Extender For Semen Cryopreservation Research Articles

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

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

The effects of purslane (Portulaca oleracea) and fennel (Foeniculum vulgare Mill) hydroalcoholic extracts on the functional parameters of human spermatozoa after vitrification.

Reactive oxygen species (ROS) are produced during cryopreservation of human sperm and impair sperm function. Antioxidant compounds, such as fennel and purslane, reduce the damaging effects of ROS. This study aimed to evaluate motility parameters, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), intracellular ROS, and DNA damage to determine the optimum concentrations of hydroalcoholic extracts of fennel and purslane for human spermatozoa cryopreservation. Twenty human sperm samples were used and divided into seven equal groups consisting of fennel hydroalcoholic extract (5, 10, and 15 mg/L), purslane hydroalcoholic extract (25, 50, and 100 mg/L), and no additive. Supplementation of 25 mg/L and 50 mg/L purslane extract and 10 mg/L fennel extract in cryopreservation extender significantly increased the motility and PMI of sperm with a significant reduction in intracellular ROS compared to control groups (p<0.05). A 50 mg/L concentration of purslane extract elevated progressive motility and MMP compared to the control group (p<0.05). No significant differences were seen for motion patterns and DNA damage of frozen-thawed human sperm in extender containing these extracts. The results showed that supplementation of 50 mg/L purslane extract and 10 mg/L fennel extract in semen cryopreservation extender has the potential to decrease intracellular ROS and subsequently elevate the motility and PMI of human sperm.

Adição de sucos de laranja, abacaxi e beterraba em diluidor para criopreservação de sêmen de carneiros

Abstract Searching for improvements in semen cryopreservation, natural substances are commonly studied focusing to improve the sperm quality. The aim of this study were evaluated the effect of adding orange, pineapple, and beet juices in different concentrations and combinations to the ram semen cryopreservation extender. Five ejaculates from five adult rams were used. The semen pool was diluted in egg yolk-based extender and mixed with the following 15 treatments (at a final concentration of 400.106 sptz/mL): orange 10% (O10) and 15% (O15); pineapple 10% (P10) and 15% (P15); beet 10% (B10) and 15% (B15); pineapple + orange 10% (PO10) and 15% (PO15); pineapple + beet 10% (PB10) and 15% (PB15); beet + orange 10% (BO10) and 15% (BO15); pineapple + beet + orange 10% (PBO10) and 15% (PBO15); and the control group (CON). Post-thaw in 0.25 mL straws semen quality analysis of cryopreserved semen was performed by CASA and flow cytometry. Analysis of variance (PROC GLM) was carried out and the averages were compared using the SNK test. Pearson's correlation test was also performed. No effect was noted in the addition of juices to the semen extender prior to cryopreservation. Post-thawed, although, statistically similar to the control group, the total motility of the B10 group reached acceptable standards of total motility. In addition, B10 group showed the highest values (p&lt;0.05) of progressive motility than control group or other treatments. The addition of 10% beet juice to the ram semen extender can improve the cryopreservation of sperm motility.

The Concentration of ProAKAP4 and Other Indicators of Cryopotential of Spermatozoa Cryopreserved in Extender with Holothuroidea Extract Addition.

Simple SummaryScientists are constantly making efforts to determine the fertilization capacity of sperm after ejaculation. The present research proves that the application of Holothuroidea extract to the semen extender improves its cryostability, and has a positive effect on the biological quality of sperm after the freezing process. Moreover, it has been proven that the proAKAP4 biomarker can be successfully used to evaluate the effectiveness of the use of various extenders for semen cryopreservation.The aim of this study was to determine the concentration of proAKAP4 and other indicators of cryopotential of spermatozoa cryopreserved in extender with Holothuroidea extract addition. Nine Holstein Friesian bulls, 3.5 years old, of known health status, were used for the study. The animals were kept and fed equally. Semen was collected once a week using an artificial vagina. The commercially available Holothuroidea extract was used as a supplement to the commercial extender (0, 2, 4 and 6 µL/mL) before the freezing/thawing process. The viability, motility, motion parameters, and acrosome integrity of the sperm were analyzed with (test) or without (control) extract samples. Furthermore, the concentration of the proAKAP4 biomarker in frozen sperm was assessed. It was shown that the addition of 4 and 6 µL of the extract may have a positive effect on the quality parameters of the sperm after thawing. The results indicate that extender supplementation with the above extract modulates (increases) the concentration of proAKAP4 in sperm at all tested levels. Additionally, this indicator has become helpful in identifying sperm of poor biological quality. Moreover, it has been proven that the proAKAP4 biomarker can be successfully used to evaluate the effectiveness of the use of various extenders for semen cryopreservation.

36&#x2003;Effect of antifreeze protein type I supplementation in the extender for semen cryopreservation in the domestic cat.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Jambul (Syzygium cumini) Pulp Extract Enhances Viability, Motility, andIn VitroFertilizability of Cryopreserved Bovine Semen

To study the ameliorative capacity of Syzygium cumini fruit Pulp Extract (SPE) on cryopreserved bovine semen, three ejaculates each of three fertile bulls (total nine samples) were collected. Each sample was mixed with semen dilution medium in 1:2 ratios, centrifuged, and 50% of the supernatant was discarded. The remaining sample was mixed with semen cryopreservation extender (SCE) in 1:4 ratios. Three aliquots (0.3 mL) of each extended semen sample were further diluted with (1) 0.7 mL SCE (SCE group), (2) 0.7 mL SCE containing SPE equivalent to 7 ppm total antioxidants (SPE7 group), and (3) 0.7 mL SCE equivalent to 14 ppm antioxidants (SPE14 group). For each aliquot, semen quality was analyzed in situ as well as after cryopreservation. Results show significant improvement in sperm membrane integrity, motility, and fertilizability in SPE7 (63.5% ± 2.04%, 56.22% ± 1.7%, 51.1%, respectively) and SPE14 (68% ± 1.12%, 63.67% ± 1.06%, 57.7%, respectively) than the SCE group (62.33% ± 1.22%, 54.9% ± 1.34%, 48.8%, respectively). Similarly, significant improvement in the mean percent number of progressively motile sperm at the expense of the mean percent twitching, shaking, and whirling spermatozoa, both before and after cryopreservation, were observed in SPE7 (72 ± 1.06, 61.2 ± 1.2) and SPE14 (78.89 ± 1.12, 67 ± 1.32) groups to that of the SCE (59.6 ± 0.97, 52.3 ± 3.5). In addition, the mean percent of progressively motile spermatozoa showing 20 μm/s or above velocity, before and after cryopreservation, were significantly higher in SPE7 (58.9 ± 4.7, 43.6 ± 1.5) and SPE14 (66.9 ± 0.8, 51.3 ± 0.8) groups than that of the SCE (42.3 ± 3.1, 30 ± 2.4). These findings indicate that SPE can significantly improve various quality parameters of the cryopreserved bovine semen.

Does supplementation of vitamin C, reduced glutathione or their association in semen extender reduce oxidative stress in bovine frozen semen?

ABSTRACT The aim of this study was to evaluate the addition of vitamin C, reduced glutathione and the association thereof to the bovine semen cryopreservation extender. The ejaculate from nine bulls were divided into four fractions, each corresponding to a treatment, namely: control group-semen diluted with Tris-yolk extender; vitamin C group-semen diluted in Tris-yolk extender supplemented with vitamin C (2.5mmol/mL); glutathione group-semen diluted in Tris-yolk extender supplemented with reduced glutathione (2.5mmol/mL) and associated group-semen diluted in Tris-yolk extender supplemented with vitamin C (1.25mmol/mL) and reduced glutathione (1.25mmol/mL). Afterwards, the semen was packed into French straws and submitted to cryopreservation using automated equipment. After cryopreservation, the semen was thawed and evaluated considering sperm motility, morphology, plasma membrane, acrosome, mitochondrial potential and oxidative stress, as well as the thermo resistance test. Extender’s supplementation with the association of vitamin C and reduced glutathione showed benefic effects on sperm motility and preservation of plasma and acrosomal membranes during semen cryopreservation, being also the group that showed higher values of reactive oxygen species. Thus, the association of both antioxidants contributed to the preservation of sperm cells in every analyzed characteristic, suggesting its use on bovine semen cryopreservation.

Efficiency of Commercial Egg Yolk-Free and Egg Yolk-Supplemented Tris-Based Extenders for Dromedary Camel Semen Cryopreservation

Simple SummaryThis study compared the efficiency of commercial egg yolk-free (AndroMed, OPTIXcell) and egg yolk-supplemented (Triladyl, Steridyl) Tris-based extenders for semen cryopreservation in dromedary camels. The camel-specific extender SHOTOR was used for reference. SHOTOR, Triladyl, Steridyl, AndroMed, and OPTIXcell can all be used for camel semen cryopreservation; however, SHOTOR and Triladyl provide the best post-thawing sperm quality.This study compared the efficiency of commercial egg yolk-free (AndroMed, OPTIXcell) and egg yolk-supplemented (Triladyl, Steridyl) Tris-based extenders for semen cryopreservation in seven adult dromedary camels. The camel-specific extender SHOTOR was used as control. The collected semen samples were evaluated and diluted with SHOTOR, Triladyl, Steridyl, AndroMed, or OPTIXcell. The diluted semen was gradually cooled and equilibrated for two hours before liquid nitrogen freezing. Semen was evaluated prior to freezing and after freeze-thawing cycles for motility, kinetics, vitality, abnormality, plasma membrane integrity, and DNA fragmentation using computer-assisted sperm analysis. In pre-freezing evaluation, progressive sperm motility was higher in SHOTOR-diluted samples (21.54 ± 1.83) than in samples diluted with Steridyl, OPTIXcell, or AndroMed (15.76 ± 1.80, 17.43 ± 1.10, and 13.27 ± 1.07, respectively). Moreover, Triladyl and SHOTOR resulted in significantly (p < 0.05) better sperm vitality and DNA integrity than all other diluents, but Triladyl resulted in a significantly (p < 0.05) better plasma membrane integrity (87.77 ± 0.31) than SHOTOR (85.48 ± 0.58). In the post-thawing evaluation, Triladyl led to significantly (p < 0.05) higher sperm motility (38.63 ± 0.81%; p < 0.05) when compared to SHOTOR, Steridyl or AndroMed (35.09 ± 1.341%, 34.4 ± 0.84%, and 31.99 ± 1.48%, respectively), with OPTIXcell being the least efficient (28.39 ± 0.86%). Progressive sperm motility was the highest when using Triladyl. Post-thawing curvilinear, straight line and average path sperm velocities were highest with Triladyl and lowest with AndroMed. Triladyl led to the highest linearity coefficient and straightness sperm coefficient, while SHOTOR to the highest DNA and plasma membrane integrity. OPTIXcell and AndroMed resulted in poor post-thawing sperm vitality, while Steridyl was less efficient than Triladyl. The highest rate of sperm abnormalities was recorded with OPTIXcell and the lowest with SHOTOR or Triladyl. In conclusion, SHOTOR, Triladyl, Steridyl, AndroMed, and OPTIXcell can all be used for camel semen cryopreservation; however, SHOTOR and Triladyl provided the best post-thawing sperm quality. Based on our findings, Triladyl is the best commercially available extender for dromedary camel semen cryopreservation to date.

Effect of different concentrations of l-carnitine in extender for semen cryopreservation in sheep

This study assessed the effect of l-carnitine (LC) in sheep semen extenders containing or not egg yolk for cryopreservation in sheep. Two extenders (TRIS-egg yolk or the commercial optiXcell™ IMV medium) were used, totaling six groups: IMV – (0, 5 and 10 mM LC) and TRIS – (0, 5 and 10 mM LC). After the freezing-thawing process and throughout incubation at 38 °C for up to 3 h, several parameters were evaluated: sperm kinetics, hypoosmotic, plasma membrane integrity, capacitation status and lipid peroxidation level. The supplementation of either 5 or 10 mM LC randomly affected some parameters and, overall, TRIS was superior (P < 0.05) than IMV extender. In the LC-groups, IMV had greater (P < 0.05) oxidative stress than TRIS. In conclusion, although LC affected isolated parameters, its supplementation in semen extender had no consistently beneficial effect on freezing-thawing ram sperm.

Low Density Lipoproteins at 2% Concentration Can Replace Whole Egg Yolk in TES-Tris-Milk Extender for Freezing Buffalo Sperm Cells

Background: Over the years, the most commonly used extenders for semen cryopreservation contain egg yolk as cryoprotectant. However, more recent studies have used the low density lipoproteins, extract of hen egg yolk which is responsible for the cryoprotective effect. Nevertheless, little was known about its required minimum concentration as well as its interaction with other extra cellular cryoprotectants, like skimmed milk. The present study aimed at investigating the effect of replacing whole egg yolk by adding low density lipoproteins at low concentrations, in TES-Tris-skim milk based extender, on the post-thaw quality of buffalo bull sperm.Materials, Methods &amp; Results: Eighteen ejaculates were collected from six buffalo bulls and diluted with TES-Tris-skim milk based extender containing LDL, extracted from hen egg yolks, at the concentrations of 2%, 4%, 8% and 14%, against a control extender containing 20% fresh egg yolk. After semen collection, analyses of subjective motility, vigor, force tourbillon, sperm concentration (Neubauer chamber) and sperm morphology (phase contrast microscopy) were performed. The diluted semen was packaged in 0.25 mL straws, and cooling was performed on computerized machine (TK 4000®), using a cooling rate of -0.25°C/min to 5°C. Semen was kept in balance at 5°C for 4 h. The straws were frozen in an ice chest, kept at 5 cm from the surface of liquid nitrogen for 20 min and then immersed in liquid nitrogen. The samples were kept in cryogenic container until thawing. Post-thaw kinetic parameters during incubation at 37°C (CASA), sperm membrane integrity (SYBR-14/PI), membrane functionality (hypo-osmotic swelling test) and DNA fragmentation (%DFI - SCSA) were evaluated after thawing. Immediately post-thaw, total motility was higher in the control (56.53 ± 9.73) than in the tested extenders; however, after 30 min the difference was no longer detected. All other kinetic parameters presented significantly better results in extenders containing 2%, 4% and 8% LDL, compared with the control. There was no difference between treatments regarding the integrity of membranes or fragmentation of the DNA after freezing/thawing procedures.Discussion: The molecules of low density lipoproteins of egg yolk have the known action of sequestering BSP ( binder of sperm proteins) protein, due to the chemical affinity of their main components, phosphatidylcholine and phosphatidylethanolamine. The BSP are responsible for removing cholesterol from the plasma membrane of the sperm, preparing it for the sperm capacitation phase. Low density lipoproteins by inhibiting the action of the BSP increase the stability of the plasma membrane during the freeze-thaw process. The milk caseins micelles have a similar ability to bind the BSP, but with lower affinity. The present study has shown that the use of purified low density lipoproteins has an advantage over the use of whole egg yolk, especially with regard to the kinetic parameters of the spermatozoa after thawing. Furthermore, it was observed that low concentrations of low density lipoproteins, such as 2%, in extenders containing skim milk, could preserve the sperm cell during the freeze-thawing process like that higher LDL concentrations and whole egg yolk. Further studies are needed to determine if the cryoprotectant action found in this study was a result of the synergistic action of skim milk with lipoproteins or even at low concentrations like 2% the lipoproteins can provide protection to the buffalo spermatozoa during freezing, as it has been reproduced in other domestic species. In conclusion, our results indicated that the extender TES-Tris-skim milk containing 2% LDL extracted from egg yolk could be used successfully in the cryopreservation of buffalo sperm cells.

Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation

ABSTRACT Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.

Cryopreservation of Semen of Mithun and Siri Bulls

The objective of the study was to assess the semen characteristics of Jersey, Mithun and Siri breeds of bulls and to compare the suitability of Tris Eggyolk Citrate and commercial soya lecithin based BIOXcell extenders for cryopreservation of semen from the these breeds of bulls. The mean semen volume was 4.55± 1.57, 4.86±1.33 and 4.83±1.72 ml in Jersey, Mithun and Siri bulls, respectively. Mean mass activity of 3, mean initial motility of 78.0±4.22, 80±4.47 and 78.33±4.08%, mean sperm concentration of 1416±484.19, 1530.91±517.41, 1122.83±293.68 x106 and mean livability of 69.20±6.81, 70.45±8.42, 76.67±8.17% was found in Jersey, Mithun and Siri bulls, respectively. There is no significant difference in all above mentioned variables among the breeds. The post-thaw motility of semen cryopreserved in Tris Eggyolk Citrate extender was found to be 45.50 ± 4.38, 46.82 ± 6.81 and 47.50 ± 8.22% in Jersey, Mithun and Siri bulls, respectively. Whereas in BIOXcell extender, it was 45.50 ± 5.99, 46.36 ± 5.05 and 49.17 ± 5.85 % in Jersey, Mithun and Siri bulls, respectively. Similarly, the plasma membrane integrity of thawed spermatozoa in Tris eggyolk extender was 17.50±2.32, 21.00±3.12 and 21.58±3.83% in Jersey, Mithun and Siri bulls, respectively. Whereas in BIOXcell extender, it was found to be 20.45±2.86, 20.86±2.281 and 22.33±3.24% in Jersey, Mithun and Siri bulls, respectively. No significant difference (p&gt;0.05) was observed in post-thaw motility as well as in plasma membrane integrity between extenders and between breeds, and there was no significant effect (p&gt;0.05) of breed x extender interaction. It can be concluded that the semen characteristics of Siri and Mithun bulls fall with the range established for other breeds of bulls. Furthermore, both Tris eggyolk and BIOXcell extenders are equally suitable for the cryopreservation of semen from these three breeds of Bulls.DOI: http://dx.doi.org/10.3329/bjvm.v12i2.21277 Bangl. J. Vet. Med. (2014). 12 (2): 143-153

Comparison of different freezing extenders for semen cryopreservation from stallions with poor and good semen freezability

s / Journal of Equine Veterinary Science 34 (2014) 60 60 agreement with a prior study [3] where stallion epididymal sperm were frozen with different extenders. The results of this study allow us to conclude that frozen semen with the extender BotucrioTM presents better sperm kinetic param- eters after thawing when compared to other commercial extenders and that the differences were more evident when stallions with poor semen cryopreservation were used.

제주흑우 정액 동결을 위한 AndreoMed와 Triladyl 희석제의 동결성 비교

This study was carried out to investigate synthetic extender for semen cryopreservation of Jeju Native Black Bull. The semen was collected using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by Tris-Egg yolk extender and contrifuged in 1,500 rpm for 15 minutes. The supernatant was removed. The pellect was diluted to final sperm concentration of 2×108/ml by doubling in every 30 minutes at 4℃ cold chamber. The semen was equilibrated for 4 hours at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm for 10 minutes. The height and duration affect the freezing speed by temperature. The frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at 37℃ water bath. Frozen-thawed sperm were evaluated sperm viability, membrane integrity and acrosome integrity. Post-thawed sperm viability has been significantly higher (p<0.05) in fresh sperm (93.27±1.62%) than frozen-thawed sperm (73.34±3.27%). However, there were no significant differences between fresh and frozen-thawed dead cell rate (7.35±2.63 vs, 13.71±2.85). In sperm motility, between Triladyl and AndroMed Extender, there was no significant different (72.86±2.83 vs, 81.47±2.48), similarly, the dead cell rates was similar (18.41±3.42% and 17.26±4.25). The results of our study suggest that AndroMed to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Jeju Native Black bull semen.

Effect of cysteine and glutamine added to extender on post-thaw sperm functional parameters of buffalo bull

Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris-egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post-thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen-thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen-thawed spermatozoa.

Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with antioxidants

The objective of this study was to evaluate the effect of addition of the antioxidants Trolox and catalase to a ram semen cryopreservation extender on lipid peroxidation and hydrogen peroxide generation on the extender and in the thawed semen. Semen was collected from 23 Santa Inês rams (one ejaculate per ram) and diluted at 32 °C to a concentration of 400 × 10 6 cells/ml in one of the following solution: Tris-egg yolk extender (control), or the same extender supplemented with either 50 μM Trolox/10 8 sperm (Trolox), 50 μg catalase/ml (Catalase) or a combination of Trolox and catalase (Tro + cat, 50 μM Trolox/10 8 sperm and 50 μg catalase/ml). The semen was loaded into 0.25 ml straws, cooled and frozen in a programmable freezer and subsequently stored in liquid nitrogen. Prior to evaluation, frozen straws were thawed in a water bath (42° C for 20 s). Lipid peroxidation (LPO), both spontaneous and catalyzed, on the semen and the extender were measured using the thiobarbituric acid (TBA) assay in accordance with the method described by Buege and Aust (1978). Hydrogen peroxide (H 2O 2) generation was measured using the horseradish peroxidase-dependent oxidation of phenol red to a derivative with absorbance at 610 nm, according to the method described by Pick and Keisari (1980). Spontaneous LPO resulted in the least production of thiobarbituric acid-reactive substances (TBARS) in the Tro + cat (1.37 ± 0.02 nMol/10 8 sperm), compared to amounts in the other treatments groups. In the catalyzed LPO experiments, the least ( P < 0.05) amounts of TBARS were observed in Trolox (2.52 ± 0.02 nMol/10 8 sperm) and Tro + cat (2.54 ± 0.02 nMol/10 8 sperm) groups, compared to the control (3.81 ± 0.02 nMol/10 8 sperm) and catalase (3.83 ± 0.02 nMol/10 8 sperm) groups. Hydrogen peroxide generation was less ( P < 0.05) in the Trolox (6.00 ± 0.18 nMol/40 × 10 6 sperm/ ± 40 min) and Tro + cat (6.08 ± 0.18 nMol/40 × 10 6 sperm/ ± 40 min) groups than in the control (6.97 ± 0.18 nMol/40 × 10 6 sperm/ ± 40 min) and catalase (6.53 ± 0.18 nMol/40 × 10 6 sperm/ ± 40 min) groups. Compared to the control group, Trolox and catalase treatment significantly reduced TBARS in catalyzed LPO and hydrogen peroxide concentrations in the samples ( P < 0.05). ROS (reactive oxygen species) generation occurred in all extenders, without sperm cells. The data presented provide evidence that ROS are produced in ram semen, both in the extender and during the freezing and thawing process. In addition, the data suggest that the antioxidants Trolox and catalase may be used to control the oxidative stress imposed on ram spermatozoa by the cryopreservation process.

Motility and viability of ram sperm cryopreserved in a Tris-egg yolk extender supplemented with anti-oxidants

The objective of this study was to evaluate the effects of the addition of the anti-oxidants Trolox and catalase to a ram semen cryopreservation extender, on the basis of post-thaw sperm motility, plasma membrane integrity, acrosomal membrane integrity and mitochondrial membrane potential. Semen from 23 Santa Inês rams was collected using an artificial vagina, and one ejaculate from each ram was evaluated and included in the study. Semen samples were diluted to a concentration of 400 × 10 6 cells/ml in Tris-egg yolk extender (control) or the same extender supplemented with 50 μM Trolox/10 8 sperm (TRO), 50 μg catalase/ml (CAT) or a combination of Trolox and catalase (TROCAT). The semen was loaded into 0.25 ml straws, cooled and frozen in a programmable freezer and subsequently stored in liquid nitrogen. Frozen straws were thawed in a water bath for sperm evaluation. Post-thaw sperm motility characteristics were determined by CASA, and plasma membrane integrity, acrosome integrity and mitochondrial membrane potential were assessed using PI/FITC-PSA/JC-1 probes. Total sperm motility was significantly higher ( P < 0.05) in the control (72.0 ± 1.5%) and in the samples in which catalase was included in the extender (CAT = 69.0 ± 1.5%), compared to samples containing only Trolox (TRO = 65.0 ± 1.5%) or Trolox in combination with catalase (TROCAT = 61.0 ± 1.5%). Progressive motility was significantly ( P < 0.05) higher in the CAT extender (31.0 ± 1.0%) than in the TRO (26.0 ± 1.1%) and TROCAT (27.0 ± 1.1%) supplemented extenders. Catalase supplementation resulted in a significantly ( P < 0.05) higher percentage of viable sperm (35.5 ± 1.3%) following cryopreservation, than the control (29.9 ± 1.3%) and TROCAT (28.1 ± 1.3%) supplementation. Anti-oxidant supplementation did not improve mitochondrial activity. The total number of sperm showing a high mitochondrial membrane potential was low in all treatments. The percentage of viable sperm was positively related with total sperm motility, progressive motility and rapid movement, while a negative correlation was observed with the percentage of slow and static sperm. Results indicate that supplementation of the extender with the anti-oxidant catalase improves sperm viability and, to a lesser extent, the progressive motility of ram sperm after cryopreservation in a Tris-egg yolk extender.

Effect of low density lipoprotein on the quality of cryopreserved dog semen

Egg yolk is included in extenders for semen cryopreservation due to its protective effect against cold shock, which is attributed to the presence of low density lipoprotein (LDL). This study evaluates how semen quality is affected by using LDL as a replacement for egg yolk in extenders for cooled and frozen dog semen. In Experiment 1, semen was extended in TRIS–glucose at 5 °C, in four treatments: 20% egg yolk (T1); 6% (T2); 8% (T3); and 10% LDL (T4). Sperm motility and membrane integrity after 24, 48, 72 and 96 h and the 50% conservation rate of motile spermatozoa (50 M) were evaluated. The 50 M was less for T1 than for the other treatments ( P < 0.01), but T2–T4 did not differ ( P > 0.05). In Experiment 2, glycerol at 10% was included in the freezing extender, in treatments similar to those from Experiment 1. Sperm motility and membrane integrity did not differ for T2, T3 and T4 at any period in Experiment 1 and after thawing in Experiment 2 ( P > 0.05), but were greater for all LDL treatments than for T1 ( P < 0.01), in both experiments. Thus, LDL can replace egg yolk in the composition of the TRIS–glucose extender for cooled or frozen dog semen.

Effect of egg yolk and seminal plasma heparin binding protein interaction on the freezability of buffalo cauda epididymal spermatozoa

Egg yolk is routinely used in most of the extenders for cryopreservation of semen, but mechanisms of protection of spermatozoa by egg yolk are not very clear. Investigations with buffalo cauda epididymal sperm have shown that seminal plasma heparin binding proteins have detrimental effects during semen cryopreservation. The present study was conducted to investigate the effect of egg yolk on the detrimental effects of heparin binding proteins during cryopreservation of buffalo cauda epididymal spermatozoa. The results indicated that egg yolk was able to reduce the heparin binding proteins mediated cryoinjury in spermatozoa. One of the mechanisms of protection of spermatozoa from cryoinjury by egg yolk may be due to the inhibition of deleterious actions of heparin binding proteins on the spermatozoa.