Freezing Hu ram semen: influence of different penetrating cryoprotectants and egg yolk level on the post-thaw quality of sperm.

OBJECTIVE: The Hu sheep is a renowned breed known for its high reproductive rate. It is in estrus all year round, and its breeding population is gradually expanding. However, the current techniques for cryopreserving semen have limited effectiveness, which hinders the continuous development of this species. The purpose of this study is to explore the effects of different penetrating cryoprotectants (CPAs) and egg yolk (EY) concentrations on the cryopreservation of Hu ram semen to determine the most effective combination. METHODS: In this study, the effects of glycerol (GLY), ethylene glycol (EG), dimethylacetamide, dimethyl sulfoxide, different proportions of GLY and EG, EY on sperm quality after thawing were investigated by detecting sperm total motility (TM), progressive motility (PM), straight-line velocity, curvilinear velocity, average path velocity, amplitude of lateral head displacement, wobble movement coefficient, average motion degree, functional integrity (plasma membrane integrity, acrosome integrity) and reactive oxygen species (ROS) level. RESULTS: When GLY and EG were added together, compared to other concentration groups, 6% GLY significantly (p<0.05) increased TM, PM, plasma membrane integrity, and acrosome integrity of thawed sperm. Additionally, it significantly (p<0.05) decreased the ROS level of sperm. In this study, the TM, PM, and membrane integrity of the 6% EG were significantly (p<0.05) higher than those of the control, 1% GLY+5% EG and 6% GLY+6% EG groups. Compared to other concentration groups, 20% EY significantly (p<0.05) improved the TM, PM, and plasma membrane integrity of thawed sperm. However, the integrity of the acrosome increased with the higher concentration of EY. CONCLUSION: In conclusion, the post-thawed Hu ram semen diluted with a diluent containing 6% GLY and 20% EY exhibited higher quality compared to the other groups.

Adjusting ram semen preservation: Exploring the impact of oxygen exposure during liquid storage.

This study investigated the effects of storage conditions on the quality of chilled ram semen stored at 4°C for 48 h, comparing aerobic and anaerobic conditions. Ejaculates from INRA180 rams were collected and stored under both conditions, with assessments at 0-, 24-, and 48-h intervals. Various sperm parameters were examined, including motility, velocity, viability, morphology, membrane integrity, and lipid peroxidation. Results showed that storage duration significantly impacted sperm quality, leading to a gradual decline from 0 to 24 h and 24 to 48 h. Notably, after the initial 24 h, progressive motility (PM) and membrane integrity (MI) demonstrated distinct responses to storage conditions. Anaerobic storage consistently improved PM and MI values compared to aerobic storage between 24 and 48 h. Anaerobic conditions also enhanced viability and reduced abnormality at the 48-h mark. Total motility remained stable throughout storage. Velocity parameters (VCL: curvilinear velocity; VSL: straight velocity and VAP: velocity average path) exhibited differences between the 24- and 48-h intervals, with anaerobic storage resulting in higher VAP and VSL values. Moreover, lipid peroxidation exhibited a progressive increase from 0 to 24 h and 24 to 48 h, independent of storage conditions. Remarkably, anaerobic storage consistently yielded lower lipid peroxidation levels compared to aerobic storage, regardless of storage duration. In conclusion, this study highlights that the anaerobic storage proved advantageous for chilled ram semen quality, particularly after the initial 24 h.

Cryopreservation of ram semen: baicalein efficiency on oxidative stress, chromatin integrity, viability and motility post thaw.

Baicalein (B) has potential antioxidant properties, but it has not been tested as a ram semen extender. This study aimed to assess the impact of B on various sperm parameters and determine its potential influence on semen quality after the freeze-thawing process. During the breeding season, ejaculates were obtained from four rams with the aid of an artificial vagina. The collected mixed semen samples were divided into four groups: control (C; 0), B0.5 (0.5 mM), B1 (1 mM), and B2 (2 mM). After semen extension, the samples were loaded into 0.25 mL straws and stored for 2 h at 4°C prior to freezing in liquid nitrogen vapor and thawed in a water bath at 37°C. Among the groups, B0.5 demonstrated the highest progressive motility results, while B1 and B2 exhibited reduced motility (p < 0.05). In terms of high mitochondrial membrane potential, plasma membrane and acrosome integrity, and viability, B0.5 showed significantly superior outcomes to the other B groups (p < 0.05), although it was not significantly better than C. B1 displayed the highest plasma membrane integrity levels (p < 0.05). Notably, B2 displayed the lowest total antioxidant status levels among the groups (p < 0.05). The findings of this study suggested that the in vitro spermatological characteristics of ram spermatozoa such as progressive motility and chromatin integrity can be protected from the freeze-thawing process by using the 0.5 mM dose of baicalein as a semen extender. The treatment of sperm freezing might benefit from further in-depth research on the role of B in the improvement of cryoinjury and its underlying processes.

In vitro characteristics of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and TLR7/8 ligand R848.

The quality of the separated fractions in sex-sorted semen is very important for the success of the artificial insemination. This study aimed to evaluate some in vitro characteristics (DNA quantity, kinematic parameters and enzymes activity) of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and toll-like receptors (TLR)7/8 ligand R848. The ejaculates from six rams were collected by artificial vagina and subjected to a computer-assisted semen analysis (CASA). Total motility and percentage of the sperms with rapid and medium progressivity or non-progressivity in whole ejaculates and in X and Y fractions were analyzed. Activity of the enzymes ALP, GGT, CK, LDH and accumulation of lactate in the seminal plasma of ejaculates and in the environmental fluid of sexed spermatozoa were measured by biochemical analyzer. DNA was isolated from precipitated spermatozoa, and its quantity was measured. For both protocols the DNA mass from X-bearing fractions was higher, than from Y-bearing fractions. The high total motility of X- and Y-bearing spermatozoa as well as greater percent sperms with progressive motility were observed after use of BSA protocol. The application of TLR7/8 ligand R848 protocol led to reducing of Y-sperm motility and enhancement of non-progressivity in both fractions, which corresponded to the determined high amount of the extracellular lactate. For both methods, the significantly reduced activity of enzymes in the X and Y spermatozoa environmental fluids was established. Both protocols produce X- and Y-sperm fractions with satisfactory quality (over 80% total motility and over 50% rapid and medium progressive spermatozoa in each fraction).

Effects of different extenders, storage temperatures, and antioxidant supplementation on chilled semen quality: a review.

The successful preservation of ram semen is essential to promote genetic variability, ensure semen transportation, and inseminate multiple ewes. Currently, either animal or plant-based lipoprotein-based extenders are used for semen preservation. Animal product-based extenders include milk and egg yolk, while soybean lecithin is a plant-based extender. Although extenders containing products of animal origin better preserve the quality of chilled semen, the in vivo efficacy after 24 h of storage is still of great concern. Storage temperature is another important and effective factor in preserving sperm quality, whereby different storage temperatures are adopted to enhance the storage life of sperm. Low temperatures (4-5 °C) better preserve sperm quality for a longer duration than high temperatures (15, 20, and 25 °C). Moreover, antioxidant supplementation has a positive impact on sperm quality during liquid storage. The current review summarizes the outcomes of various extenders, different storage temperatures, and antioxidant supplementation on the liquid storage of ram sperm.

Heat stress and ram semen production and preservation: Exploring impacts and effective strategies.

As global warming persists, heat stress (HS) continues to affect animals, particularly those raised in extensive systems such as sheep. As a result, there is a growing body of research investigating the physiological and biological consequences of HS on these animals. Recent studies have specifically examined the effects of climate change, global warming, and HS on gametes. Heat stress has been shown to affect ram semen production, resulting in decreased sperm quality and volume in both fresh and stored samples. This is attributed to the effect of heat on hormone production in the testicles, which is critical for successful spermatogenesis. Such effects can have significant consequences on the fertility of female sheep, which could affect the farmers' revenue. Therefore, farmers and researchers are utilizing various strategies and laboratory techniques to mitigate these negative effects. This review aims to comprehensively evaluate the impact of HS on ram semen production and conservation and analyze the different mitigation strategies at various levels, including management and nutritional interventions. The findings of this review will serve as a critical foundation for the development of targeted interventions and sustainable practices in sheep farming, ensuring resilient and profitable operations in the face of ongoing global climate challenges.

Astaxanthin Improved the Quality of Hu Ram Semen by Increasing the Antioxidant Capacity and Mitochondrial Potential and Mitigating Free Radicals-Induced Oxidative Damage.

The objective of this research was to investigate the effect of astaxanthin supplementations of semen extender on the quality of Hu ram semen after up to five days of preservation at 4 °C. Semen samples were collected from five healthy Hu rams using an artificial vagina during breeding season (April to August 2023) and diluted with a basic extender supplemented with control (0), 1 µM, 2 µM, 3.5 µM, or 4.5 µM of AXT. Overall, 170 semen ejaculate samples (34 repetitions) from five healthy Hu rams were used in our research study. The results revealed that the addition of AXT (3.5 µM) significantly (p ≤ 0.05) increased the sperm kinematic indexes (T.M%, P.M%, MAD%, STR%, and LIN %), sperm viability, plasma membrane integrity, acrosome integrity, total antioxidant content (T-AOC), and mitochondrial membrane potential (MMP) of the Hu rams spermatozoa after up to five days of preservation at 4 °C. Contrary to that, the addition of the best concentration of AXT (3.5 µM) to the semen extender significantly (p ≤ 0.05) reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) concentration of Hu ram semen. In conclusion, the results of the current study indicate that the addition of a semen extender with AXT improves the quality of Hu ram spermatozoa by increasing the total antioxidant capacity (T-AOC) and mitochondrial membrane potential (MMP). On the other hand, reducing free radicals induced oxidative (ROS) and per oxidative (MDA) damage to Hu ram semen.

Evaluation of Cryopreserved Ram Sperm with Nano-Ozone Solution and Post-Thaw Life Span by Flow Cytometric Analysis.

Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 µg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 µg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) (p < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.

Fortification of Extender with Basella rubra Fruit Extract Enhances the Cryosurvival of Ram Semen.

This study aimed to evaluate the impact of Basella rubra fruit extract (BR-FE) on cryopreserved ram sperm's motility, velocity, and membrane integrity. Thirty ejaculates collected from 3 fertile rams (10 from each) were diluted with semen dilution extender (SDE) in a ratio (1:2) and centrifuged to remove 50% supernatant. The remaining sample was mixed with semen cryopreservation extender (SCE) in 1:4 ratio. Then 1.2 mL of SCE diluted sample was divided in four aliquots (0.3 mL each) that were further extended with [(1) control group (0.7 mL of SCE), (2) BR-FE-0.6% group (0.7 mL of SCE supplemented with 0.6% BR-FE), (3) BR-FE-0.8% group (0.7 mL of SCE supplemented with 0.8% BR-FE), and (4) BR-FE-1.6% group (0.7 mL SCE supplemented with 1.6% BR-FE)]. All extended samples were cooled gradually from 25°C to 4°C in half an hour. The 0.1 mL sample from all aliquots was analyzed for precryopreservation sperm parameters and the remaining sample was loaded in 0.5 mL plastic semen straws, cooled gradually to -20°C, and then dipped in liquid nitrogen. After 24 hours of cryopreservation, the straws were thawed for postcryopreservation sperm evaluations. The results (analysis of variance based) showed significantly enhanced percentage of post-thaw sperm membrane integrity, progressive motility, and velocity in BR-FE-0.6% group at both pre- and postcryopreservation stages as compared with all other groups. However, analysis of covariance revealed concentration-dependent cryoprotective effect of BR-FE with maximum percentage of sperm membrane integrity in the 1.6% group. According to these results, BR-FE supplementation adds enormous sperm protective potential to ram sperm cryopreservation medium.

Effect of C-phycocyanin purified from Spirulina platensis on cooled ram semen quality and in vivo fertility.

This research sought to purify C-phycocyanin (C-PC) from Spirulina platensis and investigate its potential in enhancing the quality parameters and in vivo fertility of ram semen subjected to cooled storage at 5 °C, when using a skim milk (SM) based semen extender. The purification process of C-PC involved cold maceration, pre-purification using chitosan and activated charcoal, followed by purification through aqueous two-phase extraction (ATPE) and ion-exchange chromatography. Afterward, fifty ejaculates were collected from 4 fertile Boujaâd rams and extended using the SM extender at 37 °C, enriched with 0 µg/mL (control), 1.2 µg/mL, 2.4 µg/mL, 3.6 µg/mL, or 4.8 µg/mL of C-PC. The diluted semen was subsequently cooled to 5 °C using a controlled cooling process, with a gradual cooling rate of approximately 0.5 °C per minute, and its quality parameters were evaluated after 0, 4, 8, and 24 h of cooling storage. Then, its fertilization ability after 4 h of cooling storage was evaluated using artificial insemination. The adopted purification process yielded a grade analytical purity of 4.06. Additionally, semen extended in SM with a 2.4 µg/mL C-PC supplement displayed significant (P < 0.0001) enhancement in total motility, progressive motility, curvilinear velocity, straight-line velocity, average path velocity, viability and lipid peroxidation of ram semen at 0, 4, 8, and 24 h of cooling storage. These improvements were observed in direct comparison to both the control group and the other C-PC concentrations. Regarding fertility rates, semen extended in SM with a 2.4 µg/mL C-PC recorded a 76 % rate, a notable increment from the 63 % observed in ewes inseminated by semen extended in SM alone, although the difference was not statistically significant (p > 0.05). These findings underscore the promising potential of C-PC as a natural supplement for enhancing semen quality, warranting further investigations.

Effect of pentoxifylline on the testicular hemodynamic, volume, testosterone, nitric oxide levels and semen quality in Ossimi rams during non breeding-season.

During the non-breeding season, Ossimi rams have testicular regression, including reductions in blood flow, size and spermatogenesis. The objective was to determine the effect of pentoxifylline (PTX) on Ossimi rams during the non-breeding season. Fifteen sexually mature Ossimi rams were allotted to three groups: (1) G0 (n = 5) control group (basic diet, no PTX); (2) G1 (n = 5) 10 mg/kg BW PTX; and (3) G2 (n = 5) 20 mg/kg BW PTX. The PTX was given orally once daily for 7 weeks (wk1 to wk7), whereas ultrasonographic assessment of testes, and collection of semen and blood started 1 week before PTX and were done weekly for 8 weeks (wk0 to wk7). In G2, there was a decrease(P < 0.05) in both Doppler indices (resistive index, pulsatility index) in G2 from wk2 to wk4 and an increase(P < 0.05) in ultrasonographic testicular coloration from wk2 to wk7. Moreover, G2 had the highest (P < 0.05) testicular volume (wk5 to wk7), individual motility, sperm viability and acrosome integrity (wk4 to wk7) and sperm cell concentration (wk6 and wk7). Blood concentrations of testosterone and nitric oxide were increased (P < 0.05) concurrent with decreased Doppler indices. In conclusion, PTX enhanced testicular blood flow and volume, semen quality, and concentrations of testosterone and nitric oxide potential in Ossimi rams during the non-breeding season, with potential to ameliorate deleterious effects of heat stress and perhaps enhance ram fertility.

Methionine, cysteine, and butylated hydroxytoluene enhance cryosurvival of ram semen on post-thaw and post-incubation time points.

Despite there have been many experiments conducted about antioxidants, the best sole or combination use of antioxidants to include as a standard ingredient to freezing extenders is yet to be found. This study was designed to investigate the different doses of methionine (2.5 and 5 mM), cysteine (1 and 2 mM), and butylated hydroxytoluene (BHT) (1 and 2 mM) for ram semen cryopreservation on post-thaw and post-incubation (6 h) time points over spermatological parameters. Semen samples were collected from Kivircik rams via electro-ejaculator in breeding season. After essential spermatological evaluations, appropriate samples were pooled then split into 7 equal aliquots to create study groups (antioxidant free control, 2.5 mM methionine, 5 mM methionine, 1 mM cysteine, 2 mM cysteine, 1 mM BHT, and 2 mM BHT). Semen samples were put into French straws (0.25 mL), and freezing procedure (two-step) was conducted via a programmable gamete freezer. At both time points, motility, HOST, PSA-FITC, and TUNEL assays were made to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results compared to the control groups in terms of various spermatological parameters not only at post-thaw time point but after incubation for 6 h of time. The study demonstrated that supplementing sperm freezing extenders with previous antioxidants may create new approaches to cryopreservation procedures, and through increasing success rate of freezing, fertility results may increase to better results in near future.

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.

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, 5 H 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.

Does Rainbow Trout Seminal Plasma-Cysteine Combination Affect the Cryo-Survivability and Post-Thaw Incubation Resilience of Ram Semen?

The aim of this study was to evaluate the effect of both pure rainbow trout seminal plasma (RTSP) supplementation and RTSP-cysteine combination on cryopreservation success and post-thaw incubation resilience of ram semen in the nonbreeding season. For this purpose, different doses of RTSP (0%, 1%, 10%, and 15%) with or without cysteine supplementation were used for experiments. Ejaculates chosen for experiments were pooled and then divided into eight equal volumes for grouping (Control-ControlC, RTSP1-RTSP1C, RTSP10-RTSP10C, and RTSP15-RTSP15C). After cryopreservation, frozen-thawed semen samples were incubated for 5 hours at 37°C for determination of post-thaw incubation resistance. Motility, HOST, TUNEL, Rh123-PI, and CTC tests were performed at 0 hour and 3rd and 5th hours of post-thaw incubation to evaluate the efficacy of all experimental groups. The RTSP10 and RTSP10C groups were noted to provide the best protection on motility, plasma membrane integrity, DNA integrity, and mitochondrial function of cryopreserved ram semen. On the other hand, the best protection against cryo-capacitation was observed in RTSP15 and RTSP15C groups. The addition of cysteine was found to be effective when the higher (15%) or lower (1%) doses of RTSP were used, as well as for no use of RTSP.

Thymoquinone improves motility, plasma membrane integrity and DNA integrity of frozen-thawed ram semen.

The aim of this study was to determine the effects of thymoquinone (TQ), which is the most essential active compound of Nigella sativa, on the spermatological parameters of ram semen during cryopreservation. Ejaculates were collected from five Sonmez rams using an artificial vagina and extended with Tris-based extender not containing TQ (control, 0 µg/ml TQ) and containing 10, 25, 50 and 100 µg/ml TQ. The extended semen samples were equilibrated in a + 4°C cold cabinet for 2 h. After 2 h, the samples were loaded into 0.25 ml French straws. The straws were frozen by liquid nitrogen vapour and stored in a liquid nitrogen container (-196°C). The frozen straws were thawed in a water bath (37°C for 30 s) and evaluated in terms of motility characteristics, plasma membrane and acrosome integrity, mitochondrial reactive oxygen species levels, lipid peroxidation levels, DNA damage and biochemical alterations (oxidative stress index, malondialdehyde and glutathione). TQ100 had higher total motility (53.59 ± 3.01) and progressive motility (19.84 ± 1.44; not significantly different from TQ25 and TQ50) compared to the control and TQ10 (p Ë‚ 0.05). According to the results of the analyses on motility characteristics, there were significant differences between the groups in terms of curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and linearity (LIN; p Ë‚ 0.05). The highest DNA damage was detected in the control group (p Ë‚ 0.05). TQ50 had higher plasma membrane and acrosome integrity (59.56 ± 5.92) compared to the control and TQ25 (p < 0.05) but not significantly different from TQ10 and TQ100. The lowest mitochondrial reactive oxygen species levels were detected in TQ50 and TQ100 (p Ë‚ 0.05). There were no significant differences among the groups in terms of their oxidative stress index, lipid peroxidation, malondialdehyde and glutathione levels (p > 0.05). According to the results, it could be concluded that supplementing 50 or 100 µg/ml TQ to Tris extenders that were used for ram semen cryopreservation showed a positive effect on motility, plasma membrane integrity and acrosome integrity, and it reduced DNA damage and mitochondrial reactive oxygen species levels.

Effect of hydrated C60 fullerene on lipid, vitamin and amino acid composition in frozen-thawed ram semen.

This study was conducted to investigate the effect of different doses of hydrated C60 fullerene (C60HyFn) on freeze-thawing process-induced changes in lipid, vitamin and amino acid composition and also in motility, kinematic, sperm quality and oxidative stress parameters in ram semen. Semen was collected from seven rams twice a week for 3 weeks, so six repetitions were performed. The semen collected in each repetition was pooled. Each pooled sample was diluted with tris + egg yolk extender with (200 nM, 400 nM, 800 nM, 1 µM and 5 µM) and without (control) C60HyFn and they were frozen in mini straws. The doses of 800 nM, 1 µM and 5 µM had higher total, progressive motility, sperm membrane functionality rates, glutathione-peroxidase and catalase activities. All doses of C60HyFn significantly reduced dead and total abnormal sperm rates and malondialdehyde levels. Significant increases in vitamin A (400 and 800 nM doses), vitamin K1 (400 nM, 800 nM and 1 µM doses), total amino acid (all doses) levels, but significant decreases in vitamin D2 (800 nM, 1 and 5 µM doses), vitamin D3 (1 and 5 µM doses) and vitamin E (200 nM, 1 and 5 µM) levels were observed compared to control. In conclusion, the addition of C60HyFn to ram semen at 200 nM - 5 µM range, especially at a dose of 800 nM, provides a positive contribution to the protection of motility, vitamins A, K and total amino acid levels, and oxidant/antioxidant balance after freeze-thawing.

Supplemental dietary zinc sulphate and folic acid combination improves testicular volume and haemodynamics, testosterone levels and semen quality in rams under heat stress conditions.

This study was aimed to investigate the combined effect of zinc sulphate and folic acid (ZnF) dietary supplementation on testicular haemodynamics (TH), testicular volume (TV), plasma testosterone levels (T) and semen quality of rams under heat stress conditions. Fifteen Ossimi rams were allocated to three groups: (1) G0 (n = 5) received only basic diet; (2) G1 (n = 5) received basic diet +ZnF (Zn, 0.4 mg/kg bw; F, 0.02 mg/kg bw) and (3) G2 (n = 5) received basic diet +ZnF (Zn, 0.8 mg/kg bw; F, 0.04 mg/kg bw) daily for 60 days. TH was evaluated using colour (testicular coloration, TC) and spectral modes [resistive index (RI) and pulsatility index (PI)] Doppler of the supra-testicular arteries (proximal and distal parts, STA). Semen traits including progressive motility (PM), alive sperm % (AS), sperm viability (SV), sperm abnormalities (SA) and acrosome integrity (AI) were also assessed. The examinations were carried out one month before (D-30), the beginning of ZnF inclusion in the diet (D 0) and continued for the successive two months (D 30 and D 60). TH was significantly (p < .05) improved at D 30 and D 60, evidenced by lowering both RI and PI and increasing of TC in G1 compared to G0 and G2. In addition, both TV and serum T levels were elevated (p < .05) at D 30 and D 60 in G1 compared to other groups. Semen quality parameters (PM, AS, SV and AI) were significantly (p < .05) augmented in the same trend as TH, TV and T in G1 versus G0 and G2. A marked decrease (p < .05) in SA % was noticed at Days 30 and 60 after ZnF inclusion in G1 compared to G0 and G2. In conclusion, supplementation of the summer diet with ZnF improved the whole reproductive functions such as testicular haemodynamics and semen quality of rams housed in heat stress conditions.

The quality and fertilizing ability of the ram semen cryopreserved around 40 years ago.

The aim of the study was to verify the quality of ram semen, frozen in 1982-1983, from the historical collection of the Bank of Biological Material of the National Research Institute of Animal Production. A total of 18 ejaculates from 3 Swiniarka type rams were analyzed to assess sperm motility (subjectively), total motility, progressive motility, sperm concentration (CASA), membrane integrity (SYBR-14/PI) and chromatin structure (SCSA). In order to determine sperm fertilizing ability 49 ewes were intracervically inseminated (200×106 sperm per AI) with frozen- thawed semen 12 and 24 hours after detection of estrus. Sperm motility parameters, membrane intact spermatozoa and DFI did not differ among the analyzed rams. Spermatozoa concentration was significantly higher for ram no. 2 than for rams no. 1 and 3. The lambing rates (27.3 to 36.0%) did not differ significantly for individual rams. The ram semen, which had been stored for around 40 years, showed satisfactory quality and fertilizing capacity, allowing for its use in artificial insemination.

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.