Semen Extender Research Articles

Extend the Survival of Human Sperm In Vitro in Non-Freezing Conditions: Damage Mechanisms, Preservation Technologies, and Clinical Applications.

Preservation of human spermatozoa in vitro at normothermia or hypothermia maintaining their functions and fertility for several days plays a significant role in reproductive biology and medicine. However, it is well known that human spermatozoa left in vitro deteriorate over time irreversibly as the consequence of various stresses such as the change of osmolarity, energy deficiency, and oxidative damage, leading to substantial limitations including the need for semen examinations, fertility preservation, and assisted reproductive technology. These problems may be addressed with the aid of non-freezing storage techniques. The main and most effective preservation strategies are the partial or total replacement of seminal plasma with culture medium, named as extenders, and temperature-induced metabolic restriction. Semen extenders consist of buffers, osmolytes, and antioxidants, etc. to protect spermatozoa against the above-mentioned adverse factors. Extended preservation of human spermatozoa in vitro has a negative effect on sperm parameters, whereas its effect on ART outcomes remains inconsistent. The storage duration, temperature, and pre-treatment of semen should be determined according to the aims of preservation. Advanced techniques such as nanotechnology and omics have been introduced and show great potential in the lifespan extension of human sperm. It is certain that more patients will benefit from it in the near future. This review provided an overview of the current knowledge and prospects of prolonged non-freezing storage of human sperm in vitro.

Evaluation of Chicken Egg Yolk Plasma and Low-Density Lipoprotein Alone or Enriched with Ewe or Cow Skim Milk in Tris-Citric Acid-Based Diluent for Cryostorage of Ram Semen.

The main purpose of the current study was to find suitable and optimum levels of protectants among chicken egg yolk plasma (CEYP) and low-density lipoproteins (LDLs), alone or supplemented with ewe or cow skim milk, for cryopreservation of ram semen. In Experiments 1 and 2, the CEYP (28%) freezing extender was enriched with ewe or cow milk (2.5%, 5%, 10%, or 20%; v/v), respectively. In Experiments 3 and 4, the semen extender was prepared by varying the amounts of fresh or lyophilized LDL (lyo-LDL), respectively. Finally, ewe or cow skim milk was added to the freshly extracted LDL extender and the quality of frozen-thawed semen was examined (Experiments 5 and 6). Kinematics of spermatozoa (assessed using a computer-assisted sperm analysis system), viability, functionality of the plasma membrane, and levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) were evaluated. Results revealed that addition of ewe or cow skim milk (5%, 10%, or 20%; v/v) to the CEYP diluent enhanced kinematics, viability, and membrane integrity of spermatozoa compared with the control (p < 0.05). Moreover, fresh LDL diluent was more effective than lyo-LDL in the cryosurvival of ram spermatozoa. In addition, enrichment of fresh LDL diluent with ewe or cow skim milk improved different variables of spermatozoa compared with the control (p < 0.05). Levels of MDA and TAC were not affected by adding ewe or cow milk to the diluents (p > 0.05). In conclusion, enrichment of fresh LDL extenders with ewe or cow milk also is proposed as an approach to preserve ram semen quality against cold shock and cryodamage injuries.

Effect of Betaine and Raffinose in Cryopreservation Medium on Fertility in Kadaknath Chicken

BACKGROUND: Kadaknath is an important indigenous chicken with black pigmentation and cryopreserved semen reputably had low fertility. OBJECTIVE: The aim of this study was to evaluate the effects of betaine and raffinose in semen extenders on post thaw semen parameters and fertility. MATERIALS AND METHODS: Semen was cryopreserved in 4% dimethyl sulfoxide (DMSO) with betaine supplemented at 0.1, 0.2 and 0.4 M or raffinose supplemented at 1, 5 and 10 mM. Post thaw semen parameters and fertility were evaluated. RESULTS: Betaine at higher concentrations significantly (p &lt; 0.05) inhibited the post thaw sperm motility, live sperm and MTT dye reduction and a declining trend in the fertility with increasing betaine. Inclusion of raffinose had no effect on the post thaw in vitro semen parameters, however, the fertility was significantly (p &lt; 0.05) higher in the 10 mM raffinose supplemented group. CONCLUSION: Betaine has negative effect on post thaw semen parameters and raffinose at 10 mM concentration improves the fertility from cryopreserved semen.

Validation of a smartphone-based device to measure concentration, motility, and morphology in swine ejaculates.

Assessment of swine semen quality is important as it is used as an estimate of the fertility of an ejaculate. There are many methods to measure sperm morphology, concentration, and motility, however, some methods require expensive instrumentation or are not easy to use on-farm. A portable, low-cost, automated device could provide the potential to assess semen quality in field conditions. The objective of this study was to validate the use of Fertile-Eyez (FE), a smartphone-based device, to measure sperm concentration, total motility, and morphology in boar ejaculates. Semen from six sexually mature boars were collected and mixed to create a total of 18 unique semen samples for system evaluations. Each sample was then diluted to 1:4, 1:8, 1:10, and 1:16 (for concentration only) with Androhep Plus semen extender (n = 82 total). Sperm concentration was evaluated using FE and compared to results measured using a Nucleocounter and computer assisted sperm analysis (CASA: Ceros II, Hamilton Thorne). Sperm motility was evaluated using FE and CASA. Sperm morphological assessments were evaluated by a single technician manually counting abnormalities and compared to FE deep-learning technology. Data were analyzed using both descriptive statistics (mean, standard deviation, intra-assay coefficient of variance, and residual standard deviation [RSD]) and statistical tests (correlation analysis between devices and Bland-Altman methods). Concentration analysis was strongly correlated (n = 18; r > 0.967; P < 0.0001) among all devices and dilutions. Analysis of motility showed moderate correlation and was significant when all dilutions are analyzed together (n = 54; r = 0.558; P < 0.001). The regression analysis for motility also showed the RSD as 3.95% between FE and CASA indicating a tight fit between devices. This RSD indicates that FE can find boars with unacceptable motility (boars for example with less than 70%) which impact fertility and litter size. The Bland-Altman analysis showed that FE-estimated morphological assessment and the conventionally estimated morphological score were similar, with a mean difference of ~1% (%95 Limits of Agreement: −6.2 to 8.1; n = 17). The results of this experiment demonstrate that FE, a portable and automated smartphone-based device, is capable of assessing concentration, motility, and morphology of boar semen samples.

Effects of cysteamine supplementation on cryopreserved buffalo bull semen quality parameters

This work aimed to determine the effect of cysteamine (25, 50, 100 and 200 μM) incorporated during dilution on frozen thawed buffalo semen quality. Semen was collected twice weekly for 7 consecutive weeks from three Egyptian buffalo bulls using an artificial vagina. Semen samples were pooled and extended with a Tris-based extender, cooled, equilibrated and finally frozen in liquid nitrogen. The diluted semen was evaluated for motility, viability, morphology, plasma membrane and DNA integrity, in addition to oxidative stress and in vitro fertilizing capability. The post thaw motility and velocity parameters noticeably increased with different concentrations of cysteamine (mainly 100 μM) during different incubation periods. The post thaw sperm viability and normality significantly (p < 0.05) improved with concentrations of 50 and 100 μM. Plasma membrane integrity substantially increased at all concentrations of cysteamine. Cysteamine reduced alanine aminotransferase (at all concentrations), aspartate aminotransferase (at 25–100 μM), and creatine kinase (at 100 and 200 μM). Cysteamine at a concentration of 100 μM noticeably enhanced the total antioxidant capacity and glutathione peroxidase and decreased nitric oxide production. Cysteamine, at concentrations of 100 and 200 μM, increased the DNA intensity in the comet head (%) and decreased the DNA % in the comet tail. The comet tail length and moment substantially decreased at concentrations of 50–200 μM. Cysteamine did not affect the in vitro fertilizing capability of sperm. In conclusion, cysteamine incorporation (mainly at a concentration of 100 μM) in buffalo semen extender showed varying protective effects on different sperm parameters against cryo-damage; however, it did not affect the in vitro fertilizing capacity of sperm.

Spirulina Supplementation to the Semen Extender Influences the Quality and Antioxidant Parameters of Chilled or Cryopreserved Arabian Stallion Spermatozoa

The present study attempted to investigate the effect of various concentrations of spirulina platensis additions to the semen extender on Arabian stallion spermatozoa quality. Semen samples were collected with artificial vagina from five fertile stallions and diluted with an extender containing spirulina (2, 4, 6, and 8 mg/100 mL) or without spirulina (control). Aliquots of diluted semen were cooled (5°C, 90 minutes) and frozen (-196°C, 7 days), then physical traits of thawed spermatozoa were examined. Furthermore, antioxidant activities of superoxide dismutase (SOD), glutathione reductase (GSR), total antioxidant capacity (TAC), and malondialdehyde (MDA) were measured in post-cooling and freezing spermatozoa. The results indicated that spirulina supplemented with the extender had no effect (P >.05) on sperm quality parameters and antioxidant activities after cooling. However, adding 6 mg spirulina/100 mL to the freezing extender improved (P < .05) the speed parameters and total motility percentage of frozen/thawed spermatozoa. Besides, supplementation of freezing extender with the previous level increased (P < .05) TAC, SOD and GSR concentrations/activities (0.86 ± 0.32 mM/L, 323.70 ± 12.81 U/mL, and 38.65 ± 1.90 U/mL, respectively) compared with the control (0.70 ± 0.25 mM/L, 165.80 ± 8.12 U/mL, and 25.70 ± 1.83 U/mL, respectively). While, lipid peroxidation of the frozen-thawed semen was reduced (P < .05, 17.97 ± 1.30 µmol/ L) compared with the control (29.39 ± 1.89 µmol/ L). Accordingly, the present results revealed that additions of 6 mg spirulina/100 mL to the freezing extender improved semen quality and reduced cryodamage of the Arabian stallion spermatozoa.

Protective effect of rosiglitazone on microscopic and oxidative stress parameters of ram sperm after freeze-thawing

The purpose of this study was to investigate the effects of rosiglitazone on ram semen after cryopreservation on the quality of thawed sperm. Sperm motility, membrane functionality, viability, total abnormality, acrosome membrane integrity, mitochondrial activity, reactive oxygen species production, ATP content and apoptotic features were assessed after thawing. Rosiglitazone at concentration of 60 µM resulted in the highest (P < 0.05) total motility, progressive motility and straight-line velocity. The percentages of average path velocity and curvilinear velocity were greater in the 60 µM group. Different concentrations of rosiglitazone did not have significant effects on amplitude of the lateral head displacement, linearity and straightness. The highest amounts of membrane functionality and mitochondrial activity after freeze-thawing were observed in groups containing 60 µM. By increasing the rosiglitazone level to 80 µM, no positive effect was observed in most of the evaluated parameters. The lowest ROS concentration was recorded in 60 µM rosiglitazone group (P < 0.05). The group containing 60 µM rosiglitazone also produced the lowest significant percentage of apoptosis-like changes and dead sperm. A greater (P < 0.05) percentage of acrosome integrity in frozen-thawed spermatozoa was observed in the 60 µM rosiglitazone group. There was no significant difference between 40 and 60 µM rosiglitazone in intact acrosome of ram thawed semen. The result showed that supplementation in ram semen extender with rosiglitazone had a positive role in the regulation of ram sperm motility and had strong protective effect on the sperm membrane and acrosome integrity.

Effect of Astragalus polysaccharides on the cryopreservation of goat semen

During cryopreservation, sperm encounters oxidative stress induced by excessive reactive oxygen species (ROS), destroying the sperm plasma membrane structure and reducing its physiological functions. The present study aimed to evaluate the effect of Astragalus polysaccharides (APS) on the cryopreservation of dairy goat semen. Semen was collected from six goats, and then qualified semen with movement >80% was selected after preliminary evaluation. The semen was divided into six aliquots, diluted with dairy goat semen extender (1:10) at 37 °C, containing 0 g/L (control), 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L and 0.5 g/L APS, cryopreserved, and stored in liquid nitrogen (−196 °C). Sperm quality was assessed after freeze-thawing. The highest sperm motility, motion performance, plasma membrane integrity, acrosome integrity, and antioxidant properties (total antioxidant capacity and levels of antioxidant enzymes) were recorded (P < 0.05) in the 0.2 g/L APS group after the semen was freeze-thawed. The control and the optimal group (0.2 g/L) were then selected to analyze the effects of APS on sperm energy metabolism (mitochondrial membrane potential [MMP] and adenosine triphosphate [ATP]), sperm apoptosis, and the expression of the AMPK signaling pathway. The results showed that treatment with 0.2 g/L APS increased sperm MMP and ATP content after freeze-thawing, reduced sperm apoptosis by regulating apoptosis-related proteins, and promoted AMPK phosphorylation by activating the AMPK signaling pathway. The cleavage rate of frozen goat sperm during in vitro fertilization (IVF) was also observed to increase. These findings suggest meaningful ways to improve cryopreservation of dairy goat semen and provide new insights into the mechanism by which APS protects sperm from oxidative damage via AMPK activation.

Photostimulation Applications to Increase Spermatological Parameters

Today, it is an important issue to preserve genetic resources and transfer them to future years. The process of freezing semen, which has a wide range of uses such as reproductive biotechnology, protection of the lineage of species and clinical applications, is of great importance in this sense. Freezing the semen in a way that will provide sufficient fertility will make artificial insemination practices more practical and economical, and more accurate and reliable pedigree records will be kept. In addition, it will be possible to use animals with high genetic capacity in large populations. During the freezing-thawing of sperm, membrane lipid phase change, osmotic-mechanical stress and free oxygen radicals occur. As a result, structural deformations in membrane structures and cell organelles and breaks in DNA occur. These negative effects can be reduced by adding antioxidants, various cryoprotectants and chemical substances to semen extenders, and sperm functions are improved after solution. The developments in the light of recent research have been in the direction of increasing the fertility abilities of sperm cells in the cryopreservation process by irradiating them in different spectrum areas. On the other hand, lasers are involved in many areas of our lives due to their linearity, single wavelength, stable radiation and high power. Laser sources are gaining more and more acceptance in various industries thanks to their narrow focusing advantages. Recently, it has been shown that under intense radiation of red LED (Light Emitting Diode) lights, the resistance and capacitation of porcine sperm cells increase in vitro and fertility ability increases in vivo. Consistent results have also been obtained in species such as humans, dogs, bulls, rams and rabbits using devices and systems such as low-energy laser and visible light lamps with wavelengths ranging from 400 nm to 800 nm.

Temperature limits for storage of extended boar semen from the perspective of the sperm's energy status.

The optimum storage temperature for liquid-preserved boar semen has been empirically determined to be between 15 and 20°C. Lower temperatures provide an advantage to inhibit bacterial growth, but are regarded as critical due to the high sensitivity of boar spermatozoa to chilling injury. Higher storage temperatures are supposed to induce energy deficiency due to an insufficient depression of metabolic cell activity. However, experimental evidence for alterations of the sperm's energy status in relation to storage temperature and duration is missing. Therefore, we aimed to revisit the upper and lower storage temperature limits for liquid-preserved boar semen from the perspective of the sperm's energy metabolism. Ejaculates (n = 7 boars) were cooled down in Beltsville Thawing Solution (BTS) to 25, 17, 10, or 5°C and stored for up to 120 h. ATP and adenylate energy charge (EC) levels were assessed at storage temperature (24, 72, and 120 h storage) and after subsequent re-warming (38°C). Sperm quality and energy status remained at a stable level in samples stored at 25 and 17°C. Chilling to and storage at 10 or 5°C in BTS provoked cold shock in a subset of sperm as shown by a loss in viability and motility (P < 0.05), which was accompanied by a significant release of adenine nucleotides into the semen extender. Prolonged storage for 120 h resulted in significantly lower mean ATP concentrations in viable spermatozoa at 5 or 10°C compared to 17°C (P < 0.05). Cluster analysis revealed that the main sperm subpopulation, i.e., sperm with moderate speed and linearity, decreased from 50 to 30% (P < 0.05) in favor of slow-moving spermatozoa (5°C) or spermatozoa with a hyperactivation-like motility pattern (10°C). The results point to a sublethal imbalance in available ATP in a subset of the surviving sperm population, rather than a general decrease in available ATP in all spermatozoa. In conclusion, storing diluted boar semen at a stable temperature between 17 and 25°C is a safe procedure concerning the spermatozoa's energy status. Future concepts for hypothermic boar semen preservation below 17°C require measures which ameliorate the imbalanced energy status in viable spermatozoa.

The Quality of Bali Bull Sexed Sperm Using Soybean Extract Extender

The process of sexing spermatozoa requires a medium that is able to protect and provide an optimal environment. One of them is extender media that is used to extend the volume of semen. The extender that is commonly used is commercially but some limitation such as price and availability. Therefore, it is necessary to have an alternative extender such as soybean. The aim of this study was to know the quality of sexed sperms diluted using soybean extender. Semen of Bali bull was collected for five times and then subjected to three different extender treatments after sexing; T1 (Soybean), T2 (Tris), T3 (Tris-soybean), and T0 (Fresh semen-tris) was used as control before sexing. The parameters observed were the quality of fresh semen and after sexing. The results of the study showed that the characteristic of Bali bull fresh semen macroscopically in this study was 4.75 mL in volume, cream color, distinctive odor, pH 6.6, moderate consistency, and microscopically motility, viability, abnormality, and concentration were 94.22%, 96.06% 4.89, and 1596×106, respectively. Motility and viability after sexing were significantly (P&lt;0.05) decrease in comparison to the fresh semen (T0) at each treatment both top and bottom layers. In conclusion, the smallest decreased of the sperms motility and viability were still greater than 50%. Different extenders as one of the treatments in the present study did not showing different motility and viability of the sperms. This suggests that the use of tris and soybean or their combination can be used as semen extender for Bali bull sexed semen. Keywords: Soy extender, Bali bull, sexing, motility, viability.

Sodium dodecyl sulphate, N-octyl β-D glucopyranoside and 4-methoxy phenyl β-D glucopyranoside effect on post-thaw sperm motion and viability traits of Murrah buffalo (Bubalus bubalis) bulls

Sodium Dodecyl Sulphate (SDS), N-Octyl β-D Glucopyranoside (NOG), 4-Methoxy Phenyl β-D Glucopyranoside (4-MPG) as ice recrystallization inhibitors were added to Tris Egg Yolk Glycerol (TEYG) semen extender for cryopreservation of semen of buffalo bulls. Post-thaw sperm motion and viability traits were evaluated. Pilot study involved six semen ejaculates (2 ejaculates/bull, from three bulls); second experiment was conducted using twenty seven semen ejaculates (9 ejaculates/bull, from 3 bulls) and in third experiment three semen ejaculates (one bull) were used. Eight concentrations of SDS (2, 1, 0.5, 0.25, 0.15, 0.125, 0.0625 and 0.0312%), twelve concentrations of NOG (33, 22, 11, 5.5, 2.5, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 mM), and, eleven concentrations of 4-MPG (220, 165, 110, 55, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mM) were supplemented in TEYG semen extender to evaluate the post-thaw sperm motility and viability traits. Computer Assisted Sperm Analysis (CASA) was used to measure the kinetic and functional parameters for sperm motion traits, Hypo Osmotic Swelling Test (HOST) for sperm plasma membrane integrity, Eosin Nigrosin staining for viability and Rose Bengal staining for sperm abnormalities for all the experiments except for pilot study where only Total Motility (TM) and Rapid Progressive Motility (RP) were evaluated. Three freezing protocols; i) Normal P24 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −40 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); ii) Moderate P25 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −50 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); and iii) Rapid P26 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −60 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C) were evaluated using SDS 0.125% in TEYG semen extender. SDS ≤0.125%, NOG ≤0.0625 mM and 4-MPG ≤ 3.125 mM in TEYG buffalo semen extender improved significantly (p < .05) the kinetic and functional parameters as compared to the other Ice Recrystallization Inhibitors (IRIs) concentrations used for cryopreservation of buffalo bull semen in the pilot study. SDS 0.125% supplementation was the best IRI among all which resulted in improved kinetic and functional parameters of bull semen in second experiment. Conclusion was drawn that buffalo bull semen cryopreservation using sodium dodecyl sulphate, 0.125% as IRI in TEYG semen extender along with freezing protocol P 25 revealed optimum kinetic and functional parameters for post-thaw spermatozoa.

A retrospective study of the relationship between canine age, semen quality, chilled semen transit time and season and whelping rate and litter size

The objectives of this study were to investigate the effect of the dog’s age, semen quality, and the duration and season of semen transit on whelping rate and litter size after insemination with transported chilled extended semen. The sperm rich fraction was collected from 43 dogs of 18 breeds, which were presented at the Clinic for chilled semen transport, in the period from 2017 to 2021. Immediately after collection, the total sperm concentration and count, motility, membrane integrity (HOS test), the percentage of live spermatozoa and sperm morphology (eosin nigrosin staining) were evaluated. The sperm rich fraction was centrifuged and diluted with Tris – fructose - citrate extender with the addition of 20% (v/v) egg yolk, then chilled and prepared for shipping. A dose consisted of at least 200x106 live, motile, morphologically normal spermatozoa. The data on the dog’s age, chilled sperm transit time, the season of transit, and the whelping rate and litter size after insemination were recorded. The whelping rate was 55.8% with a mean (±SEM) litter size of 4.71±0.58 pups. The total number of spermatozoa was higher in artificial insemination (AI) that resulted in whelping compared to unsuccessful AI (P&lt;0.05). No difference was observed for any of other sperm quality parameters tested, such as the dog’s age or season of transit regarding whelping rate or litter size. Transit time significantly affected the whelping rate (P&lt;0.01), at (mean±SEM) 21.50±1.28 and 37.00±5.59 h in successful and unsuccessful AI, respectively. In conclusion, analysis of the factors related to the dogs identified total sperm count and transit time as factors that significantly affected whelping rates in bitches inseminated with transported chilled extended semen.

Determining the Optimal Dosage of Lecithin Nanoliposome in Rooster Semen Freezing Medium and Fertility Potential.

Introduction: Lecithin nanoliposome (nano-LPO), with its cryoprotective properties, is considered to enhance the performance of a traditional semen cryoprotectant. Objective: To determine the optimal dose of lecithin nano-LPO added to the rooster semen extender. Materials and Methods: Semen samples collected weekly from eight broiler breeder roosters were mixed and aliquoted into five equal subsamples, during the five successive weeks. The subsamples were then diluted with a semen extender containing 0%, 0.5%, 1%, 1.5%, or 2% of lecithin nano-LPO. Post-thawed semen quality attributes, including sperm motility and velocity parameters, plasma membrane functionality, mitochondrial membrane potential (MMP), apoptosis-like changes, and fertility potential, were evaluated. Results: Total motility and velocity parameters, including curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity μm/s (VAP), straightness (STR), linearity (LIN), lateral head displacement (ALH), and wobble (WOB) were quadratically (p < 0.01) influenced by graded levels of lecithin nano-LPO, such that the highest values were obtained when 1% of lecithin nano-LPO was used. Treatments had no significant effect on plasma membrane functionality; however, MMP (p < 0.08) and percentages of live and dead spermatozoa (p < 0.05) quadratically responded to increasing levels of lecithin nano-LPO, where the best outcome was found when about 1% of lecithin nano-LPO was used in the semen extender. The percentage of apoptotic spermatozoa cubically responded to increasing levels of lecithin nano-LPO (p ≤ 0.07). No significant trend of fertility rate was found in response to addition of lecithin nano-LPO levels. Conclusions: Supplementing an extender with 1.10% of lecithin nano-LPO is shown to be the optimal dose associated with the most improvement in post-thawed rooster sperm velocity measurements.

Effect of supplementation of freezing media with isoespintanol on functional integrity of equine semen

Abstract Adding antioxidants to semen extender has become one of the major strategies to reduce the damage caused by freezing to stallion sperm. Isoespintanol (Iso) is a natural antioxidant that is known as a good free radical scavenger. The aim of this research was to evaluate the effect of supplementation of freezing media with Iso on functional integrity of equine semen. Semen from five Colombian Creole horses was frozen with 0, 10, 20, 30, 40 and 50 µM Iso. Post-thawing motility and kinetics, structural membrane integrity (SMI), normal morphology (NM) and functional membrane integrity (FMI) of sperm were evaluated. Seminal quality (SQi) and seminal velocity (SVi) indexes were calculated. Through a flow cytometric analysis, mitochondrial membrane potential (ΔΨM), viability and DNA integrity of frozen-thawed semen were assessed. General linear models were adjusted, and means comparisons were performed using Tukey’s test. The significance level was P&lt;0.05. No differences for post-thaw sperm quality parameters were found, except for total motility, which showed a reduction by adding 40 µM Iso (P&lt;0.05). However, the estimation of indexes, showed that supplementation with 20 or 30 µM Iso before semen freezing, can increase post-thaw SQi and SVi values of some horses. A higher proportion of high-∆ΨM sperm for 20 µM Iso was found. It is concluded that Iso can improve the quality and mitochondrial activity of frozen-thawed equine semen, however its effect can vary according to the horse and in high concentrations it could have detrimental effects.

Roselle Calyx Extract as an Antioxidant Supplement on Tris-Base Extender for Goat Semen

In this study, we used extract from Roselle (Hisbiscus sabdariffa L.) Calyx to determine its ability as an antioxidant supplement to improve motility, progressive score and abnormality of goat’s sperms. Pooled semen from four male goats with average of live body weight 34.4 ± 0.6 kg and body condition score 2.5 ± 0.1 were aliquot into two groups: Control group; 0 mg/ml and Treatment groups; (Treatment 1:13 mg/ml, Treatment 2: 20 mg/ml, Treatment 3: 33 mg/ml, Treatment 4: 43 mg/ml of Roselle’s extract. All the semen parameter was observed at 0 hour, 0.5 hour, 1.0 hour, 1.5 hours after semen collection. The supplementation of extender with 13 mg/ml, 20 mg/ml, 33 mg/ml, 43 mg/ml of extract from Roselle Calyx does not affect sperm motility, progressive score and morphology of goat (p&gt;0.05). However, all sperm, regardless with supplementation and without supplementation of Roselle extract were maintained in good quality (60%) up to 1.5 hours. It is quite difficult to conclude that there are no effect of Roselle extract on sperm quality due to arise possibility of factors that may contribute to inefficiency effect of Roselle extract on sperm parameter. There were high possibility that the causes are due to inefficiencies of extraction method and/or degradation of anthocyanin pigment during extraction and storage process. Unsuitable extraction method could results in low yield and unstable of anthocyanin pigment. Moreover, temperature, pH and light causes the pigment to become unstable during processing and storage leading to degradation. Thus we suggest anthocyanin content monitoring in extract Roselle Calyx are required for future study.

Effect of Aqueous Extract of Maca Addition to an Extender for Chilled Canine Semen.

Simple SummaryThe wide use of artificial insemination in dogs justifies the development of new strategies to prevent the reduction of fertilizing ability of stored semen. In recent years, the use of plant antioxidant supplementation has become increasingly popular. Maca (Lepidium meyenii) is an Andean edible root with antioxidant properties. The effectiveness of the oral supplementation of Maca in improving fresh semen quality and quantity and cooling or freezing ability has already been reported. This is the first in vitro study on the effects of aqueous extract of Maca on canine spermatozoa. The addition of low concentrations of aqueous extract of Maca to the canine chilled extender had positive effects only until 24 h of storage, increasing hyperactivation of sperm cells and preserving DNA integrity of spermatozoa in short-term storage. Meanwhile, a high concentration of Maca had an immediately deleterious effect on semen quality. Antioxidant supplementation has been proposed as a new strategy to improve the long-term preservation of semen. The aim of this study was to evaluate the effect of Maca supplementation of semen extender on quality-related canine semen parameters during cooling. Ejaculates from nine dogs were cooled for 7 days in the absence (control group) or in the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca. Sperm were evaluated for sperm viability, motility, DNA fragmentation and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage. The addition of 10 μL/mL of Maca preserved sperm DNA and plasma membrane integrity at 3 h and increased sperm curvilinear velocity after 24 h. Treatment with 20 and 50 μL/mL of Maca increased the percentage of hyperactivated sperm after 3 h. Moreover, semen treated with 20 μL/mL of Maca decreased lipid peroxidation at 24 h. A significant reduction of sperm DNA and plasma membrane integrity as well as of kinetics parameters between 3 and 24 h of refrigerated storage with the higher concentration tested was observed. Although Maca was not able to protect canine semen with extended refrigeration storage time, it increased hyperactivation and preserved DNA integrity in short-term storage.

The effects of caffeine on the motility and viability of stallion spermatozoa at different temperature conditions.

The purpose of this study was to evaluate the dose- and time-dependent effect of caffeine treatment on the motility and viability of stallion spermatozoa at different temperatures. Six dose groups (A to F) were established with changing caffeine concentrations (from 0.625 to 10 mg/mL). The control samples were prepared by diluting the ejaculate only with physiological salt solution. The samples were examined after 0, 1, 2 and 3 h of incubation at 5 °C and 37 °C. The motility parameters were evaluated by Computer Assisted Semen Analyzer (CASA) system, and the viability was assessed by the mitochondrial toxicity test at the end of the incubation. A positive effect of the lowest tested caffeine concentration on the motility parameters was observed throughout the incubation period at 5 °C. At the end of the 3h incubation, the viability in every sample in these groups, treated with any caffeine concentration, showed lower values compared to the control. At the higher incubation temperature (37 °C), caffeine positively affected the motility in samples B (P < 0.05) and D, E, F (P < 0.001) after 3 h of incubation; however, the viability showed a slightly decreasing tendency. Our results suggest that caffeine, in an optimal concentration, may be used as a component of stallion semen extenders.

Shielding effect of melatonin improves seminal quality and oxidative stress indices during chilled storage of ram semen.

Supplementation of antioxidant to semen extender maintains seminal quality by reducing oxidative stress during preservation time period. The main aim of this study was to investigate the effect of different concentrations of melatonin supplementation on liquid storage of Magra ram semen. This study was performed on adult Magra ram (n = 8), and seminal ejaculates (48) were collected and evaluated for various macroscopic and microscopic seminal quality parameters for further processing. After preliminary evaluation, ejaculates of each collection session were mixed and divided into four equal aliquots. All the aliquots were diluted (1:10) with Tris-citric fructose egg yolk extender contained sans melatonin served as control, whereas the other three aliquots were supplemented with 0.5, 1 and 2mM MLT which were grouped as MLT0.5, MLT1 and MLT2, respectively. Thereafter, the samples were stored at 4 ºC for 72h, and various seminal parameters (individual sperm progressive motility, viability, abnormalities, plasma membrane functionality) along with oxidative stress parameters (total antioxidant capacity (TAC), malondialdehyde (MDA)) were evaluated at 0, 24, 48 and 72h of preservation. The results indicated that the mean percent values for progressive sperm motility, viability, plasma membrane functionality and TAC were significantly higher (p < 0.05) in treated groups with the highest values in MLT1 group. Significantly lower (p < 0.05) percentage of total sperm abnormalities and MDA level was observed in treatment groups compared to control group. The results report that supplementation of 1mM melatonin efficiently maintains the seminal quality and ameliorates oxidative stress during preservation at 4 ºC to 72h.

The replacement of fresh egg yolk by lyophilized egg yolk in Tris-base extender in cryopreserved Boer and Saanen semen.

Egg yolk is a common cryoprotectant that can be used as a semen extender to protect the spermatozoa from damage during cryopreservation. Therefore, the aim of this study was to investigate the efficacy of fresh and lyophilized egg yolk, as a Tris-base extender, on the quality of cryopreserved goat semen. Semen from 10 rams of two different breeds (Boer and Saanen) was collected using an artificial vagina. Each ejaculate sample was divided into four equal aliquots, which contained 20% of the fresh egg yolk (a control group), and then 10%, 15%, and 20% of the lyophilized egg yolk as a Tris-base extender. Sperm motility and kinetic parameters were determined using a computer-assisted semen analyser. The results showed that the addition of 20% of the fresh egg yolk in Tris-base extender exhibited significantly higher progressive motility, progressive fast motility, distance curve line, and beat-cross frequency parameters in the post-thaw Boer and Saanen goat sperm when compared with the addition of 10%, 15%, and 20% of the lyophilized egg yolk. The percentage of total motility and immotile parameters in the post-thaw Boer and Saanen goat sperm were not significantly different between the control and 10%, 15% as well as 20% of the lyophilized egg yolk groups. Moreover, the percentage of viability parameter in the Boer and Saanen goat sperm was not significantly different between the control and 10% of the lyophilized egg yolk group but showed significant difference between the control group and 15% and 20% of the lyophilized egg yolk groups. Furthermore, the interaction between the two breeds was significantly different in terms of head activity and straightness parameter. In conclusion, the treatment with 20% of fresh egg yolk in Tris-base extender is superior to the lyophilized egg yolk. However, an addition of 10% of the lyophilized egg yolk in Tris-base extender presented the percentage of total motility and viability parameters showing no difference with 20% of fresh egg yolk. Therefore, 10% of the lyophilized egg yolk in Tris-base extender provided detail of the lyophilized egg yolk protocol in cryopreserved goat semen as an example of an alternative extender to 20% of fresh egg yolk for situations where an animal's origin represents a microbiological risk.