Tris-egg Yolk Extender Research Articles

Effect of adding different concentrations of L-arginine to Tris-yolk extender on the quality of sub-fertile ejaculates in buffalo.

To investigate the effect of supplementation of L-arginine (AR) on sub-fertile buffalo-bulls' ejaculates, 25 ejaculates of poor motility (40 to 55%) were collected by artificial vagina from 5 buffalo-bulls and extended with Tris-yolk extender (1:10) supplemented with different concentrations of AR (0, 3, 4, 5, and 6mM). Semen was cooled gradually to 4°C within 2h and incubated at 4°C for additional 2h. Incubated semen samples were evaluated by computer-assisted semen analysis. Results showed that addition of 5mM AR increased (P < 0.05) total sperm motility and rapid progressive motility percentages, while decreased (P < 0.05) non-motile sperm and static sperm percentages compared with AR-free (control) extender. Increasing the AR level to 6mM increased (P < 0.05) the percentages of sperm progressive motility and rapid and slow progressive motilities, while decreased (P < 0.05) the non-progressive sperm motility percentages compared with AR-free extender. Supplementation of 5mM AR improved (P < 0.05) sperm straight linear, curve linear, and average path velocities (36 ± 0.13, 20.6 ± 5.3, and 33.2 ± 8.5, respectively) in comparing with control and other AR treatments. Addition of AR (5 and 6mM) improved (P < 0.05) the percentages of vitality (89.8 ± 1.9 and 80.0 ± 3.4, respectively), normality (44.3 ± 3.6 and 44.8 ± 1.5, respectively), and functional sperm (20.4 ± 8.6 and 21.0 ± 0.61, respectively), and decreased abnormal neck and tail percentages compared with AR-free extender. All AR levels decreased (P < 0.05) the abnormal neck and tail percentages. Addition of all AR levels had no significant (P > 0.05) effect on the activity of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in semen extender. Supplementation of Tris-yolk extender with L-arginine (5 or 6mM) can improve sperm motility, velocity, vitality, and functional sperm and can decrease tail and neck abnormalities of sub-fertile buffalo ejaculate after 4h incubation at cool temperature.

Supplementation of Snakehead Fish Extracts into Tris Egg-Yolk Extender on Mortality and Motility of Limousin Bull Sperm

Supplementation of Snakehead Fish Extracts into Tris Egg-Yolk Extender on Mortality and Motility of Limousin Bull Sperm

Effect of Different Antioxidant Sources Added to Buffalo Semen Extender During Cryopreservation on Freezability and Fertility of Buffalo Spermatozoa

| There are several types of antioxidants which can be use in buffalo semen preservation. The current study was performed to study the influence of antioxidants viz., Coenzyme Q10 (CoQ10), L-Carnitine (LC), or N-Acetyl-Cysteine (NAC) added to the extender of buffalo semen on freezability and fertility of spermatozoa. Semen of Egyptian buffalo bulls (n=5) were pooled and extended with tris (1:10) without addition of antioxidant (E1), or with antioxidants i.e., 0.03 mM CoQ10 (E2), 3.0 mM LC (E3), and 1.0 mM NAC (E4). Then the diluted semen was frozen at -196oC in liquid nitrogen. Results showed that the progressive motility, livability, membrane integrity, and head-head agglutination percentages increased, while sperm abnormality and acrosomal damage decreased by all antioxidant supplements (P<0.05), being the best for E2. Pregnancy rate was higher (P<0.05) for buffalo cows inseminated with semen of E2 and E4 (81.8% for each) versus 72.7 and 63.6% for E3 and E1, respectively. In conclusion, tris-egg yolk extender supplemented with 0.03 mM CoQ10 can improve sperm characteristics of cryopreserved semen and increase pregnancy rate of inseminated buffalo cows.

Does resveratrol preserve the integrity of the sperm membrane in goats after thawing?

This study aimed to determine the effects of resveratrol in the trisaminomethane (TRIS)-egg yolk extender and its optimal inclusion level for goat semen cryopreservation. Five ejaculates of three Anglo Nubian goats were used, each divided into four 200 µL aliquots for use in four treatments: 0.00 (control), 0.04, 0.08 and 0.12 mg mL-1 resveratrol in the TRIS-egg yolk extender. We evaluated progressive sperm motility and sperm vigor post-dilution, post-cooling, and post thawing; membrane integrity (HOST); and acrosomal integrity and performed a slow thermoresistance test (STT). The data were submitted to a regression analysis at a 5% probability. There was no difference in progressive motility or sperm vigor in the post-dilution (89.5, 89.0, 88.7 and 88.3, and 4.9, 5.0, 4.9, and 4.9) or post-cooling (81.0, 82.0, 83.0, and 78.3; and 4.3, 4.3, 4.2, and 4.2) experiments (P &gt; 0.05), or in the complementary acrosomal integrity test (42.0, 47.4, 42.2 and 38.2) (P &gt; 0.05). However, the motility and vigor parameters decreased linearly in the post-thaw phase, as well as during the 2 hours of incubation on STT (P &lt; 0.05). These factors increased quadratically when resveratrol was added to HOST, to an optimal level of 0.039 mg mL-1 resveratrol for a plasma membrane integrity of 52.55% (P &lt; 0.05). The inclusion of resveratrol was effective in maintaining sperm viability; in particular, it was effective in maintaining plasmatic membrane integrity during the cryopreservation process up to 0.039 mg mL-1, meaning that it could be an alternative to conventionally used seminal extenders in goats.

Percentage Viability Spermatozoa in Post-Thawing Boer Buck Semen with Addition of Sweet Orange Essential Oil to Tris Yolk Extender

Percentage Viability Spermatozoa in Post-Thawing Boer Buck Semen with Addition of Sweet Orange Essential Oil to Tris Yolk Extender

Exploring the role of E.coli derived enzyme, Oxyrase, as an oxygen scavenger to improve the cryotolerance of spermatozoa of Sahiwal bull

The current study intended to optimize the concentration of Oxyrase in the semen dilutor and to evaluate its effect on freezability of spermatozoa of Sahiwal bulls. Supplementation of Oxyrase at 0.125 IU/mL concentration significantly reduced dissolved oxygen (DO) in the dilutor to 4 ppm in 16–18 min at 35 °C. For supplementation studies, a total of 24 ejaculates were categorized into poor and good ejaculates categories (n = 12 each) based on their initial progressive motility. Each ejaculate was further divided into two aliquotes. The first aliquote was diluted with tris-egg yolk extender without Oxyrase (control group) whereas, in the treatment group, Oxyrase was supplemented at the concentration of 0.125 IU/mL of extender. The parameters evaluated include cholesterol and plasma membrane phospholipids (PMP) at fresh, while IPM, acrosomal and plasma membrane integrity, cholesterol, PMP and oxidative stress parameters like lipid peroxidation (LPO), total antioxidant capacity (TAC) and reactive oxygen species (ROS) were evaluated at pre-freeze and post-thaw stages. The IPM and acrosomal intactness were higher (p < 0.05) in treatment group at post-thaw stage in good ejaculates. Oxyrase supplementation resulted in lower (p < 0.05) cholesterol leakage in both categories and lower (p < 0.05) LPO in good ejaculates at post-thaw stage. No statistical difference in ROS was observed between control and treatment groups at all stages whereas, level of TAC was higher (p < 0.05) in the treatment group compared to control group at post-thaw stage of both categories. Therefore, Oxyrase as an oxygen scavenging agent could preserve the post-thaw quality of Sahiwal bull spermatozoa.

Total Bacteria of Post-Thawing Boer Buck Semen with Addition of Sweet Orange Essential Oil to Tris Yolk Extender

Aims: To determine the effect of the addition of sweet orange essential oil to the extender (tris yolk extender without the addition of antibiotics/antibacterial) to total bacteria Boer Buck frozen semen.&#x0D; Study Design: Randomized Block Design.&#x0D; Place and Duration of Study: Sample: Laboratory Reproduction of Loka Penelitian Kambing Potong Sei Putih Indonesia, between January and March 2019.&#x0D; Methodology: The research procedure starts with the preparation of semen extender, collection of fresh semen, dilution of semen, equilibration, freezing of semen, and thawing. This research obtained conducted using a Randomized Block Design consisting of 5 treatment levels and five replications. Semen storage using 3 Boer buck, which done for three days. As a treatment is the addition of sweet orange essential oil as much as (P0) 0%, (P1) 0,25%, (P2) 0,5%, (P3) 0,75% and (P4) 1% on the tris yolk extender. The observed variables was total bacteria evaluated before freezing and after freezing (Post-Thawing).&#x0D; Results: The results showed that the addition of sweet orange essential oil had a very significant effect (P &lt;0.01). The results of adding sweet orange essential oil to the extender (Post-Thawing) were 78.6 x 102 CFU (P0), 76 x 102 CFU (P1), 73,2 x 102 CFU (P2), 71 x 102 CFU (P3) and 68.6 x 102 CFU (P4).&#x0D; Conclusion: The best values the total bacteria 68.6 x 102 CFU (P4). It can conclude that the best result of adding sweet orange essential oil to tris yolk extender is (P4) 1%.

Cryopreservation of Semen from Domestic Livestock: Bovine, Equine, and Porcine Sperm.

In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5°C, packaging of insemination doses in straws, freezing at a defined cooling rate in liquid nitrogen vapor, cryogenic storage, and thawing. Two-step dilution approaches, with commonly used diluents, are presented, namely, TRIS-egg yolk (TEY) extender for bull sperm, skim milk (INRA-82) extender for stallion sperm, and lactose-egg yolk (LEY) extender for boar sperm. Furthermore, simple methods are presented for cooling and freezing of sperm at defined cooling rates.

Escherichia coli membrane‐derived oxygen‐reducing enzyme system (Oxyrase) protects bubaline spermatozoa during cryopreservation

The objective of this study was to determine the effectiveness of deoxygenation of semen extender using Escherichia coli membrane-derived oxygen scavenger (Oxyrase) on post-thaw quality of buffalo (Bubalus bubalis) spermatozoa. Sixteen semen ejaculates, four each from four bulls, were each divided into five equal fractions, diluted using Tris-egg yolk extender supplemented with different concentrations of Oxyrase (0, 0.3, 0.6, 0.9, and 1.2 U/ml), designated as treatments T1, T2, T3, T4, and T5, respectively, and cryopreserved. Immediately after thawing, Oxyrase did not improve sperm kinetics and motility; however, it improved the keeping quality (significantly lower deterioration of post-thaw sperm motility after incubation for 120 min) in T3. Further, T3 reduced (p < .05) cholesterol efflux and protected the intactness of the sperm plasma membrane. Flow cytometry with Fluo-3 AM/propidium iodide (PI) dual staining revealed the highest (p < .05) proportion of live spermatozoa with low intracellular calcium in T3. Oxyrase supplementation protected spermatozoa from premature capacitation which was confirmed by low expression of tyrosine-phosphorylated proteins (32, 75, and 80 kDa) and a relatively lower percentage of F-pattern (uncapacitated spermatozoa) in chlortetracycline assay. Importantly, the Oxyrase fortification decreased superoxide anion in a dose-dependent manner indicating reduced availability of oxygen at sperm mitochondrial level. Similarly, in Oxyrase-fortified sperm, malondialdehyde concentration, an index of lipid peroxidation, is also reduced in a dose-dependent manner. In conclusion, we demonstrate that deoxygenation of buffalo semen by Oxyrase has the potential of improving post-thaw sperm quality by overcoming the problem of cryocapacitation and oxidative damage during cryopreservation process.

Effects of reduced glutathione on ram sperm parameters, antioxidant status, mitochondrial activity and the abundance of hexose transporters during liquid storage at 5 ℃

The objective of this study was to evaluate the effects of reduced glutathione (GSH) supplementation on sperm motility, kinematics, mitochondrial activity and the abundance of sperm hexose transporters during liquid storage of ram semen at 5 ℃. Semen samples from eight rams were pooled and diluted with Tris-egg yolk extender supplemented with 0 (control), 50, 100 and 200 mM GSH, and they were assessed at different processing periods (fresh diluted, stored for 24, 48 and 72 h at 5 ℃). Sperm motility and kinematic parameters were analyzed by computer-assisted sperm analysis (CASA). The hypo-osmotic swelling test (HOST) was used to evaluate the functional integrity of sperm membrane. Eosin-nigrosin and JC-1/PI staining methods were used to assess sperm viability and mitochondrial activity, respectively. The antioxidant status of sperm was examined using biological assays. The changes of sperm hexose transporters (GLUT 3, 5 and 8) were determined using western blot analysis. As compared to the control group, supplementation with 100 and 200 mM GSH improved (P ＜ 0.05) sperm total motility, membrane integrity, motion parameters and mitochondrial activity at 72 h of storage, as well as resulted in the higher sperm antioxidant status after storage from 24 to 72 h. The cooling induced a reduction of GLUT 3 and 8, and GSH treatment at 200 mM retarded the gradual decline of these proteins during the prolonged storage time. Our data suggest that GSH can reduce the decline of semen quality by influencing the antioxidant status and energy metabolism of liquid stored ram spermatozoa. The concentration of 200 mM could be appropriate for ram semen during liquid storage at 5 ℃.

Field friendly method for wild feline semen cryopreservation

The aim of this study was to develop a field-friendly method for free-living jaguar and cougar semen cryopreservation. Six captive Jaguars Panthera onca and three captive Cougars Puma concolor were chemically restrained with a combination of medetomidine (0.08–0.1 mg/kg) and ketamine (5 mg/kg). Semen was collected through a tomcat urinary catheter with an open end, diluted for a final concentration of 50 x 106 sperm/mL in a TRIS-egg yolk extender and packaged into 0.25 mL straws. We compared two cooling methods: CoolA - in which straws were placed in a glass tube that was placed in a glass bottle containing water (600mL at 38°C) and transferred to a polystyrene container (12L) containing an 11cm column of ice and water at room temperature; CoolB – where the glass bottle – straws kit was transferred to a 4.26L cooler containing nine blocks (81cm3) of Ice Foam recyclable ice, previously frozen in liquid nitrogen. The sperm volume varied from 2 to 720 µl for the jaguars and from 80 to 140 µl for the cougars. Sperm concentration varied from 224 to 5,115 x106 sperm/mL for the jaguars and from 485.7 to 562.5 x 106 sperm/mL for the cougars. Concerning the cooling treatments, there was no difference in frozen-thawed sperm quality between the methods, in both species. Thereby, the cooling method using recyclable ice frozen in liquid nitrogen can be used for semen cryopreservation in wild felines, eliminating the need for electric energy.

The effect of pineapple [Ananas comosus (L.) Merr.] juice in extender on Garut ram (Ovis aries L.) spermatozoa quality postcryopreservation

This study was intended to determine the effect of various concentrations of pineapple [Ananas comosus (L.) Merr.] juice in extender on garut ram (Ovis aries L.) spermatozoa quality 24 h postcryopreservation. Semen samples were collected from five garut rams once a week using an artificial vagina. The samples were diluted in Tris-egg yolk extender with pineapple juice at concentrations of 0 % (KK), 5 % (KP 1), 10 % (KP 2), 15 % (KP 3), and 20 % (KP 4). The diluted semen samples were loaded into mini straws (0.25 mL) with 50 million cells/ml dosage. Samples were equilibrated at 5 °C for 2 h, and then frozen and stored in liquid nitrogen. Parameters evaluated were motility, viability, membrane integrity, acrosome integrity, and morphological abnormality. One-way ANOVA followed by Duncan’s test showed significant differences (P < 0.05) between KK and KP 3 in the percentage of motile spermatozoa and membrane integrity. Pineapple juice at 15 % could minimize the reduction of spermatozoa quality based on the percentage of motile spermatozoa (52.19 ± 5.32 %) and membrane integrity (38.52 ± 4.85 %).

Spermatozoa Abnormality Test of Boer Goats Frozen Semen with the Addition of Sweet Orange Essential Oil and Streptomycin in Tris Yolk Extender

This research aims to to determine the percentage value of spermatozoa abnormalities in Boer Goat frozen semen by adding a combination of streptomycin with sweet orange essential oil to tris yolk extender. The ingredients used in this study were fresh semen from Boer Goat, tris yolk extender, streptomycin, and sweet orange essential oil. Tris yolk extender is made using Tris (hydroxymethylaminomethane) (3.32 g), citric acid (1.86 g), fructose (1.37 g), glycerol (6 ml), egg yolk (20 ml), aquabides (100 ml). The study design was a non factorial completely treatments given were the addition of sweet orange essential oil at 0%, 0.25%, 0.5%, 0.75% and 1%. The results showed that the addition of a combination of streptomycin and sweet orange essential oil into the tris yolk extender had a highly significant effect (P &lt;0.01) on the percentage value of spermatozoa abnormalities of Boer Goats before and after freezing. The best average value of spermatozoa abnormalities is the addition of sweet orange essential oil at 1% (P4) with the percentage of spermatozoa abnormalities before freezing at 3% and after freezing at 6%.

Influence of butylated hydroxytoluene addition to cryodiluents on freezability and DNA integrity of Boer and Zaraibi buck spermatozoa

Objective: To evaluate the cryoprotective effect of butylated hydroxytoluene on buck frozen semen. Methods: Semen was collected from Boer (n=6) and Zaraibi (n=6) bucks by electroejaculator for 5 weeks. Semen aliquots were diluted at 38 °C in Tris-buffer with egg yolk 15.0% (vol/vol) (Tris- egg yolk extender) or soya lecithin 2.5% (weight/vol) (Tris-soya lecithin extender) supplemented with butylated hydroxytoluene at 0.0 (as the control), 0.5, 1.0, 2.0 and 4.0 mM. Post-thawing motility (at 400x magnification), plasma (hypo-osmotic swelling test), acrosome (Trypan blue/Giemsa dual staining) membranes, DNA (comet assay), and lipid peroxidation (by malondialdehyde concentration) were assessed. Results: Spermatozoa motility was enhanced by butylated hydroxytoluene in Tris-soya lecithin extender at 0.5 mM in the two breeds, and in Tris-egg yolk extender at 1.0 mM in Boer and at 2.0 mM in Zaraibi bucks for up to 3 h post-thawing. Plasma and acrosome membranes and DNA integrity of the two breeds were maximally high with butylated hydroxytoluene at 1.0-2.0 mM in Tris-egg yolk extender and at 0.5-1.0 mM in Tris-soya lecithin extender. Lipid peroxidation was minimal with butylated hydroxytoluene at 1.0-2.0 mM in Tris-egg yolk and soya lecithin extenders in the two breeds. Butylated hydroxytoluene at 4.0 mM deteriorated spermatozoa motility, and plasma and acrosome membranes. Conclusions: The consequence of butylated hydroxytoluene on buck frozen-thawed spermatozoa varies with the levels of supplementation, buck breed, and phospholipid source in the extender. Semen parameters of Boer buck are better in their response to butylated hydroxytoluene than Zaraibi buck. Butylated hydroxytoluene at 1.0 and 2.0 mM in Tris-egg yolk extender, and at 0.5 mM in Tris-soya lecithin extender represents the best concentrations and profitably improves the semen quality of buck semen.

The effect of siam orange (Citrus nobilis Lour.) and pineapple (Ananas comosus (L.) Merr.) juices combination in extender on Garut Ram (Ovis aries L.) spermatozoa quality post-cryopreservation

The research aimed to find out the effect of various concentrations of siam orange (Citrus nobilis Lour.) and pineapple (Ananas comosus (L.) Merr.) juices combination (1:1) in extender on garut ram (Ovis aries L.) spermatozoa quality 24 hours post-cryopreservation. Semen samples were collected from five Garut Rams once a week using an artificial vagina. The samples were diluted in Tris-egg yolk extender with siam orange and pineapple juices combination 0 %, 5 %, 10 %, 15 %, and 20 %. Diluted semen samples were loaded into mini straws (0.25 mL) with 50 million cells/ml dosage. Samples were equilibrated at 5°C for two hours, then frozen and stored in liquid nitrogen. The one factor ANOVA and continued with Duncan test showed a significant differences (P<0.05) between control and sample with 10 % juices combination in the percentage of spermatozoa motility and membrane integrity, a significant difference (P<0.05) between control and sample with 5 % along with 10 % juices combination in the percentage of spermatozoa viability. Siam orange and pineapple juices combination 10% in extender was able to minimize the reduction of spermatozoa quality post-cryopreservation based on the percentage of spermatozoa motility (68.06 ± 2.34 %), viability (41.47 ± 6.64 %) and membrane integrity (46.00 ± 5.65 %).

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.

Inclusion of Sericin in Tris Egg Yolk Extender Improves Bovine Sperm Quality during Cryopreservation (-196°C)

Inclusion of Sericin in Tris Egg Yolk Extender Improves Bovine Sperm Quality during Cryopreservation (-196°C)

The in vitro investigation of lycopene effects on post-thawed ram sperm

This study was conducted to evaluate the effects of lycopene on quality parameters of cryopreserved ram semen. Semen samples were collected from three Santa Inês rams to form a seminal pool (n = 8). Aliquots from each pool were diluted in Tris-egg yolk extender to create experimental groups containing 0 µM (control), 0.1 µM, 1 µM and 5 µM lycopene. The samples were evaluated for sperm kinetics, integrity of plasma and acrosomal membranes, intracellular reactive oxygen species (ROS) production and lipid peroxidation immediately after thawing and after incubation for two hours at 37 °C. The addition of lycopene had no effect on the parameters that were evaluated at the time of thawing when compared with the control. However, after incubation, the groups with added lycopene showed a decrease in progressive motility. All experimental groups showed a significant reduction in linearity and straightness following incubation. Furthermore, the 5 µM lycopene group showed a decrease in wobble and an increase in amplitude of lateral head displacement. In conclusion, the addition of lycopene to the freezing extender of ram semen affected the kinetics parameters of cryopreserved spermatozoa after a two-hour incubation period.Keywords: carotenoid, flow cytometry, kinetic, sheep

Effects of supplementation of tris-egg yolk extender with different sugars and antioxidants on freezability of ram semen

This study was designed to determine the effects of the combined addition of different levels of certain sugars (trehalose, sucrose and raffinose) and antioxidants (vitamin E, C and taurine), in Tris-egg yolk extender on frozen-thawed ram semen parameters. Semen samples were collected from five healthy, mature and fertile Iranian Afshari rams, twice a week for 8 weeks. Selected samples were pooled and diluted with a Tris-egg yolk extender containing different levels of sugars and antioxidants. In Experiment 1, different levels of trehalose (0, 50 and 100 mM) were tested with different levels of taurine (0, 25 and 50 mM), vitamin E and C (0, 1 and 2 mM). In Experiment 2, different levels of sucrose (0, 60 and 80 mM) were tested with different levels of taurine (0, 25 and 50 mM), vitamin E and C (0, 1 and 2 mM). In Experiment 3, different levels of raffinose (0, 5, 10 mM) were tested with different levels of taurine (0, 25 and 50 mM), and vitamin E and C (0, 1 and 2 mM). In Experiment 4, the selected extenders of experiments 1, 2 and 3 were compared statistically with control (no selected sugar and antioxidant) extender. The results of experiments 1, 2 and 3 revealed that the highest frozen–thawed sperm parameters were recorded for the selected extenders containing 100 mM trehalose +2 mM vitamin E (T100E2), 60 mM sucrose + 2 mM vitamin E (S60E2) and 10 mM raffinose + 2 mM vitamin E (R10E2), respectively. The results of experiment 4 revealed that the post-thaw sperm total motility in T100E2 (62.41 ± 2.41%), S60E2 (59.52 ± 1.91%) and R10E2 (58.33 ± 2.00%) was higher than that of the control extender (46.00 ± 1.79%; P ≤ 0.05). Similarly, the progressive sperm motility in T100E2 (57.18 ± 1.96%), S60E2 (57.49 ± 1.94%) and R10E2 (55.03 ± 2.99%) was also higher than that of the control extender (41.20 ± 1.70%; P ≤ 0.05). Post-thaw sperm viability in selected extenders of T100E2 (65.20 ± 2.67%), S60E2 (62.00 ± 2.07%) and R10E2 (61.80 ± 2.46%) was higher than that of control extender (51.00 ± 1.88%; P ≤ 0.05). In conclusion, the addition of 100 mM trehalose, 60 mM sucrose and 10 mM raffinose combined with 2 mM vitamin E in Tris-egg yolk extender significantly improved frozen-thawed ram semen parameters.

“Effect of the addition of different catalase concentrations to a TRIS-egg yolk extender on quality and in vitro fertilization rate of frozen-thawed bull sperm’’

The aim of this study was to investigate the effects of different concentrations of catalase in a TRIS-egg yolk extender on sperm quality and embryonic development after in vitro fertilization of frozen-thawed bull sperm. For this purpose, from each of 7 bulls 2 ejaculates were collected and diluted with a TRIS-egg yolk extender containing 0, 5, 10, 15 or 20 IU catalase/mL. Sperm quality was evaluated 0, 3, 6, 12 and 24 h after thawing by using computer assisted analysis of motility and by flow cytometric assays. Embryonic development was determined after in vitro fertilization of bovine oocytes. Semen diluted with TRIS-egg yolk extender containing different concentrations of catalase showed more motile sperm, more sperm with intact plasma membranes, acrosomes and DNA, a high mitochondrial membrane potential, a high esterase activity, a low calcium level, a lower amount of synthesis of reactive oxygen species and lower degree of lipid peroxidation of sperm compared to semen frozen without catalase (P < 0.05), but not before 3 h after thawing. There was a dose-response relationship with the most prominent effect of 20 IU catalase/mL. However, the improvement of sperm quality had no effect (P ≥ 0.05) on embryonic development after in vitro fertilization with 20 IU catalase/mL. In conclusion, the addition of catalase to the sperm extender improved sperm quality with no obvious effect on in vitro fertility.