Hypo-osmotic Test Research Articles

Anethole improves mitochondrial activity and quality parameters in fresh and frozen-thawed ovine semen

Anethole, an antioxidant found in plants, appears to improve the survival of spermatozoa during semen cryopreservation. This study assessed the effects of commercial trans-anethole in ram semen cryopreservation. Thirty ejaculates from six rams were diluted in media containing anethole at the following concentrations: CONT (0 μM), AN10 (10 μM), AN50 (50 μM), and AN100 (100 μM). Semen was slow-frozen, preserved in liquid nitrogen, and thawed. Anethole at 10 μM or 50 μM did not compromise any studied sperm quality parameter but increased pre-freezing functionality of membrane and mitochondrial activity. At 10 μM, anethole reduced post-thawing spermatozoa lipoperoxidation. At 50 μM, anethole sustained higher mitochondrial activity after thawing, reduced minor defects in sperm, and increased the number of sperm binding to perivitelline membrane, while keeping lipoperoxidation levels as in control. Anethole at 100 μM promoted higher pre- and post-freezing mitochondrial activity and higher number of sperm binding to perivitelline membrane, in comparison to control. Additionally, some post-thawing kinematic parameters were enhanced by anethole at 100 μM. Of note, mitochondrial activity and lipoperoxidation were higher with anethole at 100 μM in comparison to 50 μM, not differing from control. At the hypoosmotic test, the highest concentration (100 μM) tested reduced sperm osmotic resistance. The results of this study indicate that using anethole in cryopreservation media promoted mostly positive effects on the fresh and post-thawed ram semen, and the advantages vary according to its concentration.

Quality and redox state of bovine sperm cryopreserved with resveratrol use of resveratrol in bovine semen

AbstractThe use of antioxidants for semen preservation prevents oxidative damage caused by reactive oxygen species (ROS). One of the most promising natural antioxidants is resveratrol, a phytoalexin derived from plants, grapes, berries, peanuts and red wine. To evaluate the effect of resveratrol on the quality and redox status of cryopreserved bovine semen. Five bulls were subjected to electroejaculation to obtain 15 ejaculates. Each ejaculate was extended with a tris‐egg yolk‐glycerol‐based medium and divided into six aliquots supplemented with 0 (control), 10, 20, 30, 40 and 50 μM of resveratrol. Semen was frozen with liquid nitrogen vapours. Post‐thawing, motility and kinetics were evaluated using a computer‐assisted sperm analysis (CASA) system, membrane integrity using the hypoosmotic test (HOST), morphology by staining with eosin–nigrosin, sperm vitality by fluorescence microscopy with the SYBR14/IP probes. Total antioxidant capacity (TAC) was evaluated using the ABTS•+ assay and ROS was evaluated using spectrofluorimetry with the H2DCFDA probe. For the statistical analysis linear models were adjusted and means were compared using the Tukey test. All concentrations of resveratrol reduced post‐thawed motility and kinetics of sperm. Supplementation with 40 and 50 μM of resveratrol reduced sperm kinetics, and between 30 and 50 μM of resveratrol alterations in the sperm membrane and morphology were observed. However, using resveratrol at 50 μM increased TAC and at 20 μM, it reduced ROS production of cryopreserved bovine semen. Resveratrol appears to have a dose‐dependent effect in which higher doses produce greater sperm alterations, however, it can increase semen TAC during freezing. It is concluded that resveratrol can increase antioxidant capacity and reduce ROS production in cryopreserved bovine semen. However, its use between 10 and 50 μM reduces post‐thawing semen quality.

Effect of a dietary antioxidant supplementation on semen characteristics of the Colombian horse

Cooling equine semen increases production of reactive oxygen species (ROS). Elevated concentrations of ROS have detrimental consequences by causing changes in membrane permeability. Although there are reports of antioxidant supplementation to improve sperm quality in horses, there are few reports on the Colombian breed. The objective of this study was to evaluate the effect of nutritional supplementation with a variety of antioxidants on semen characteristics of Colombian horses. Six (n=6) Colombian Creole stallions aged between 5 and 15 years were collected 3 times a week using a Missouri artificial vagina for 20 weeks. Stallions were randomly allocated into a control (C) or a supplementation group (SG) with Natural Fertility®. Cobalamin 0.36 mg/day; pyridoxin 16 mg/day; retinoic acid 366799 IU/day; alpha tocopherol 1600 IU/day; ascorbic acid 4000 mg/day; folic acid 1200 mg/day; lysine 640 mg/day; methionine 960 mg/day; threonine 400 mg/day; arginine 400 mg/day; L-Carnitine 10000 mg/day; omega 3 3200 mg/day; zinc 1200 mg/day; copper 44 mg/day, chromium 6 mg/day and selenium 8 mg/day, was added to the basal diet (hay, mineralized salt, 2 kg of grain and water). Repeated measures design with a wash-out period of 2 weeks between treatments was used. During a periodof 8 weeks, semen was analyzed twice a week for motility and kinetics using a Sperm Class Analyzer (SCA®) system; sperm vitality (SV) and acrosomal Integrity (AI) by fluorescent microscopy, abnormal morphology (AM) by supravital stain, and plasmatic membrane Integrity by hypoosmotic test (HOST). Reactive oxygen species and total antioxidant capacity (TAC) by ferric reducing antioxidant power assay (FRAP) were assessed by spectrofluorimetry. Comparison of means was performed using Tukey's test with p≤0.05; using SAS program 3.81. SG increased the beat cross frequency of semen with 36.68 Hz ± 0.89 vs. 32.67 Hz ± 0.73 (C). In addition, a higher AI was found for SG (82.92 ± 0.72 %) compared to C (78.51% ± 0.83). SG also increased the TAC of seminal plasma (15.35± 0.92 μmol Trolox/L) compared to C (18.86± 1.75 μmol Trolox/L). In the individual analysis it was found that SG increased the total and progressive motility of the semen of two horses, however it reduced both parameters for two other individuals, compared to C. In conclusion, supplementation of the diet of horses with a supplement that provides vitamins, minerals, amino acids, and omega 3 can increase the antioxidant capacity of seminal plasma, as well as sperm quality, however this may be subject to a differential effect on each horse.

Comparison of different cryoprotectants for freezing donkey jack (Equus asinus) semen

The objective of this study was to extrapolate a rapid sperm freezing technique developed in horses to Remonta Argentino donkey semen, evaluating, in vitro and in vivo, the effect of freezing media that included two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) at two different concentrations (5 and 7%). Twenty-four ejaculates from 8 fertile jacks (n=8; r=3) were processed with 8 extenders: BotuSemen Gold with 5% or 7% MF and 5% or 7% DMF and EDTA-glucose with 5% or 7% MF and 5% or 7% DMF, all containing 11% lactose, 20% egg-yolk and Equex and frozen: to -15°C (10-12°C/min) then to -120°C (25-40°C/min.), then plunged in N2. Post-thaw evaluations included sperm motility (Computer Assisted Semen Analysis; AndroVision, Minitube, Tiefenbach, Germany), morphology (Diff Quick stain), membrane function and acrosome status (combined hypoosmotic test and Coomassie blue stain). Sperm data were analyzed using Kruskal Wallis and pregnancy data using Chi-Square test. Values are mean ± SD. Differences were observed in the post-thaw progressive sperm motility between individuals (p<0.05) resulting in three subgroups (low: 9.23 ± 0.16; intermediate: 22.92 ± 9.81 and high: 41.33 ± 5.41). Within each subgroup, there were no in vitro differences among the 8media used for any of the sperm characteristics evaluated. Samples in BotuSemen Gold with 5% DMF, however, tended to show highest percentages (P>0.05) of sperm with acrosomes and functional membranes (DMF: 5%: 53.67 ± 22.01; 7%: 33.92 ± 23.4; MF: 5%: 44.5 ± 20.46; 7%: 38.75 ± 27.4). In vivo, 300 × 106 PM sperm were deeply inseminated post-ovulation in 30 mares: 15 with BotuSemen Gold 5% DMF and 15 with BotuSemen Gold 7% DMF. A 46% (7/15) pregnancy rate was obtained using the extender with 5% DMF and no pregnancies (0%; 0/15) were obtained using the extender with 7% DMF (P=0.003). Perhaps uterine inflammatory response post-insemination was too high when using 7% DMF or sperm chromatin was damaged, affecting in vivo results. In conclusion, the rapid semen freezing technique was successfully applied in jacks. Regarding the extenders, no significant in vitro differences were observed using either BotuSemen Gold or EDTA-glucose media, whether adding MF or DMF at either 5% or 7%. However, when inseminating mares, pregnancies were only obtained using 5% DMF. It would be interesting to analyze sperm DNA and peroxidation characteristics as well as the uterine response to insemination to determine possible reasons for the failure in fertility and the variability observed among individuals.

Effect of post-thaw addition of seminal plasma to cryopreserved epididymal equine spermatozoa

Seminal plasma (SP) has been reported to increase the immediate motility of frozen-thawed epididymal sperm and appears to be important for plasma membrane remodeling, capacitation, acrosomal reaction and fertilization. The objective of this study was to evaluate the effect of incubating equine epididymal frozen-thawed sperm with different concentrations of seminal plasma. Sperm samples were obtained from testes after castration of 40 young Silla Argentino stallions (24 to 36 months of age). Testicles were cooled (5°C) and after 24 h, the cauda epididymis were dissected and flushed (retrograde) with Kenney extender. Epididymal sperm were cryopreserved in EDTA-glucose with 5% DMF, 11% lactose, 20% egg-yolk and Equex, using a rapid temperature-descent curve. The samples were thawed at 37°C for 1 min and then incubated 10 minutes with different concentrations of SP (10%, 50%, 100%) vs. a control (0% SP). To create a pool of SP, Silla Argentino stallions of proven fertility were used. Seminal plasma was separated and stored at -18 °C until use. Semen parameters evaluated were sperm motility (Computer Assisted Semen Analysis; ISAS v1, Proiser), morphology (Diff Quick stain), viability and membrane function (combined hypoosmotic test and Coomassie blue stain). Data were analyzed using Kruskal Wallis test with P<0.05considered significant. Post-thaw incubation with increasing concentrations of seminal plasma significantly decreased sperm total and progressive motility (0%: 33.06 ± 2.02; 26.71 ± 1.90; 10%: 24.06 ± 1.74; 18.85 ± 1.59; 50%: 19.44 ± 1.71; 14.92 ± 1.53; 100%: 14.97 ± 1.57; 11.66 ± 1.48 respectively. Values are mean +/- SEM). However, the values of Beat-Cross Frequency, Linearity and Straightness were significantly higher when the spermatozoa were incubated with SP (BCF: 0%: 10.24 ± 1.47a; 10%: 10.67 ± 3.13a,b; 50%: 10.63 ± 2.31b; 100%: 11.45 ± 2.73b; STR: 0%: 0.59 ± 0.07a; 10%: 0.62 ± 0.09a,b; 50%: 0.64 ± 0.09b; 100%: 0.64 ± 0.12b; LIN: 0%: 0.23 ± 0.05a; 10%: 0.25 ± 0.06b; 50%: 0.26 ± 0.07b; 100%: 0.26 ± 0.07b). There was no effect on membrane function, acrosome status and sperm morphology, regardless of the concentration of SP used (P>0.05). The results indicate that there is no evidence of a beneficial effect of post-thaw supplementation with SP at the concentrations used in this study. It has to be considered that the main role of SP is not associated with sperm physiology but as a modulator in the female reproductive tract. Hence we are continuing in vivo studies to assess whether SP addition can improve pregnancy rates after artificial insemination with frozen-thawed epididymal sperm.

Metabolomics profile of equine seminal plasma

Seminal plasma (SP) is the product of testes, epididymis, and accessory sex glands released during ejaculation, making up 98% of the volume of the stallion ejaculate. Analyzing the entire metabolome allows to assess the final products of the biological system metabolism. The present study aimed to investigate the metabolomics of stallion SP. Twenty-four Criollo stallions with a known reproductive history and ≥30 inseminated mares were once collected during the breeding season. Pregnancy rates (day 16 after artificial insemination) ranged from 20.2% to 95.6%. Two groups were formed: High Pregnancy (HP; pregnancy rate per cycle ≥50%), and Low Pregnancy (LP; pregnancy rate of ≤49%). Microscopic semen analysis was performed using a computer-assisted sperm analysis system. Membrane physical integrity was assessed by fluorescent probes, and the hypoosmotic test analyzed membrane functional integrity. After centrifugation at 400xg for 10min centrifugation, SP plasma was transferred to a 2mL microcentrifuge tube and centrifuged again (10.000xg, 60min, 4°C). The supernatant was mixed 1:1 with deionized water after filtering with 0.45μm filters to remove cell sediment. Each sample was again filtered through a 0.22μm syringe filter before profile acquisition. The metabolic profile was acquired using liquid chromatography coupled with time-of-flight mass spectrometry. A total of 18 target metabolites were identified after analysis. The metabolites were tryptophan, lactic acid, ornithine, stearic acid, oleic acid, palmitic acid, melatonin, acetylcarnitine, carnitine,phenylalanine, isoleucine, isovaleryl, citric acid, hippuricacid, 1,3-dioxan-5-ol, taurine, phosphoric acid, and fructose. Groups were considered independent factors while the metabolite profile was considered a dependent factor. The Shapiro-Wilk Test was used to evaluate data distribution of SP metabolomics. The Kruskal Wallis test for nonparametric data and ANOVA and Tukey test for parametric data were used. Pearson's correlation was performed with a significance level of P<0.05. Four metabolites: oleic acid, isoleucine, taurine, and phosphoric acid, presented a difference (P<0.05) between the HP and LP groups. The oleic acid and isoleucine were higher in the LP group, while taurine and phosphoric acid were higher in stallions from the HP group. Phosphoric acid presented a relevant negative correlation with immobile spermatozoa (r=-0.7206) and a positive correlation with functional integrity (r=0.6162), physical integrity (r=0.6306), fertility (r=0.6471), progressive motility (r=0.5145), total motility (r=0.7157), VCL (r=0.6034), VSL (r=0.5491), VAP (r=0.5629), BCF (r=0.7055), ALH (r=0.5945), the total volume of the ejaculate (r=0.5941) and total protein/ejaculated (r=0.5182). Metabolites are products of cell signaling pathways and can be used as biological markers. Phosphoric acid contributes to the energy metabolism of sperm by breakdown of an inorganic pyrophosphate molecule. In conclusion, phosphoric acid in stallion SP should be considered as a marker of fertility.

Cooling of porcine semen in an extender supplemented with Carvacrol.

The addition of antioxidants in boar semen is an alternative to mitigate the reduction of sperm quality during preservation. To evaluate the effect of carvacrol on cooling of boar semen. Fifteen ejaculates from five boars were extended in MR-A® with 0, 5, 10, 15, 15, 20, 25 and 30 μM of carvacrol (C), and were cooled for five days at 16°C. Sperm motility and kinetics were evaluated with computer-assisted semen analysis (CASA). At 0 and 96 h, membrane functionality was determined by hypoosmotic test; reactive oxygen species (ROS) production and total antioxidant capacity (TAC) by spectrofluorimetry; and mitochondrial membrane potential (Δ¥M) by flow cytometry. Linear models, regression analysis and comparison of means by Duncan test, were conducted. The addition of carvacrol did not influence sperm motility, but at low concentrations decreased ROS production, whereas 30 μM C reduced the membrane functionality and 25 μM C decreased Δ¥M. In addition, regression coefficients showed that C produced a lower rate of decrease in different parameters of sperm motility and kinetics. During cooling there is a reduction in sperm quality due to the excessive production of ROS, generating oxidative stress and affecting cell permeability and functionality. In this study, it was possible to demonstrate the protective activity of C as a molecule capable of neutralizing free radicals. In addition, it has been proposed that C is also capable of reducing peroxyl radicals, superoxide radicals, hydrogen peroxide and nitric oxide. Carvacrol can mitigate the reduction of boar semen quality during the storage period under cooling conditions. Likewise, it can reduce ROS production and modulate the mitochondrial activity of porcine sperm.

Evaluación de la longevidad espermática en semen de Búfalo (Bubalus bubalis) refrigerado a 5 oC

The semen of 11 buffalo bulls, Murrah, aged from 5 to 6 years, were collected by artificial vagina and submitted physical and morphological analysis of the characteristics of the semen. The objective of the experiment was to test, in vitro, the efficacy of four different extensors (Control -TRIS with 10% LDL- and the same extender at concentrations of 0.5, 1 and 2% of soy lecithin) on the sperm longevity of buffaloes in the refrigeration process at 5 °C for 1, 24, 48, 72, 96 and 120 hours. After collection, each ejaculate was fractionated in 4 aliquots to obtain 50×106 SPTZ/mL, packed in 0.5 mL vats, refrigerated for 4 hours, obtaining a cooling curve of 0.25o C/min. The reeds were kept refrigerated at 5° C and sequentially evaluated, the contents reheated at 37° C/30 seconds prior to the evaluation of sperm motility (total and progressive) using the computerized system. Samples were taken for evaluation by the hypo-osmotic test (HOST) at times 1, 24, 48 and 72 hours. For the statistical analysis, the STATA 12.0 package was used, and the values obtained were submitted to the Friedman test (P &lt; 0.05). Progressive spermatozoa and plasma membrane integrity were higher (P &lt; 0.05) in the TRIS extender with 10% LDL plus 0.5% soybean lecithin (67.2% and 67.9%, respectively) in the refrigeration process for 48 hours. In conclusion, a comparative evaluation of the sperm parameters revealed that the TRIS extender with 10% LDL plus 0.5% soy lecithin can be effectively used in the preservation of refrigerated buffalo semen in AI and IATF for up to 48 hours.

Dimethylacetamide - an alternative to glycerol as cryoprotectant of Malabari buck semen

The key role of a cryoprotectant is to minimize the chemical and physical stress which occurs during cooling, freezing and thawing of semen. The difference between the cryoprotectant (CPA) occurs in their permeability coefficient and the structural model of the cryogenic agent. The beneficial effect of dimethylacetamide (DMA) as a cryoprotectant especially for sperms had been observed in several studies. The aim of the study was to study the cryoprotective effect of DMA in freezing the Malabari buck semen compared to glycerol. Ten ejaculates were taken from fourMalabaribucks . After preliminary evaluation sample split technique was followed with Tris based extender containing glycerol (6.7 per cent) as cryoprotectant (control) and Trisextender containing DMA (3 per cent) as cryoprotectant (treatment group). The semen straws (0.25mL) after filling were subjected for equilibration and manual freezing. Sperm kinetics was studied using computer-aided sperm analyzer. Pre-freeze and post-thaw evaluation included sperm viability, sperm abnormality, hypo osmotic test, acrosome integrity test and DNA fragmentation. Results indicated that inclusion of 6.7 per cent glycerol had significantly higher (p&lt;0.05) post-thaw values than DMA. From our study we conclude that 6.7 per cent glycerol was better than 3 per cent DMA in cryopreservation of Malabari buck semen.

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

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

Resfriamento de sêmen suíno em um diluente suplementado com isoespintanol

ABSTRACT: Spermatozoa experience oxidative, osmotic, chemical, and thermal stresses when cooled, which degrade the quality and fertilizing capacity of the cells. Adding antioxidants to the sperm extender mitigates these alterations. This study evaluated the effect of isoespintanol (ISO) on boar semen subjected to cooling. Fifteen ejaculates from five boars (Susscrofadomestica) were extended in Beltsville thawing solution (BTS) supplemented with 0 µM (control), 5 µM (ISO5), 10 µM (ISO10), 15 µM (ISO15), 20 µM (ISO20), 25 µM (ISO25), and 30 µM (ISO30) of ISO, which were then cooled for five days at 16 °C. Sperm kinetics, total motility (TM), and progressive motility (PM) were evaluated every 24 h using an IVOS computer-assisted sperm analysis (CASA) system. On day 1 and day 5 of cooling, a hypoosmotic test, spectrofluorometry, and flow cytometry were performed to evaluate the following: membrane functionality, measured as a function of hypoosmotic swelling (HOS); total antioxidant capacity (TAC); reactive oxygen species (ROS); and mitochondrial membrane potential (Δ¥M). Regression analysis and comparison of means using the Duncan test were performed. The ISO added had a slight impact on sperm motility, as evidenced by a reduction in TM at 24 h of cooling (but not prior) with the addition of 20 µM of ISO. Similarly, no effect of the ISO on the kinetics and functional integrity of the sperm membrane was observed at 96 h of cooling; however, the regression coefficients indicated that the ISO lowered the rate of decrease in sperm motility and the proportion of rapid spermatozoa relative to the concentration of ISO used. The ISO did not affect the TAC of the cooled semen; however, different concentrations of ISO lowered ROS production in the semen after 96 h of cooling. ISO also impacted the Δ¥M of the spermatozoa at 0 h of cooling, increasing the proportion of low Δ¥M cells and decreasing the proportion of high Δ¥M cells. In conclusion, ISO can reduce the loss of quality and oxidative stress occurring in boar semen during cooling and can modulate the mitochondrial activity of sperm.

Is seminal quality worsening? A 20-year experience in Córdoba, Argentina.

To assess the possible variations in semen quality during the last 20 years in Córdoba, Argentina, and to identify possible causal lifestyle or genitourinary factors. Retrospective study of 23,130 patients attending an andrology laboratory. The 20-year period (2001-2020) was divided into four quinquenniums. Seminal parameters (sperm concentration, motility, morphology, viability, and membrane functional integrity) were classified as normal or abnormal according to WHO, and results were expressed as percentage of patients abnormal for each parameter per quinquennium. In addition, the percentage of patients per quinquennium exposed to the different risk factors (daily alcohol and/or tobacco consumption; occupational exposure to heat or toxics; history of parotitis or varicocele; and high body mass index, BMI) was reported. Patients included in our study did not show impairment in seminal quality over time. Beyond a transient decrease in normozoospermia in the second and third quinquennium, possibly explained by a parallel increase in teratozoospermia, other important parameters of the spermogram did not change. In fact, abnormalities in sperm concentration (oligozoospermia), total sperm count, viability and response to hypoosmotic test showed a decreasing trend over time. On the other hand, parotitis, varicocele, morbid obesity and regular exposition to heat/toxics were the factors more frequently associated with semen abnormalities; the last two increased their frequency over the study period. The population included in this study did not show a clear impairment in semen quality during the last 20 years. The decreasing patterns found were associated with high BMI and exposure to heat/toxics.

Development of a microfluidic system structured on a modified polydimethylsiloxane device for the selection of bovine epididymal spermatozoa

Microfluidic systems are on the rise in several studies that evaluate reproductive cells. However, the material used for manufacturing can still be considered relatively expensive. The objective of this study was to develop a new microfluidic device, using a modified polydimethylsiloxane ((PDMS) Silpuran®), test its viability and carry out a selection of bovine sperm. Sperm was collected from epididymis (n = 10) and evaluated at different incubation times (60 min, 120 min, 180 min) to assess polydimethylsiloxane toxicity, where a tube was used as a control and the microfluidic device as treatment. An additional ten epididymis were used for the sperm selection test, which utilized four types of solutions: in vitro maturation medium (IVM) with and without oocyte, progesterone and saline solution (SS). The Percoll gradient was used as a control and the microfluidic device as treatment. The kinetic parameters of sperm were evaluated using the computer-assisted semen analysis (CASA). Morphology was performed with Bengal Rose, the integrity, and viability of the sperm using the hypoosmotic test and fluorescent microscopy probes, respectively. Mann-Whitney test was used in the first experiment, Kruskall-Wallis variance analysis tests with post hoc and Student-Newman-Keuls used in the second experiment. Regarding the non-toxic effects, most motility parameters demonstrated the superiority of the microfluidic device compared to the control. In the second experiment, the sperm showed equivalence between the microfluidic device and the Percoll gradient Silpuran® PDMS was not toxic to the cells and can be efficient for selecting bovine sperm, achieving better results in a medium for IVM with or without oocytes.

Cryoprotective Effects of Ergothioneine and Isoespintanol on Canine Semen.

Simple SummaryCryopreserving dog semen allows the long-term availability of male gametes for future artificial insemination and other assisted reproductive techniques. However, freezing causes irreversible damage to sperm that can affect its ability to fertilize and generate a viable pregnancy. Sperm alterations are partly attributed to oxidation produced by reactive oxygen species (ROS); therefore, antioxidants have been included as extenders for seminal cryopreservation. The unconventional natural antioxidants might reduce deleterious changes in cryopreserved dog sperm; therefore, we evaluated the effects of cryopreservation with the antioxidants ergothioneine and isoespintanol on thawed canine sperm. Various concentrations of both antioxidants improved the movement capacity and structure of thawed spermatozoa, possibly by reducing ROS production. The unconventional antioxidants isoespintanol and ergothioneine improved the quality of cryopreserved canine semen and hence improved assisted canine reproduction.Sperm undergo oxidative stress due to excessive production of reactive oxygen species (ROS) during cryopreservation. Some unconventional natural antioxidants can reduce ROS-induced changes in cryopreserved canine sperm. This study aimed to identify the cryoprotective effects of ergothioneine and isoespintanol on the quality of thawed canine semen. Twelve ejaculates from six dogs were cryopreserved in a tris-yolk extender without (control) or with 50 (E50), 100 (E100), or 150 (E150) µM ergothioneine or 20 (I20), 40 (I40), or 60 (I60) µM isoespintanol. We evaluated the motility and kinetics of thawed sperm using computerized analysis; determined morphology by eosin-nigrosin staining; functional membrane integrity using hypoosmotic tests, and structural membrane and acrosome integrity; mitochondrial membrane potential by fluorescence microscopy; and ROS production by spectrophotometry. Data were statistically analyzed using mixed models and Tukey tests. E100 increased total (60.6% vs. 49.6%) and progressive (26.4% vs. 20.1%) motility, straight line velocity (41.3 vs. 35.9 µm/s), and rapid sperm (17.6% vs. 12.3%) compared with controls. However, E150 reduced the numbers of hyperactive sperm. E100, I40, and I60 reduced the abnormal morphology and ROS production, and all concentrations of both antioxidants increased acrosomal integrity. We concluded that ergothioneine and isoespintanol reduce deleterious sperm alterations and oxidative stress in thawed canine semen.

Micromechanical properties and functional activity of granulocytes when simulating exogenous loading with ATP in vitro

The micromechanical properties of leukocytes make a certain contribution to the blood flow velocity in the microcirculatory bed, while the micromechanical properties themselves change under the influence of a complex network of purinergic signals.The aim of the work was to study the micromechanical properties and functional activity of granulocytes in normal conditions and in patients with acute lymphoblastic leukemia when simulating exogenous loading with ATP in vitro.Materials and methods. Leukocytes were isolated from the blood of patients with acute lymphoblastic leukemia and healthy people. Each sample was divided into a test sample and a control sample. In the test samples, the loading with ATP in vitro was simulated. Leukocytes of the control samples were incubated in the culture medium without the addition of ATP. Young’s modulus and adhesion force were measured using an atomic force microscope in the force spectroscopy mode. The cell surface potential was measured in an atomic force microscope in the Kelvin mode. To assess the functional activity of granulocytes, hypoosmotic tests in vitro and determination of migration activity were used.Results. In tests with exogenous ATP, both in samples from healthy people and from patients with acute lymphoblastic leukemia, a decrease in the rigidity and potential of the plasma membrane surface, an increase in the adhesive properties of leukocytes and migration activity were found. At the same time, the responses of granulocytes to the osmotic loading were different: for example, in the group of healthy people, the loading with ATP caused cell contraction and a decrease in the use of the membrane reserve by the cell in a hypotonic environment, and in patients with acute lymphoblastic leukemia, it caused an increase in the volume and more intensive use of the membrane reserve in volume regulation.Conclusion. The revealed effects indicate the leading role of the ATP molecule in the signal transduction mechanisms between blood cells in the microvasculature. The increase in the adhesive properties of the cell surface of granulocytes revealed in the study, in parallel with the increase in their migration activity under the influence of the ATP molecule, can contribute to the development of inflammation in the vessel wall.

34 Cellular effects of antifreeze proteins type I and III in extender for sheep semen cryopreservation

Antifreeze proteins (AFP) have been included in extenders for sperm cryopreservation to prevent ice crystal formation. Thus, this study assessed the effects of supplementing semen extender with two concentrations of AFP types I and III on the quality of frozen–thawed ram sperm. The hypothesis is that various types and concentrations of AFP enhance cryopreservation of ram sperm. Semen was collected from 4 rams, pooled in 6 replicates, and allocated into 1 of 5 treatments: Control (CONT, without AFP); AFP type I [0.1 (AFPI-0.1) or 0.5 (AFPI-0.5) µg±mL−1]; or AFP type III [0.1 (AFPIII-0.1) or 0.5 (AFPIII-0.5) µg±mL−1]. Straws were placed on a metal wire net frame at 37°C and placed in a refrigerator for 2h to cool them to 5°C (−0.25°C/min). After 2h for stabilisation, straws were cooled in liquid nitrogen vapor (−15.3°C/min) and subsequently immersed (−196°C). After thawing, samples from each treatment were evaluated microscopically (sperm kinetics, plasma membrane integrity, capacitation, hypoosmotic test, acrosome status and mitochondrial activity, chromatin condensation, morphology, binding to egg perivitelline membrane, and lipoperoxidation quantification). The normal distribution of residuals was determined by Shapiro-Wilk test and homoscedasticity by Levene’s test. Normally distributed variables were analysed with one-way analysis of variance (ANOVA), followed by Tukey’s test. The non-normally distributed were analysed by Kruskal–Wallis and Dunn’s test. The repeated-measures ANOVA in general linear model (GLM) was used to effects of concentration for each AFP type in paired samples. The Greenhouse-Geisser test was applied when sphericity was not considered, followed by the Sidak test. Values of P&amp;lt;0.05 were considered significant. Treatments affected (P&amp;lt;0.05) kinetic parameters, plasma membrane integrity, and morphology. Linearity was greater in AFPI-0.1 (56.6±3.1%, mean±s.e.m.), AFPI-0.5 (56.9±2.2%), and AFPIII-0.5 (64.7±6.2%) than in CONT (36.8±3.0%). Straightness was greater in all AFP-supplemented extenders (overall mean, 78.6±2.8%) than in CONT (63.2±0.8%). Plasma membrane integrity was greater in AFPI-0.1 (49.1±4.6%) and AFPI-0.5 (36.6±7.3%) compared with CONT (13.0±4.4%). All AFP groups had a greater percentage of normal sperm (overall mean: 74.3±1.3%) than CONT (65.3±1.9%). There were no significant differences in percentage of sperm with functional membrane (overall mean: 16.1±3.3%), normal acrosome (11.5±4.5%), mitochondrial activity (24.5±6.5%), chromatin condensation (98.8±0.4%), perivitelline membrane binding rate (194.0±44.5 sperm/mm2), and lipoperoxidation (556.7±20.5 TBARS ng±mL−1). In conclusion, the use of AFP, predominantly type I, had potential as a cryoprotectant for ram sperm, increasing sperm cell protection, with no adverse effects on potential fertilization capacity and did not increase reactive oxygen species. This research was funded by FAPERJ, CNPq, and CAPES (Finance Code 001).

Addition of antifreeze protein type I or III to extenders for ram sperm cryopreservation

Antifreeze proteins (AFP) play an important role in cellular survival at sub-zero temperatures. This study assessed the effect of AFP type I or III in semen extender (TRIS-egg yolk) for ram sperm cryopreservation. Pooled semen of four rams were allocated into five treatments: Control (CONT, without AFP); AFP Type I [0.1 (AFPI-0.1) or 0.5 (AFPI-0.5) μg/mL]; or III [0.1 (AFPIII-0.1) or 0.5 (AFPIII-0.5) μg/mL], and then frozen in six replicates. Treatments affected kinetic parameters, plasma membrane integrity and morphology (P < 0.05). The AFPIII-0.1 presented lesser total motility. Linearity was greater in AFPI-0.1, AFPI-0.5 and AFPIII-0.5 and straightness was greater in all AFP-supplemented extenders. Plasma membrane integrity was greater in AFPI-0.1 and AFPI-0.5. All AFP groups had greater percentage of normal sperm than CONT. No differences (P > 0.05) were observed in hypoosmotic test, sperm acrosome status, mitochondrial activity, chromatin condensation, perivitelline membrane binding rate and lipoperoxidation. In conclusion, the use of AFP, predominantly type I, may increase sperm cell protection during cryopreservation, with no adverse effect on potential fertilization capacity or increase in reactive oxygen species, being a potential cryoprotectant to ram sperm.

Effect of low-density lipoproteins and trehalose on the quality of cryopreserved bovine semen

Background: In artificial insemination, chicken egg yolk is added to bovine semen to protect it during the cryopreservation process, although it contains substances that can affect the microbiological quality and metabolism of sperm. Objective: To evaluate post-thaw quality of bovine cryopreserved semen added with centrifuged and non-centrifuged egg yolk, low-density lipoproteins (LDL), and trehalose (T). Methods: Ten ejaculates from five bulls were cryopreserved under the treatments T1: pure egg yolk (PEY) at 20% v/v, T2: centrifuged egg yolk (CEY) at 20% v/v, T3: LDL at 8% v/v, T4: T at 100 mM, and T5: T at 100 mM plus LDL at 8% v/v (TLDL). Spermatic motility and kinetics, functional membrane integrity (FMI), structural membrane integrity (SMI), sperm vitality (SV) and abnormal morphology (AM) were assessed using the Sperm Class Analyzer (SCA®) system, hypoosmotic test (HOST), SYBR/PI probes, and eosin–nigrosin staining, respectively. A completely randomized design was used. Normal distribution of the variables was validated through the Kolmogórov– Smirnov test. A generalized linear model was used to determine sources of variation. Means were compared using the Tukey test. Results: Inclusion of CEY or LDL had a similar effect on sperm protection, and were superior for motility, kinetics and membrane integrity compared to the other treatments (p&lt;0.05). CEY was superior for progressive motility (p&lt;0.05). The cryoprotective action of LDL was similar to TLDL for motility and kinetics, SMI, SV, and AM (p&lt;0.05). Inclusion of PEY and T resulted in the lowest semen quality (p&lt;0.05). The use of T resulted in a reduction in FMI and SMI (p&lt;0.05). No differences in AM between treatments were found (p&gt;0.05). Conclusions: Egg yolk can be replaced by centrifuged egg yolk or low-density lipoproteins in the freezing extender used for bovine semen used in artificial insemination.

Comparative Evaluation of Different Methods for Assessing Stallion Sperm Membrane Integrity

Comparative evaluation of three different methods for assessing stallion sperm-membrane integrity, including the eosin staining technique, the fluorochrome staining (SYBR-14 + PI) method, and the hypo-osmotic test, has been made in order to reveal the most efficient method of measurement for this item. The stallion sperm showed high coefficients of correlation between sperm motility and intact membrane proportion with all the methods of measurement. The best coincidence result was attained with the eosin staining technique, assessed by a correlation coefficient of 0.91. This technique has the advantages of simplicity, accessibility, and efficiency. Accurate assessment requires performing a method of subtracting a number of unstained damaged sperm cells, which are nonfunctional, from a total number of unstained spermatozoa. The proportion of motile sperm does not coincide with the proportion of sperm intact membranes during the stallion-semen storage process according to all the methods of measurement. A significant sperm count remains with intact cell membranes at the loss of total motility at the end of a storage process. This indicates that the sperm cells become immotile because of layout of the energetic material in flagellar mitochondria, which is not associated with membrane integrity. Therefore, it is recommended to assess the stallion sperm membranes with the use of fresh or thawed sperm. It is suggested to use assessment of membrane integrity in clarifying the sperm motility, particularly, in especially doubtful cases.

Effectiveness of tes-tris or tris association with low density lipoprotein on in vitro longevity of refrigerated buffalo semen

ABSTRACT This study investigated in vitro the efficacy of four different extenders (TES-TRIS and TRIS with LDL low-density lipoprotein at concentrations of 10 or 5%) on the longevity of buffalo sperm in the refrigeration process at 5ºC. Sperm motility was assessed every 24 hours up to 72 hours of incubation using computer assisted sperm analysis and sperm membrane integrity was examined by the hypoosmotic test (HOST) at T1, T24, T48 and T72 hours. Eleven buffaloes (1 ejaculate per buffalo) of the Murrah breed were used, ranging in age from 4 to 5 years. Immediately after collection, each ejaculate was fractionated into 4 aliquots, and each aliquot was diluted in one of four diluents to obtain 50x106SPTZ/mL. The samples were packed in 0.5mL straws and refrigerated (-0.25°C/min) to 5°C and maintained at this temperature until evaluation. Prior to evaluation the samples were heated at 37°C for 30 seconds. The statistical package used for analysis was STATA 12.0 "Statistical Analysis Software" and means were compared by the Friedman test (P&lt;0.05). The results of sperm kinetics and HOST indicate that the TRIS diluent with 10% LDL could be a promising alternative for semen refrigeration at 5ºC, to be used in conventional and fixed time artificial insemination.