-thaw quality of ram sperm replacing egg yolk on Tris-Glucose extender with different concentrations of LDL (2 or 8%), associated with the addition of 10 mM non-enzymatic antioxidants (ascorbic acid, hydroxytoluene butylate, ascorbyl palmitate, and trehalose). Semen samples were collected from six rams, split into different treatments, and frozen. After thawing, kinematic (CASA), structural (propidium iodide and carboxyfluorescein diacetate) and functional (hypoosmotic test) sperm membrane integrity was assessed. Total motility, VCL, and LIN were also assessed in thawed samples during 3 h of incubation (38 °C). The results showed that hydroxytoluene butylate at 10 mM in Tris-Glucose extender with 8% LDL improved velocity parameters immediately post-thaw compared with Tris-Glucose egg yolk extender, as well as prevented the reduction of total motility and VCL after incubation. There was no benefit of adding ascorbic acid and trehalose. Moreover, for the first time, it was shown the motility impairment promoted by ascorbyl palmitate to ram sperm.

Influence of hypoxanthine concentrations on capacitation, acrosome reaction and In vitro fertilization in Ram Spermatozoa

The aim of the study was to evaluate the effect of hypoxanthine concentrations and incubation time on motility, hyperactivity (HA) and acrosome reaction (AR) in ram sperm in vitro. Semen samples (n= 40) were collected using artificial vagina from 3 mature Barki rams. Freshly ejaculated spermatozoa were pooled and subjected to swim up technique in modified sperm Tyrode's albumin lactate pyruvate (S-TALP) medium supplemented with different concentrations of hypoxanthine (5, 10, 15, 20 mg/ml). Motility, hyperactivity percentage and acrosomal status were recorded at 0, 1, 2, 3 and 4 h post-incubation. They were examined for ability to fertilize oocyte in vitro. The results showed that hypoxanthine concentrations and incubation time for 2 h did not significantly affect the motility. However, increase in incubation time for >2 h significantly (P<0.05) decreased the motility of spermatozoa as compared to control. Treatment of spermatozoa with 20 mg/ml hypoxanthine significant (P<0.05) increased in HA after 2 h of incubation compared to control (5.2±0.3 vs. 1.0± 0.1%). Significant (P<0.05) increase in total AR was associated to the increase in concentration of hypoxanthine with maximum at 20 mg/ml at 3h of incubation (82.0±2.2%). The fertilization rate of oocytes treated with 20 mg/ml hypoxanthine was higher as compared with control (42.8 vs. 26.0%). In conclusion, treatment of ram spermatozoa with 20 mg/ml hypoxanthine for 2 to 3 h was considered the best concentration for in vitro fertilization in sheep.

A Pilot Study on the Uptake of Propidium Iodide and YO-PRO-1 Iodide through the Pannexin Channels in Wallachian Frozen-Thawed Ram Spermatozoa.

Propidium iodide (PI) and YO-PRO-1 (YPI) dyes are routinely used to determine sperm viability in many livestock species. It is commonly accepted that these dyes penetrate only sperm cells with damaged plasma membranes. Recently, however, the mechanism of dye uptake unrelated to damaged plasma membranes, but instead related to pannexin channels in dog and stallion sperm cells was demonstrated. This pilot study aimed to evaluate the role of pannexins in the uptake of PI and YPI dyes on Wallachian frozen-thawed ram spermatozoa by flow cytometry using probenecid, a specific inhibitor of pannexin channels. Additionally, the expression of pannexins in Wallachian sperm was evaluated directly (by qRT-PCR). The results demonstrate the active role of pannexin channels in the uptake of PI and YPI dyes on frozen-thawed Wallachian ram sperm. In conclusion, when using the PI or YPI exclusion assay to determine Wallachian frozen-thawed ram sperm viability, the danger of overestimating the number of spermatozoa with the damaged plasma membrane must be considered. The observed breed-specific, and more importantly, individual differences in gene expression as well as in dye uptake indicate the need for further studies.

The Addition of Opuntia ficus-indica Ethanolic Extract to a Skimmed Milk-Based Extender Impacts Ram Sperm Quality.

Recently, researchers have focused on the use of natural antioxidants to improve semen quality as a key element for successful artificial insemination. In this context, the first aim of this study was to determine the antioxidant activity and composition (minerals, vitamins, and sugars) of Opuntia ficus-indica cladode ethanolic extract (ETHEX). A further purpose of the study was to investigate the effect of ETHEX supplementation on the quality of liquid ram semen extended with skim milk (SM) at 5°C. The antioxidant activity of ETHEX was studied using free radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH•) assay. The mineral composition and the sugar and vitamin contents of ETHEX were determined using an inductively coupled plasma optical emission spectrometry (ICP-OES) and HPLC-DAD-RID analytical instruments. As a second part, semen was collected from five Boujaâd rams with an artificial vagina. The ejaculates with more than 70% motility were pooled, extended with skim milk (SM) extender without (control) or supplemented with 1-8% of ETHEX (37°C; 0.8 × 109 sperm/mL). Sperm quality parameters were assessed at 8, 24, 48, and 72 h. The results showed that ETHEX had a higher antioxidant activity compared to those of ascorbic acid and butylated hydroxytoluene (BHT). Furthermore, ETHEX contains a considerable amount of minerals, vitamins, and sugars. The inclusion of 1 or 2% ETHEX in SM increased the sperm motility, viability, and membrane integrity and decreased the abnormality of spontaneous and catalyzed lipids peroxidation (p < 0.05) up to 72 h. In addition, semen diluted with 1 and 2% ETHEX decreased the level of DNA fragmentation compared to the control group (p < 0.05). In conclusion, the ETHEX could be recommended to improve the quality of liquid ram spermatozoa. However, its effects on artificial insemination should be further studied.

The effect of a single dose of selenium with vitamin E on oxidative stress and fertilization indicators of ram sperm over the course of spermatogenesis

The aim of the experiment was to determine the effect of a single subcutaneous administration of selenium (Se) + vitamin E on the ejaculate volume, sperm count and viability, level of apoptosis and oxidative stress (OS) and Se concentration in ejaculates and blood of rams with respect to the time course of the spermatogenic cycle. The experimental group (EG; n = 6) was treated with a single injection of Selevit at a dose of 5 ml per animal (11 mg of sodium selenite and 125 mg of vitamin E/per animal). The control group (CG; n = 4) was treated in the same way, only with saline. Samples of blood and ejaculates were collected from each ram prior to application of Se and on the 1st, 14th, 26th, 38th, 50th and 62nd day after injection. Results showed that the Se concentration in the blood of EG was significantly higher only 24 h after application. Selenium concentration in the ejaculates of the EG was higher during the whole duration of the experiment (62 days), but significantly so only until day 14. The level of OS was significantly reduced on day 1, 14, and 62 after application of Selevit. There were no significant differences in the other analysed indicators. The results showed that one subcutaneous injection of Selevit had a positive effect on Se concentration and OS level in ejaculates, but was not sufficient to improve other monitored sperm quality indicators.

Effect of mitochondria-targeted antioxidant Mito-TEMPO addition to the sperm herbal extender on ram chilled sperm quality

The aim of this study was to evaluate the sperm quality preservation in sheep using mitochondria-targeted antioxidant Mito-TEMPO during chilling process. In this experiment, semen samples were collected from five Zandi rams and added to the extender containing 0, 0.5, 1, 5, 50 and 500 µM Mito-TEMPO and stored at 4 ˚C until 48 hours. Sperm quality was evaluated at 0, 24 and 48 hours after chilling and total motility, progressive motility, viability, membrane integrity, mitochondrial activity and lipid peroxidation were assessed. No difference was observed between treatments at time 0 of cooling. During 24 and 48 hours cooling periods, using 5 and 50 µM Mito-TEMPO increased total motility, progressive motility, viability, membrane integrity, mitochondrial activity and decreased lipid peroxidation in ram chilled sperm. In conclusion, using 5 and 50 µM Mito-TEMPO in chilling herbal extender could be a suitable method to conserve ram sperm quality during chilling process.

Effects of Extenders Supplementation with Gum Arabic and Antioxidants on Ram Spermatozoa Quality after Cryopreservation

Semen cryopreservation is very important in animal agriculture to maximize the number of daughters of genetically superior males and to distribute the cryopreserved semen of good males all over the world. However, the freezing process generates some damage to sperm that reduce their fertilizing ability after thawing. Moreover, egg yolk, which is the most common animal-origin cryoprotectant used in semen dilution, is considered a source of biosecurity risk. In the current study, we aimed to compare the replacement of egg yolk in the extender by gum arabic (5%) along with supplementation with antioxidant cysteine or ascorbic acid on semen quality and freezability in Noemi rams in vitro. Semen from six rams were collected with an artificial vagina two times per week. Semen evaluation parameters such as color, volume, pH, general motility, percentage motility, concentration and cell viability ratio were assessed. Spermatozoa motility and concentration were estimated with the computer-assisted semen analysis system. The semen samples were frozen using a Tris extender containing either 15% egg yolk or 5% gum arabic. For antioxidant-supplemented extenders, cysteine or ascorbic acid was dissolved at concentrations of 0.10, 0.50 or 1.0 mM in egg yolk or gum arabic extender. The semen from each ejaculate of each ram were resuspended with a specific extender with glycerol (5%); the final volume after dilution was 1 mL semen to 4 mL extender. The samples were then cooled to 4 °C for 120 min, loaded into 0.5 mL straws and frozen in liquid nitrogen for 7 days. Supplementation of gum arabic or egg yolk extenders for ram semen with antioxidants such as cysteine or ascorbic acid has beneficial effects on semen quality after cold storage or cryopreservation. However, supplementation of a 5% gum arabic extender with cysteine at 0.5 or 1 mM concentration or ascorbic acid at 0.5 mM concentration improved the quality of spermatozoa postcryopreservation. It could be concluded that gum arabic is a good alternative for egg yolk in Noemi ram semen extenders. Antioxidants are necessary to support the addition of gum arabic to the extender to help the ram spermatozoa to survive freezing-thawing and oxidative stresses.

Changes on proteomic and metabolomic profiling of cryopreserved sperm effected by melatonin

Cryopreservation may reduce sperm fertility due to cryodamage including physical-chemical and oxidative stress damages. As a powerful antioxidant, melatonin has been reported to improve cryoprotective effect of sperm. However, the molecular mechanism of melatonin on cryopreserved ram sperm hasn't been fully understand. Give this, this study aimed to investigate the postthaw motility parameters, antioxidative enzyme activities and lipid peroxidation, as well as proteomic, metabolomic changes of Huang-huai ram spermatozoa with freezing medium supplemented with melatonin. Melatonin was firstly replenished to the medium to yield five different final concentrations: 0.1, 0.5, 1.0, 1.5, and 2.0 mM. A control (NC) group without melatonin replenishment was included. Protective effects of melatonin as evidenced by postthaw motility, activities of T-AOC, T-SOD, GSH-Px, CAT, contents of MDA, 4-HNE, as well as acrosome integrity, plasma membrane integrity, with 0.5 mM being the most effective concentration (MC group). Furthermore, 29 differentially abundant proteins involving in sperm functions were screened among Fresh, NC and MC groups of samples (n = 5) based on the 4D-LFQ, with 7 of them upregulated in Fresh and MC groups. 26 differentially abundant metabolites were obtained involving in sperm metabolism among the three groups of samples (n = 8) based on the UHPLC-QE-MS, with 18 of them upregulated in Fresh and MC groups. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the abundance changes of vital proteins and metabolites related to sperm function. Particularly, several proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine may be potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa. In conclusion, a dose-dependent replenishment of melatonin to freezing medium protected ram spermatozoa during cryopreservation, which can improve motility, antioxidant enzyme activities, reduce levels of lipid peroxidation products, modify the proteomic and metabolomic profiling of cryopreserved ram spermatozoa through reduction of oxidative stress, maintenance of OXPHOS and microtubule structure. SignificanceMelatonin, a powerful antioxidant protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner, with 0.5 mM being the most effective concentration. Furthermore, sequencing results based on the 4D-LFQ combined with the UHPLC-QE-MS indicated that melatonin modifies proteomic and metabolomic profiling of ram sperm during cryopreservation. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the expression changes of vital proteins and metabolites related to sperm metabolism and function. Particularly, several potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa were acquired, proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine.

In vitro approach points to a chemotactic effect of melatonin on ram spermatozoa

Sperm orientation mechanisms, such as chemotaxis, are essential for the sperm to reach the oocyte and fertilize it. Melatonin is secreted by the cumulus cells and is also present in the follicular fluid in mammals. The presence of membrane receptors for melatonin in ram spermatozoa, and its proven involvement in the sperm functionality, may suggest a possible role in the guided movement towards the oocyte. Hence, the objective of the present work is to study the in vitro potential chemotactic action of melatonin on ram spermatozoa, analysing the influence of the season (breeding and non-breeding) and the sperm capacitation state. The first experimental approach consisted in the inclusion of melatonin in the upper layer of a swim-up selection method. During the non-breeding season, the presence of melatonin at 100 pM and 1 μM concentrations significantly increased the cell recovery rate, and induced changes in the sperm location of the MT2 melatonin receptor, compared with the standard swim-up. Moreover, the selected sperm population with 100 pM melatonin presented a higher percentage of capacitated spermatozoa. The greater recovery rate obtained with melatonin could be due to the stimulation of sperm movement in random directions, i.e., a chemokinetic effect, or due to a guided movement (chemotaxis) towards the gradient of the melatonin. To elucidate this issue, together with the study of the influence of the sperm capacitation status, we performed a second experimental approach which consisted in the use of chemotaxis chambers and an open-source software (Open-CASA) that analyses the sperm trajectories towards the hormone gradient and calculates a chemotaxis index (SL index). There was a significant difference between the SL index in the presence of 1 μM melatonin and the control without hormone. This effect was only observed in capacitated spermatozoa with cAMP-elevating agents (Cap-CK samples) obtained during the non-breeding season. These results would point to an in vitro chemotactic effect of melatonin on ram spermatozoa, although chemokinesis cannot be ruled out. Nonetheless, the inclusion of this hormone in the swim-up procedure could enhance the sperm recovery rate.