Dilution rate, dilution temperature and storage time have been recognized as vital steps in the processing of semen for storage before artificial insemination. The objective of this study was to ...determine optimal dilution and dilution temperature with an ostrich-specific semen extender for chilled storage. Four preselected ostrich (Struthio camelus var. domesticus) males, known for their ease of collection and specific semen quality parameters, were collected using the “dummy” female method. Dilution of 384 semen samples, at rates of 1:1, 1:2, 1:4 and 1:8 semen/diluent ratio with a diluent set at 5, 21 and 38 °C was performed and stored for 48 h at 5 °C. In vitro sperm function tests were conducted to evaluate treated semen during different storage intervals of 1, 5, 24 and 48 h. Motility and kinematic parameters were measured by the Sperm Class Analyzer®, the percentage live sperm measured by fluorescence SYBR14®/PI (LIVE/DEAD®), the percentage of sperm able to resist the hypo-osmotic swelling (HOS) stress test and sperm morphology determined by Nigrosin-Eosin staining. Progressive motility (PMOT), motility (MOT), sperm kinematics, LIVE and HOS were best (P < 0.05) maintained at a higher dilution of 1:4–1:8. The beneficial effect (P < 0.05) of a higher dilution temperature (21 °C) was prominent in terms of PMOT at a higher dilution. Storage of chilled semen at 5 °C requires dilution, at interpolated rates of 1:6–1:7, together with an extender temperature of 21 °C, to maintain optimal sperm function with minimal loss over a 48 h storage period.
•An interpolated dilution rate of 1:6–1:7 maintained sperm function best.•Sperm cell membrane integrity was most affected by dilution rate.•Higher dilution temperatures of 21 °C were optimal for 24 h storage at 5 °C.•Ostrich sperm demonstrated a degree of thermal sensitivity with higher cooling rates.
BACKGROUND Semen quality is taken as a surrogate measure of male fecundity in clinical andrology, male fertility, reproductive toxicology, epidemiology and pregnancy risk assessments. Reference ...intervals for values of semen parameters from a fertile population could provide data from which prognosis of fertility or diagnosis of infertility can be extrapolated. METHODS Semen samples from over 4500 men in 14 countries on four continents were obtained from retrospective and prospective analyses on fertile men, men of unknown fertility status and men selected as normozoospermic. Men whose partners had a time-to-pregnancy (TTP) of ≤12 months were chosen as individuals to provide reference distributions for semen parameters. Distributions were also generated for a population assumed to represent the general population. RESULTS The following one-sided lower reference limits, the fifth centiles (with 95th percent confidence intervals), were generated from men whose partners had TTP ≤ 12 months: semen volume, 1.5 ml (1.4–1.7); total sperm number, 39 million per ejaculate (33–46); sperm concentration, 15 million per ml (12–16); vitality, 58% live (55–63); progressive motility, 32% (31–34); total (progressive + non-progressive) motility, 40% (38–42); morphologically normal forms, 4.0% (3.0–4.0). Semen quality of the reference population was superior to that of the men from the general population and normozoospermic men. CONCLUSIONS The data represent sound reference distributions of semen characteristics of fertile men in a number of countries. They provide an appropriate tool in conjunction with clinical data to evaluate a patient's semen quality and prospects for fertility.
There is a lack of information regarding lipid peroxidation and antioxidant capacity in cryopreserved ram semen, and cryopreservation is associated with the production of reactive oxygen species ...(ROS) which lead to lipid peroxidation (LPO) of sperm membranes, resulting in a loss of motility, viability and fertility of sperm. The aim of this study was to determine the influence of certain additives and their different doses on standard semen parameters, lipid peroxidation and antioxidant activities after the cryopreservation/thawing of ram semen. Ejaculates collected from four Akkaraman rams, a native breed of sheep, were evaluated and pooled at 33 °C. Semen samples which were diluted with a Tris-based extender containing additives including trehalose (50, 100 mM), taurine (25, 50 mM), cysteamine (5, 10 mM), and hyaluronan (0.5, 1 mg/ml), and an extender containing no additives (control) were cooled to 5 °C and frozen in 0.25 ml French straws, being stored in liquid nitrogen. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The use of a Tris-based extender supplemented with 50 mM trehalose, 25 mM taurine, and 5 and 10 mM cysteamine led to higher percentages of post-thaw motility, in comparison to the control group (P < 0.01). No significant differences were observed in the percentages of acrosome and total abnormalities, and the hypoosmotic swelling test upon the supplementation of the freezing extender with antioxidants after the thawing of semen. In biochemical assays, the addition of antioxidants did not cause significant differences in levels of malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-Px), after thawing, when compared to groups with no additives. In this study, catalase (CAT) activities were higher in the group that was applied 25 mM taurine as an antioxidant, than in all of the other groups (P < 0.001). Compared to the controls, antioxidant treatment with 100 mM trehalose, 50 mM taurine, 5 mM cysteamine and 0.5 mg/ml hyaluronan, significantly elevated vitamin E (vit E) levels in samples (P < 0.001).
This study sought to determine whether sperm irradiation using a light emission diode (LED) at 620–630 nm affects the motility, membrane integrity (viability), mitochondrial activity and ...intracellular levels of reactive oxygen species (ROS) in fresh diluted and liquid-stored donkey semen. With this purpose, sixteen ejaculates (eight fresh diluted and eight cooled-stored) were collected from eight adult jackasses. Fresh semen samples were diluted in Kenney extender and stimulated with red-light after collection, whereas cooled semen was stored at 4 °C for 24 h after dilution and then irradiated. In all cases, semen samples were packed into 0.5-mL transparent straws, which were then randomly divided into control and 19 treatments: six consisted of single red-light exposure, and the other 13 involved irradiation at light-dark-light intervals. Upon irradiation, sperm motility, membrane integrity mitochondrial membrane potential (MMP) and intracellular levels of superoxide anion (·O2−) and hydrogen peroxide (H2O2) were evaluated. While specific light-patterns increased both sperm motility and mitochondrial activity, they did not affect sperm membrane integrity and had no clear impact on intracellular ROS levels. The effects of irradiation patterns differed between fresh and cooled semen since, whereas 1 and 4 min patterns induced the greatest increments in the total and progressive motility of fresh semen, 4 min, 4-1-4 and 4-4-4 were the most suitable for cooled-stored samples. In both fresh diluted and cooled-stored semen, the motility increase observed after light-stimulation for 4 min was concomitant with changes in the percentages of spermatozoa with high mitochondrial membrane potential. In summary, this study shows, for the first time, that specific irradiation patterns increase sperm motility and mitochondrial activity in the donkey. Furthermore, the precise effect of red-light appears to depend on the specific functional status of cells, with separate effects on fresh and cooled samples.
•Red-light stimulation increases donkey sperm motility.•Red-light stimulation increases mitochondrial membrane potential of donkey sperm.•The effects of red-light stimulation differ between freshly diluted and cooled semen.•Single exposure to red-light for 4 min works for both fresh and cooled donkey sperm.
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.
Our objectives were to study the effect of trehalose in extender on (1) antioxidant enzymes profile during cryopreservation (after dilution, before freezing, and after thawing), (2) in vitro quality ...(after thawing), and (3) in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 20) from four buffalo bulls were diluted in Tris-citric acid–based extender having different concentrations of trehalose (0.0, 15, 30, 45, and 60 mM) and frozen in French straws. At post dilution, profile of sperm catalase (U/mL) was higher (P < 0.05) in extenders containing 15, 30, and 45 mM of trehalose as compared to control. Although profiles of superoxide dismutase (U/mL) and total glutathione (μM) were higher (P < 0.05) in extenders containing 15 and 30 mM of trehalose as compared to control. At prefreezing, sperm catalase, superoxide dismutase, and total glutathione profiles were higher (P < 0.05) in all the treatment groups as compared to control. At post thawing, the profiles of catalase and total glutathione were higher (P < 0.05) in extender containing 30-mM trehalose as compared to other treatment groups and control. Whereas, profile of superoxide dismutase was higher (P < 0.05) in extenders containing 30, 45, and 60 mM of trehalose as compared to control and 15mM group. Post thaw total sperm motility (%) was higher (P < 0.05) in extender containing 30-mM trehalose as compared to control and 15 and 60-mM groups. Although sperm progressive motility (%), rapid velocity (%), average path velocity (μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), plasma membrane (structural and functional, %), acrosome (%), and DNA (%) integrity were higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment groups and control. The fertility rates (61% vs. 43%) were higher (P < 0.05) in buffaloes inseminated with semen doses cryopreserved in extender containing 30 mM of trehalose than the control. It is concluded that addition of 30-mM trehalose in extender improves the semen antioxidant enzymes activity, post thaw quality, and fertility in Nili Ravi buffaloes.
This comprehensive review delves into the evolving landscape of assisted reproductive technologies (ARTs) in bovine species, particularly focusing on the pivotal roles of semen additives in the ...cryopreservation of buffalo and cattle semen. In developing nations, where ARTs are still emerging, these techniques significantly influence bovine reproductive strategies. In contrast, developed regions have embraced them as primary approaches for dairy buffalo and cattle breeding. Semen cryopreservation, while offering advantages like extended storage and genetic propagation, also presents challenges. These include diminished sperm quality due to reactive oxygen species (ROS) production, alterations in sperm structure, and temperature fluctuations. Further, the effect of cryopreservation differs between cattle and buffaloes, with the latter exhibiting poorer semen viability and fertility due to inherent lipid composition susceptibilities. The generation and implications of ROS, especially hydrogen peroxide, contribute significantly to sperm DNA damage and functional impairments. To counteract these challenges, research has intensified on semen additives, aiming to bolster semen quality and protect against oxidative stress-induced damage. As the field advances, the review emphasizes the need for optimized cryopreservation techniques and tailored antioxidant strategies to harness the full potential of ARTs in bovine breeding programs. Doi.org/10.54680/fr24410110112.
Inhibiting oxidative stress is key for ensuring sperm motility during semen cryopreservation. The aim of this study was to investigate the effect of adding alpha-lipoic acid (ALA) as an extender in ...rooster semen cryopreservation. Different concentrations of ALA were added to the frozen diluent of rooster semen; subsequently, computer-aided semen analysis was used to determine membrane functional integrity, acrosome integrity, antioxidant capacity (based on T-AOC, GSH-Px, SOD, CAT, and MDA contents), and mitochondrial integrity. The frozen sperm ultrastructure was observed using transmission electron microscopy. The results showed that the addition of different concentrations of ALA partially to greatly improved the quality of frozen sperm; in particular, 8 μg/mL ALA significantly improved multiple parameters of sperm quality, including sperm motility and antioxidant enzyme activity, after freeze–thaw. The results of this study provide empirical and theoretical support for effective rooster semen cryopreservation and can inform the development of new protective agents in the field of livestock reproduction.
Abstract
BACKGROUND
Numerous studies have reported declines in semen quality and other markers of male reproductive health. Our previous meta-analysis reported a significant decrease in sperm ...concentration (SC) and total sperm count (TSC) among men from North America–Europe–Australia (NEA) based on studies published during 1981–2013. At that time, there were too few studies with data from South/Central America–Asia–Africa (SAA) to reliably estimate trends among men from these continents.
OBJECTIVE AND RATIONALE
The aim of this study was to examine trends in sperm count among men from all continents. The broader implications of a global decline in sperm count, the knowledge gaps left unfilled by our prior analysis and the controversies surrounding this issue warranted an up-to-date meta-analysis.
SEARCH METHODS
We searched PubMed/MEDLINE and EMBASE to identify studies of human SC and TSC published during 2014–2019. After review of 2936 abstracts and 868 full articles, 44 estimates of SC and TSC from 38 studies met the protocol criteria. Data were extracted on semen parameters (SC, TSC, semen volume), collection year and covariates. Combining these new data with data from our previous meta-analysis, the current meta-analysis includes results from 223 studies, yielding 288 estimates based on semen samples collected 1973–2018. Slopes of SC and TSC were estimated as functions of sample collection year using simple linear regression as well as weighted meta-regression. The latter models were adjusted for predetermined covariates and examined for modification by fertility status (unselected by fertility versus fertile), and by two groups of continents: NEA and SAA. These analyses were repeated for data collected post-2000. Multiple sensitivity analyses were conducted to examine assumptions, including linearity.
OUTCOMES
Overall, SC declined appreciably between 1973 and 2018 (slope in the simple linear model: –0.87 million/ml/year, 95% CI: –0.89 to –0.86; P < 0.001). In an adjusted meta-regression model, which included two interaction terms time × fertility group (P = 0.012) and time × continents (P = 0.058), declines were seen among unselected men from NEA (–1.27; –1.78 to –0.77; P < 0.001) and unselected men from SAA (–0.65; –1.29 to –0.01; P = 0.045) and fertile men from NEA (–0.50; –1.00 to –0.01; P = 0.046). Among unselected men from all continents, the mean SC declined by 51.6% between 1973 and 2018 (–1.17: –1.66 to –0.68; P < 0.001). The slope for SC among unselected men was steeper in a model restricted to post-2000 data (–1.73: –3.23 to –0.24; P = 0.024) and the percent decline per year doubled, increasing from 1.16% post-1972 to 2.64% post-2000. Results were similar for TSC, with a 62.3% overall decline among unselected men (–4.70 million/year; –6.56 to –2.83; P < 0.001) in the adjusted meta-regression model. All results changed only minimally in multiple sensitivity analyses.
WIDER IMPLICATIONS
This analysis is the first to report a decline in sperm count among unselected men from South/Central America–Asia–Africa, in contrast to our previous meta-analysis that was underpowered to examine those continents. Furthermore, data suggest that this world-wide decline is continuing in the 21st century at an accelerated pace. Research on the causes of this continuing decline and actions to prevent further disruption of male reproductive health are urgently needed.
Graphical Abstract
Graphical Abstract
Sperm count is declining at an accelerated pace globally.
The use of antioxidants is an important aspect in the preservation of boar semen. Oligomeric proanthocyanidins (OPC) are an effective natural antioxidants that scavenges free radicals and reactive ...oxygen species (ROS). This study was designed to investigate the antioxidative effect of OPC on boar semen quality during liquid preservation at 17 °C. The effects of different concentrations of OPC on sperm quality variables, antioxidant effects and fertility were analyzed in this experiment. Semen collected from six Guanzhong-Black boars and was diluted with Beltsville thawing solution (BTS). During the process of liquid preservation at 17 °C, the variables assessed were measured and analyzed every 24 h. The addition of OPC improved boar sperm motility, acrosome integrity, membrane integrity, and mitochondrial membrane potential as compared with that of the control group (P < 0.05). Meanwhile, malondialdehyde content (MDA) and ROS content was less after adding OPC, thereby improving the total antioxidant capacity (T-AOC) (P < 0.05). Different concentrations of OPC have different degrees of protective effects on boar semen quality. The results indicate that 50 μg/mL of OPC was the optimum concentration, and that the conception rate, litter size, and survival rate increased at this concentration as compared with that of the control group (P < 0.05). In summary, the addition of OPC to BTS diluents can improve the quality of boar semen at 17 °C during liquid preservation. Further research is needed to understand the mechanism by which OPC provides protection to boar semen during preservation in vitro.
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