Tris-based Extender Research Articles

Impact of Algerian date palm pollen aqueous extract on epididymal and ejaculated rabbit sperm motility during in vitro incubation

Date palm pollen (DPP) has been widely tested in vitro in preservation of spermatozoa of different farm animals except in rabbits. The objective of this study was to evaluate the effect of various concentrations (20, 40, 80 mg/mL) of DPP aqueous extract on epididymal and ejaculated rabbit spermatozoa during in vitro incubation at 37 °C during 120 min. NaCl Pollen Extender (NPE) and TRIS Base Extender (TPE) were respectively used in epididymal and ejaculated sperm preservation. Sperm motility parameters were objectively analysed by a Computer Aided Sperm Analyser (CASA). The results revealed that in epididymal sperm at 0 minute, sperm motility showed significant higher values (p ≤ 0.05) for all doses of DPP in comparison to the control group. After 30, 60 and 120 min, the motility rate was higher (p > .05) in all the experimental groups compared to the control. Concerning velocity traits (VCL and VAP), and trajectories parameters (LIN, ALH, BCF), the doses of 40 mg/mL and 80 mg/mL showed the highest values than Control. In ejaculated samples, the motility rate at 0 minute improved only with the dose of 80 mg/mL and DPP showed protective effect at all doses (20, 40, 80 mg/mL) up to 60 min of incubation. DPP enhanced spermatozoa velocity (VCL and VAP) and the trajectory of sperm (LIN, ALH, BCF) even from 20 mg/mL at any points of storage. In conclusion, aqueous extract of date palm pollen is a suitable supplement to the extender for rabbit sperm by protecting and improving sperm motility parameters. HIGHLIGHTS The addition of DPP extract improves the quality of epididymal and ejaculated rabbit sperm. The extender supplemented with 80 mg/mL of DPP in NPE has a beneficial effect on kinetic traits of epididymal sperm. Nevertheless, in TPE extender, 20 mg/mL of DPP was enough to improve sperm motility traits in ejaculated rabbit semen.

Spirulina platensis extract addition to semen extender enhances cryotolerance and fertilizing potentials of buffalo bull spermatozoa.

The objective of this study was to investigate the effect of Spirulina platensis extract (SPE) addition to the freezing extender on freezability, lipid peroxidation, ultrastructure alterations and fertilizing potentials of frozen-thawed buffalo bull spermatozoa. Semen samples were collected with artificial vagina from five adult fertile bulls and diluted with Tris-base extender containing SPE (1, 5, 10 and 20 μg/mL) or without SPE (control). Diluted semen was cooled to 4 °C throughout one hour and frozen in 0.25 mL straws: prior to being stored in liquid nitrogen. Cryopresreved spermatozoa were assessed for post-thawing sperm motility, viability, acrosomal integrity, ultrastructure changes, antioxidant activities, lipid peroxidation and fertility rate. The current results clearly indicated that adding 10μg/mL SPE to the freezing extender significantly improved (P< 0.05) post-thawing motility and decrease the percentage of acrosomal damage (51.67±6.02% and 16.33±1.46%, respectively) compared with the control (28.33±4.41% and 26.33±1.77%, respectively). Moreover, addition of 10 μg/mL SPE to the semen extender significantly diminished (P< 0.05) MDA concentration (10.66±2.40 nmol/109) compared with the control (22.66±4.26 nmol/109). Therefore, the present results revealed that addition of 10μgl/mL SPE to the freezing extender might improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.

Soybean Lecithin as an Alternative to Egg Yolk in Tris-Based Extender of Cryopreserved Apri Rabbit Semen

The present study was conducted to demonstrate whether soy lecithin could be used as an alternative to egg yolk (EY) in tris-extender for cryopreservation of APRI rabbit semen. In tris-extender (E1), 18% EY was replaced by 1% (E2), 1.5% (E3), or 2% (E4) SBL (1:4). Semen was evaluated visually after cryopreservation stages and by CASA only post-thawing. Results showed that visual sperm motility, livability, abnormality, acrosome integrity, and membrane integrity were improved (P<0.05) by E3 after all cryopreservation stages. Slow, total progressive, and total motility were the highest (P<0.05), while non-motility and non-progressive motility were the lowest (P<0.05) with E3 compared with other extenders. In comparing with E1, E3 showed the best dynamic sperm parameters, in terms of the highest VCL, STR (P<0.05), VSL (P≥0.05), and the lowest (P<0.05) WOB. Prolificacy of rabbits showed the highest (6.68 kits/doe, P<0.05) by E3 compared with 4.96, 4.72, and 2.8 kits/doe for E1, E2, and E4, respectively. The correlation coefficient was the lowest (r=0.691, P<0.05) between acrosome integrity and litter size, and the highest (r= 0.921, P<0.001) between membrane integrity and prolificacy. Progressive motility and slow motility correlated with pregnancy (r=0.811and r=0.778, P<0.01) and prolificacy (r=0.898, P<0.001 and 0.801, P<0.01). In conclusion, addition of soybean lecithin at a level of 1.5% as an alternative to 18% egg yolk in tris extender may has a beneficial impact on motility parameters of cryopreserved APRI-rabbit spermatozoa, and prolificacy of doe rabbits.

The Eff ect of Gallic Acid Addition to Tris-Based Extender on Frozen Bull Semen

The Eff ect of Gallic Acid Addition to Tris-Based Extender on Frozen Bull Semen

Effect of Monthly Variations on the Plasma Membrane, Acrosome and DNA Integrities of Spermatozoa in Friesian Holstein Bulls Born in Iraq

This study was aimed to evaluate the influence of months of the year on the quality of semen in Holstein bulls. A study carried out at artificial insemination centre/ Abou-Ghareeb/ western of Baghdad. A total of 160 ejaculates were collected from 15 bulls born in Iraq via the artificial vagina. The age of the bulls ranged between 4 to 5 years and the study period were December to March and September. The semen samples were diluted with Tris base extender. The semen was packed in a straw according to the program of artificial insemination centre. Semen characteristics (plasma membrane, acrosome, and DNA integrities) were evaluated. The results revealed a significant decrease (P≤0.01) in the plasma membrane and acrosome integrity in September as compared with December, January, February, and March. There was a significant decline (P≤0.05) in DNA integrity in September as compared with December, January, February, and March in fresh and frozen semen. In conclusion, the September month had a negative effect on the plasma membrane, acrosome, and DNA percentage in all bulls.

The proteomic characterization of ram sperm during cryopreservation analyzed by the two-dimensional electrophoresis coupled with mass spectrometry

The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality.

Addition of butylated hydroxytoluene (BHT) in tris-based extender improves post-thaw quality and motion dynamics of dog spermatozoa

The aim of this study was to evaluate the interaction of different concentrations of butylated hydroxytoluene (BHT) in a tris-based extender on semen quality parameters in post-thawed dog semen. Twenty-four ejaculates were collected from eight male Beagle dogs using an artificial vagina. Pooled semen was diluted with a tris-based extender supplemented with 0 (control), 0.5, 1.0, 1.5, and 2.0 mM BHT, at a final concentration of 200 × 106 spermatozoa/mL. After thawing, sperm samples were assessed for motility parameters (CASA), membrane integrity (SYBR-14/PI), acrosome integrity (FITC-PNA), mitochondrial activity (JC-1/PI), malondialdehyde (MDA) concentration, and glutathione peroxidase (GPx) activity. The total motility, progressive motility, and average path velocity of the frozen-thawed sperm were significantly higher in the BHT1.5 group than in the control and the other sample groups (P < 0.05). Higher values of straight-line velocity, curvilinear velocity, amplitude of the lateral head displacement, and linearity were observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (P < 0.05). The BHT1.0 and BHT1.5 groups had higher percentages of straightness and acrosome integrity than the other groups (P < 0.05). Beat cross frequency, plasma membrane integrity, and GPx activity of the BHT1.5 and BHT2.0 groups were higher than those of the control (P < 0.05). A lower concentration of MDA was observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (BHT0) (P < 0.05).Our results indicate that 1.5 mM BHT is the optimal concentration for improving the post-thaw quality of canine spermatozoa.

Efficiency of Tris-Based Extender Steridyl for Semen Cryopreservation in Stallions.

Simple SummaryThe cryopreservation and long-term storage of semen is one of the methods for accelerated improvement of the genetic qualities of animals. However, horse breeders prefer to use fresh or chilled semen, as the fertilizing capacity of frozen equine semen is much lower. It is important to find extenders, or a combination of extenders, that will improve semen survival after freezing. It is also important that the extender can be easily and simply prepared for use. Steridyl is a concentrate to which you just need to add sterilized water. This extender was developed for ruminants. In this study we tested Steridyl for freezing stallion semen. The motility, morphology, energy metabolism, DNA damage, and fertility of sperm frozen in Steridyl were evaluated. As a result, Steridyl was shown to be a good extender for equine semen freezing.The fertilizing ability of stallion sperm after freezing is lower than in other species. The search for the optimal extender, combination of extenders, and the freezing protocol is relevant. The aim of this study was to compare lactose-chelate-citrate-yolk (LCCY) extender, usually used in Russia, and Steridyl® (Minitube) for freezing sperm of stallions. Steridyl is a concentrated extender medium for freezing ruminant semen. It already contains sterilized egg yolk. Semen was collected from nine stallions, aged from 7 to 12 years old. The total and progressive motility of sperm frozen in Steridyl was significantly higher than in semen frozen in LCCY. The number of spermatozoa with normal morphology in samples frozen in LCCY was 60.4 ± 1.72%, and with Steridyl, 72.4 ± 2.10% (p < 0.01). Semen frozen in Steridyl showed good stimulation of respiration by 2.4-DNP, which indicates that oxidative phosphorylation was retained after freezing–thawing. No differences among the extenders were seen with the DNA integrity of spermatozoa. Six out of ten (60%) mares were pregnant after artificial insemination (AI) by LCCY frozen semen, and 9/12 (75%) by Steridyl frozen semen. No differences among extenders were seen in pregnancy rate. In conclusion, Steridyl was proven to be a good diluent for freezing stallion semen, even though it was developed for ruminants.

Decreasing glycerol content by co-supplementation of trehalose and taxifolin hydrate in ram semen extender: Microscopic, oxidative stress, and gene expression analyses

This study aimed to evaluate the comparative effects of taxifolin hydrate and trehalose on the quality of frozen-thawed ram spermatozoa for the first time. Ejaculates collected from six mature rams were pooled, and divided to eight equal aliquots to extend them with different concentrations of glycerol (%5 and %3), taxifolin hydrate (10, 100, and 500 μM), and trehalose (60 mM) as eight groups (G5T0, G5T10, G5T100, G5T500, G3T0, G3T10, G3T100, and G3T500). After freeze-thawing process of cryopreservation, microscopic and oxidative stress parameters, and gene expression levels were investigated for understanding of possible impacts of taxifolin hydrate and trehalose. The study showed that G3T10 resulted in the highest post-thawed viability and mitochondrial activity. Moreover, all extenders with taxifolin hydrate reduced DNA fragmentation in comparison to G5T0, but DNA damage was prevented at the highest rate in presence of G5T10. The level of LPO significantly decreased in the groups G5T500 and G3T100, and the expression levels of NQO1, GCLC, and GSTP1 genes significantly increased in the groups G5T100, G5T500, G3T10, and G3T100 compared to the group G5T0. Finally, co-supplementation of tris-based extender having 3% glycerol with 60 mM trehalose and 10 μM taxifolin hydrate in cryopreservation extender may be recommended to improve the quality of post-thawed ram spermatozoa. However, further in vivo and in vitro studies are suggested to evaluate fertility rates of frozen-thawed ram spermatozoa co-supplemented with trehalose and taxifolin hydrate.

Use of phosphatidylcholine in Tris-based extender with or without egg yolk to freeze Bapedi ram semen

Traditionally, egg yolk is a protective agent that is used to freeze semen in various species. However, the addition of egg yolk in extender risks the introduction of disease. Therefore, an alternative cryoprotective agent should be found to preserve ram semen. The aim of this study was to evaluate the effect of phosphatidylcholine (PC) as a protective agent in extender with or without egg yolk on semen characteristics and acrosome integrity of frozen then thawed Bapedi ram semen. Semen was collected from four mature Bapedi rams, in the Agricultural Research Council (ARC) Germplasm Conservation Programme, using an artificial vagina. Following collection, semen samples were randomly diluted into Tris-based extender (1: 2), with and without egg yolk, and supplemented with four concentrations of PC liposome (0 mg/ml), 0.25 mg/ml, 0.5 mg/ml and 0.75 mg/ml). Supplementation of PC liposome in extender with or without egg yolk did not improve the semen total motility (TM), progressive motility (PM) and rapid motility (RM) rate. The sperm cell membrane integrity in extender with or without egg yolk was not influenced by the supplementation of PC liposome after thawing (P &gt;0.05). The addition of PC liposome to Tris-based extender with egg yolk had a similar result to control (Tris-based extender with egg yolk) on sperm cell acrosome integrity. In conclusion, supplementation of PC liposome to Tris-based extender without egg yolk had lower sperm cell viability and motility rates compared with the extender with egg yolk, regardless of concentration.Keywords: acrosome, cryoprotectant, liposome, membrane, motility

EFFECT OF LEVEL AND TIME OF L-ARGININE ADDITION TO SEMEN EXTENDER ON THE FREEZABILITY AND FERTILIZING POTENTIALS OF BUFFALO SPERMATOZOA

The objective of this study was to investigate the profit of L-arginine addition to the freezing extender on freezability, lipid peroxidation, and fertilizing potentials of frozen-thawed buffalo spermatozoa. Semen was collected with artificial vagina from five adult fertile bulls and diluted with Tris-base extender containing different L-arginine concentrations (0, 0.5, 1, 5 and 10 mM) added during the dilution or after equilibration for 2 h. Diluted semen was cooled to 4oC throughout one hour, equilibrated for 2 h and then frozen in 0.25 ml straws, prior to be stored in liquid nitrogen. Cryopreserved spermatozoa were assessed for post-thawing sperm motility, viability index, acrosomal integrity, lipid peroxidation and fertility rate. The current results clearly indicated that adding 0.5 mM L- arginine to the freezing extender after the equilibration period significantly (P< 0.05) improved post-thawing motility, viability index and maintain acrosomal integrity (61.66±10.14%, 130.83±9.62 and 19.33±4.82%, respectively) compared with the control semen (33.33±4.40%, 88.33±11.03 and 33.67±3.28%, respectively). Moreover, addition of 0.5 mM L- arginine to the freezing extender after equilibration period significantly diminished (P< 0.05) lipid peroxidation (11.33±3.28 nmol/109) compared with the control (26.67±3.18 nmol/109). All of these previously enhanced semen characteristics were reflected positively on its fertilizing potentials. The present results revealed that addition of 0.5 mM L- arginine to the freezing extender after 2 h equilibration (short time exposure) might improve semen quality, preserve the fertilizing potentials and reduce cryodamage of the buffalo spermatozoa.

Influence Of Combinations Of Some Permeable Cryoprotectants With Chicken And Duck Egg Yolks On Freezability And Dna Integrity Of Buffalo Spermatozoa

The present study aimed to investigate the cryoprotective effect of different combinations of glycerol (G), dimethyl sulphoxide (DMSO) and dimethylformamide (DMF) with chicken egg yolk (CEY) and duck egg yolk (DEY) on freezability of buffalo spermatozoa. Semen samples were collected from 4 buffalo bulls and diluted with Tris-based extender supplemented with 7% (V/V) cryoprotectant including G, DMSO, DMF or a mixture of 3.5% G + 3.5% DMSO, 3.5% G + 3.5% DMF and 3.5% DMSO + 3.5% DMF. All the fore-mentioned aliquots were divided into two equal portions; one is supplemented with chicken's egg yolk (CEY) and the other with duck's egg yolk (DEY). Post-thawing sperm motility, viability and acrosomal, plasmatic membrane and DNA integrities were assessed. The results clearly indicated that adding a mixture of 3.5% G + 3.5% DMSO with DEY to Tris extender significantly improved (p < 0.01) post-thawing sperm motility, viability index and percentage of spermatozoa with intact acrosomes, plasma membrane and DNA (58.33%±1.66%, 173.33±5.46, 69.00±1.58%, 65.00±1.15% and 96.91±0.13%, respectively) compared with the use of 7% G and CEY (51.50±1.44%, 146.25±6.49, 61.00±2.30, 58.00±1.15% and 91.40±0.17, respectively). In conclusion, motility and functional integrity of cryopreserved buffalo spermatozoa could be enhanced by using Tris extender containing a mixture of 3.5% glycerol + 3.5% DMSO with duck’s egg yolk.

Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process

This study investigates the effects of cryopreservation and supplementation of buffalo's semen with Caffeic acid. It studies the effects of different Caffeic acid concentrations on cryopreservation capacity of the buffalo and evaluates their influence on various sperm parameters like motility, viability, progressive motility, sperm plasma membrane integrity, and antioxidant status. Twenty-four semen samples were collected with an artificial vaginal from three adult water buffalos. The semen samples were evaluated and the qualified ejaculates were separated and were diluted in a Tris-based extender. The resulting samples were classified into 5 groups: No antioxidant (control), Control sham (NaOH), Caffeic acid 50 μM, Caffeic acid 100 μM, and Caffeic acid 200 μM. The semen samples encountered cryodamage and the quality was deteriorating during the cryopreservation (P < 0.05). The semen evaluation after thawing showed that the groups of samples receiving 100 μM Caffeic acid had higher viability, total motility, and lower abnormal sperm and better linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and path velocity and higher intact plasma membrane (P < 0.05) compared to other groups. It is notable that adding 100 μM Caffeic acid to freezing extenders enhances the CAT, GPx, SOD, and GSH and also ameliorates total antioxidant capacity of spermatozoa after thawing. It is notable that the addition of 100 μM Caffeic acid decreases the amount of Malondialdehyde. These reactions lead us to conclude that 100 μM Caffeic acid enhances the semen quality of water buffalo after thawing.

Ram semen preserved at 0°C with soybean lecithin Tris-based extender substituted for egg yolk.

ObjectiveThe present study evaluated the preservation of ram semen at 0°C using soybean lecithin with a Tris-fructose extender.MethodsSemen was collected by artificial vagina ejaculation from six rams with proven fertility. High quality ejaculates were diluted by soybean lecithin (0.25%, 0.5%, 0.75%, 1.0%, 1.25%) using Tris-fructose extender and control (Tris-fructose egg yolk extender), respectively. The ejaculates were diluted to a concentration of 5×108 sperm/mL, followed by cooling to 0°C in 90 min and maintaining the temperature for 12 days. The diluted semen samples were examined and recorded for sperm progressive motility, acrosome integrity at 0, 24, 72, 144, 216, 288 h, respectively. Two hundred and twenty-three ewes were inseminated for 216 h with optimal soybean lecithin concentrated semen or control via trans-cervical insemination.ResultsThe results showed that there were no differences in sperm progressive motility at 0, 24, 72, and 144 h (p>0.05). After 216 h, the sperm progressive motility in the control group and 0.5% concentration groups was significantly higher when compared to 0.25% concentration (p<0.05). The 0.5% concentration group demonstrated the highest survival rate and had no difference with the control group (p>0.05). At 216 h, the sperm progressive motility of all groups was still above 50%. The acrosome integrity of all groups was decreased with prolongation of storage time, but there was no difference at each time point (p>0.05). There was no significant difference in the lambing rate and pregnancy rate between the 0.5% concentration group and the control group (p>0.05).ConclusionThese results suggest that ram sperm is capable of fertilization after preservation at 0°C with 0.5% of soybean lecithin in Tris-based extender substituted for egg yolk and produce normal offspring after insemination.

The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN2). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05).The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05).In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.

Soybean Phospholipids-Based Extender as an Alternative for Bull Sperm Cryopreservation

This current work investigated the usage of soybean phospholipid (SP) in extenders for bull sperm cryopreservation. The sperm was obtained from Friesian Holstein (FH) bulls using an artificial vagina method. Furthermore, semen samples were pooled and diluted in a Tris egg yolk-based extender (control group; CG) or Tris extender supplemented with SP at different concentrations (G1 = 0.5%, G2 = 1%, G3 = 1.5%, G4 = 2%, G5 = 2.5%) for a final concentration of 25×106 spermatozoa/0.25 mL. Subsequently, they were packed in straws (0.25 mL), cryopreserved using liquid nitrogen vapor for 10 minutes, then stored in liquid nitrogen (-196°C). As a result, control group presented significantly higher values in motility, viability and membrane integrity at the stage of dilution, equilibration and post-thawing compared to treatment group (P<0.05). After thawing (37°C/30s), no significant difference was observed between G4 and control group for all parameters observed using Computer Assisted Sperm Analysis (CASA) (P>0.05). In conclusion, the addition of 2% soybean phospholipid in Tris-based extender can be used for freezing bull semen, producing a satisfied fertility rate in post-thawing stage.

Semen collection methods and cooling rates affect post-thaw sperm motility and kinematic parameters of Saanen goat

Objective: To evaluate the effects of semen collection methods and different cooling rates on cooled and frozen-thawed Saanen goat semen. Methods: Twenty bucks were divided into two groups of 10 animals per group based on semen collection methods: artificial vagina and electro-ejaculator. Samples from each collection method were pooled and diluted with Tris-based extender. The pooled semen was divided into two cooling methods: slow cooling and fast cooling. Sperm motility and velocity, sperm plasma membrane integrity, sperm viability, mitochondrial membrane potential and DNA integrity were evaluated before dilution, immediately after cooling at 4 °C and at 24 h after freezing by using computer assisted sperm analyser method and epifluorescence microscope. Results: Mean values for total motility, rapid-speed and progressive motile spermatozoa were significantly higher in semen collected with artificial vagina than electro-ejaculator (P 0.05). Conclusions: The use of artificial vagina and slow cooling rate in cryopreservation protocol improves the cooled and post-thaw sperm quality of Saanen buck semen.

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.

Kısa Süreli Saklanan Koç Spermasına İlave Edilen Sığır Serum Albumininin (BSA) Farklı Dozlarının Sperma Kalitesi ve DNA Hasarı Üzerine Etkisi

In the present research the aim was emphasized to find out the effects of bovine serum albumin (BSA) on quality of sperm and DNA damage at 0, 24 th, 48 th and 72 th hours in Merino ram semen at short term preservation (5°C). Ejaculates were obtained from five Merino Rams which were collected by artificial vagina and all the samples were pooled at 37°C for detection. All the procedures were applied six times concurrently. Different concentrations (0, 2, 4 and 6 mg/ml) of BSA with Tris-based extender was used for dilution on semen samples. Sperm motility, abnormality, plasma membrane integrity, acrosome integrity and % DNA in tail (COMET assay) content were measured and analyzed. The extender with 4 mg/ml of BSA had both higher motility percentages and plasma membrane integrity, when compare with the control, up to 72 hours (P<0.05). At the 24th and 48th hours of the study, DNA damage was lower in all doses of BSA than in the control group. In addition, in terms of abnormal spermatozoon rates, all doses of BSA were lower than the control group at 24, 48 and 72 hours (P<0.05). In conclusion, it was found to be especially useful to add 4 mg / ml BSA for the ram semen parameters in short term storage.

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.