Tris Extender Research Articles

Evaluation of used Purslane extracts in Tris extenders on cryopreserved goat sperm

This study aimed to evaluate the comparative effects of Purslane aqueous extract (PAE), Purslane methanolic extract (PME) and Purslane ethanolic extract (PEE on the quality of frozen-thawed goat spermatozoa. Collected semen with motility >75% and sperm concentration >1.0 × 109 sperm/ml was pooled and divided into 10 equal aliquots and supplemented by basic extender containing 25, 50 or 100 μg/ml of Purslane aqueous extract (PAE25μg/ml, PAE50μg/ml, PAE100μg/ml, respectively), basic extender containing 25, 50 or 100 μg/ml of Purslane methanolic extract (PME25μg/ml, PME50μg/ml, PME100μg/ml, respectively), basic extender containing 25, 50 or 100 μg/ml of Purslane ethanolic extract (PEE25μg/ml, PEE50μg/ml, PEE100μg/ml, respectively). Control diluent contained no additives. For the determination of sperm quality, frozen straws were thawed and then the sperm characteristics were assessed. Results indicated that higher (P < 0.05) percentages of total motility, viability, mitochondrial activity and lower percentages of malondialdehyde (MDA) for PAE50μg/ml, PME50μg/ml and PEE50μg/ml than those of the control. In addition, PME50μg/ml resulted in the highest) P < 0.05) total motility and the lowest (P < 0.05) MDA levels compared to other treatments. Compared to the control group, PME50μg/ml resulted in higher integrity (P < 0.05) of plasma membranes and in lower amounts of apoptotic and dead spermatozoa. PME50μg/ml and PAE50μg/ml showed higher (P < 0.05) percentages of progressive motility, DNA integrity and live post-thawed spermatozoa than those of the control. No significant differences in the motility, viability, mitochondrial activity and number of live sperms were observed between PME50μg/ml and PAE50μg/ml treatments. In conclusion, the results of this study indicated that 50 μg/ml purslane extracts could be used for the cryopreservation. However, the results of methanolic extract was more beneficial compared to other extracts.

Effect of Addition of Moringa Oleifera Extract to Tris Extender on the Preservability of Cattle Bull Semen

Moringa oleifera extract is a strong natural antioxidant that when was added to the semen extenders, it induced a cryoprotection to spermatozoa effect through elimination of the excess free radicals. So, the existing study intended for clarification of the consequence of extract of Moringa leaves (MLE) on bull spermatozoa after chilling and cryopreservation. MLE concentrations were 0% (control), 10, 20, 30, 40 and 50% (v/v) [MLE: TCF (Tris-citric-fructose diluent)] then 20% egg yolk was added, then the extended semen was assigned to the freezing protocol. Then, it was evaluated for (motility, alive, abnormality %, sperm membrane integrity % before and after freezing). Sperm motility was kept high with the concentration 10, 30 and 40% of MEEY till 8 days of chilling. The concentration 20% maintained sperm motility high till 7 days of chilling. Addition of MLE to TCF significantly (P&lt;0.002) improved sperm motility in all concentrations except the 50% moringa enriched extender with egg yolk (MEEY) where sperm motility was maintained as the control. The use of MEEY maintained % of alive sperms and % of normal spermatozoal membrane (HOST%) as good as the control. In conclusion: moringa as a herbal supplement to semen diluents enhanced preservation in cooled and cryopreserved cattle bull semen.

EFFECT OF ADDING PENTOXIFYLLINE AND NITRIC OXIDE TO TRIS EXTENDER ON SOME POST-CRYOPRESERVED SEMEN ATTRIBUTES OF HOLSTEIN BULLS

This study was aimed to explore the effect of adding pentoxifylline (PTX) and nitric oxide (NO) to Tris extender on some post-cryopreserved semen attributes of Holstein bulls for different preservation periods (cooling at 5◦C, 48 hrs., 1, 2 and 3 months post cryopreservation, PC). Seven Holstein bulls of 2.5-3 years old were used in the current study during the period from 20th November, 2017 to 20th August, 2018. Pooled semen was equally divided into three groups within one experiment. PTX (0.192 g / 100 ml extender) and NO (0.018 g / 100 ml extender) were added to Tris extender and comparisons in response were made with the control group (Tris extender, C). The PTX group exhibited greater (P≤ 0.01) sperm's cell individual motility percentage as compared with the C group at cooling (5ºc) and 48 hr PC periods, while, PTX and NO groups were superior in these percentages at the remaining PC periods than C group. Excluding data of 2 months PC, greater (P≤ 0.01) live sperm percentage was observed in PTX and NO groups in comparison with the C group at all preservation periods. Lesser (P≤ 0.01) abnormal sperm percentage were noticed for PTX and NO groups as compared with the C group at all preservation time periods. The PTX and NO groups exhibited greater (P≤ 0.01) acrosome and plasma membrane integrity percentages in comparison with the C group at all preservation time periods. In conclusion, adding PTX and NO to Tris extender enhanced some of PC semen characteristics of Holstein bulls at different preservation periods.

Red pine (Pinus brutia Ten) bark tree extract preserves sperm quality by reducing oxidative stress and preventing chromatin damage.

This study aimed to investigate the effectiveness of using red pine bark tree extract (P; Pinus brutia Ten) as a TRIS extender in an attempt to prevent oxidative stress in bull spermatozoa after freezing. Semen specimens were obtained from Simmental bulls via an artificial vagina and pooled. They were separated into five specimens and diluted with Tris extender consisting of P (200, 100, 50 and 25µg/ml) and P free (control; C) up to a final concentration of 16×106 per straw. All specimens were equilibrated for a period of 4hr at a temperature of 4°C, following which they were filled in 0.25-ml French straws and frozen. Addition of P resulted in favourable tail length in comparison with C (p<.05). The lowest malondialdehyde levels and the highest glutathione levels were detected in all P groups (p<.05). Supplementation with P did not show advanced results in terms of total, progressive sperm motility and total abnormality in comparison with C (p>.05). In conclusion, it has been shown that although P added to a Tris extender does not have a positive effect on sperm motility, it prevents chromatin damage by reducing oxidative stress, in addition to reducing head abnormalities when used at the amount of 50μg/ml.

The quality of sexed sperm separated using bovine serum albumin column and extended using tris aminomethane at different temperatures

Sexing sperms is one of the reproductive technologies that is considered as a promising alternative in the effort of reproductive efficiency to produce offspring according to the desired sex. The method for maintaining and extending the sperms can be done by using bovine serum albumin (BSA) column with Tris Buffer at room temperature and waterbath without damaging the quality of the sperms. The purpose of this study was to determine the effect of BSA - Tris Buffer extender on the quality of sexed sperms after thawing at 27°C and 37°C. The study was divided into two treatments; P1 was Tris extender in the temperature of 27°C and P2 was Tris extender in the temperature of 37°C with 6 replications. Those treatments were compared using a Paired Sample test (T-Test). The parameters observed in the present study were motility, viability, abnormalities, and intact plasma membrane. The results showed that motility, viability, abnormalities, and intact plasma membrane of the sperms X and Y at 27°C and 37°C after sexing did not differed significantly (P>0.05). Likewise, after equilibration, the motility of sperms X at 27°C and 37°C did not differed significantly (P>0.05), however, sperm Y at 27°C and 37°C showed a significant difference (P<0,05). Viability and abnormalities of sperms X at 27°C and 37°C differed significantly (P<0,05) but sperms Y at 27°C and 37°C did not show any significant differences. After thawing process, motility, viability, abnormalities and intact plasma membrane of the sperms X and Y at 27°C and 37°C did not show any significant differences. However, it can be conluded that the quality of sexed sperms in the present study is still good because it has motility above 40% and abnormalities below 20%.

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.

Quality of Garut Ram’s Sperm in Tris Extender Supplemented with Ethylene Diamine Tetraacetic Acid Preserved at Low Temperature (3–5°C)

Ethylenediaminetetraacetic acid (EDTA) is a chemical substance that can bind the heavy metal and other toxic compounds so it is suitable to use for protecting sperm during preservation at 3-5°C. The research aimed to evaluate the role of EDTA to maintain the quality of Garut ram sperm in low-temperature preservation. Semen was collected by an artificial vagina. Fresh semen was evaluated and divided into 3 tubes and diluted with tris extender contain 20% egg yolk (TEY) without EDTA (T1), TEY+0.01% EDTA (T2) and TEY+0.02% EDTA (T 3). Semen was evaluated both macroscopically and microscopically before preserved in a refrigerator at 3-5°C. During storage, semen was also evaluated on daily basis (four consecutive days) that included variables relate to sperm motility (SM), sperm viability (SV), sperm membrane integrity (SMI) and sperm acrosome intact (SAI). The research design used was Completely Randomized Design with three treatments and five replications. Differences between treatments were tested with the least significant difference test. The results showed that the average of each variable gained of fresh semen was 0.88mL for volume, cream for color, thick for consistency and 7 for pH. While the mass movement of sperm was +++ and sperm concentration was 3,962 million cells/mL. The average percentages of SM, SV, abnormality, SMI, and SAI were 75.00%, 86.60%, 4.80%, 84.40%, and 84.80%, respectively. On the day-4 of storage, percentages of SM, SV, SMI, and SAI in control (T1) decreased to 48.75%, 66.25%, 64.75%, and 63.5%, respectively, but still met the requirement of sperm quality for artificial insemination purposes. Moreover, there was no significant difference between T2 (45.00%, 65.00%, 63.25% and 65.25%) and T3 (40.00%, 65.67%, 60.75% and 62.50%) of those variables measured. Finally, it was concluded that the addition of EDTA in Tris extender could maintain the quality of Garut ram sperm.

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos.

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P<0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.

Modified cryopreservation of semen of young bulls with reduced freezability

The aim of the study was to improve cryopreservation of young bulls’ semen. Bioxcell and Tris extenders were modified by adding α-linoleic acid (ALA) or linoleic acid albumin (LAA). Non-modified Bioxcell extender was used as control. Semen diluted in modified, glycerol-free Tris extender was subjected to a longer equilibration (26-28 hours at +4°C). Thirty-one ejaculates collected from 12 bulls aged 12-17 months were used in this research. After collection, fresh semen was divided into 7 portions, and each portion was diluted in one of experimental diluents, as well as in the control diluent. After equilibration, semen was frozen in a freezer. Subsequently, the semen was thawed and its motility was assessed (approximately and by CSA) along with cell membrane integrity (SYBR-14 and PI). The highest motility was observed in Bioxcell diluent supplemented with α-linoleic acid (ALA), and it was significantly higher (p ≤ 0.5) than in Bioxcell diluent (control). No significant differences in cell membrane integrity were observed between experimental groups that were subjected to a 2.5 hour equilibration. The extension of the equilibration time to 26-28 hours caused a significant (p ≤ 0.01) decrease in both the motility and the cell membrane integrity of spermatozoa compared to semen frozen in Bioxcell diluent (control) as well as to all other experimental diluents. In the case of semen that did not meet qualitative standards (motility below 50%) when frozen in the control diluent, significantly better results (p ≤ 0.01 and p ≤ 0.05) in terms of motility (assessed approximately and by SCA) were observed when the semen was diluted in Bioxcell diluent supplemented with α-linoleic acid (ALA) as well as in Bioxcell diluent supplemented with linoleic acid albumin (LAA) (p ≤ 0.05). No significant differences in cell membrane integrity were observed. The extension of equilibration time caused a significant (p ≤ 0.01) decrease in both the motility and the cell membrane integrity of spermatozoa. The study confirmed the possibility of improving cryopreservation of young bulls’ semen by modification of freezing diluents.

An Investigative Immunoassay Targeting Two Osteopontin Epitopes in Boar Semen

The identification of superior sire is largely dependent on the semen quality evaluations, currently through computer-aided sperm class analyser (CASA). Immunoassays present a viable method for analysis of proteins associated with fertility, such as osteopontin. We have targeted osteopontin at an upstream epitope at position 249 - 267 amino acids and a downstream epitope at position 59 - 74 amino acids of full osteopontin protein in raw, BTS, Kobidil, Tris and Citrate liquid preserved Large white and Kolbroek boar semen. The Genetool analysis software was used to quantify the amount of osteopontin detected. Three boars per breed were used and experiments were repeated five times. Our results revealed that the downstream 15 amino acid epitope was more sensitive to osteopontin antibody. Between total motility and osteopontin, Kolbroek boar semen preserved in Kobidil, Citrate, BTS and Tris extenders revealed positive correlations of 0.564, 0.471, -0.960 and 0.330, respectively. Large white boars semen showed correlations of -0.043, 0.655, 0.778, and 0.202 Kobidil, Citrate, BTS and Tris, respectively. For rapid motility and osteopontin, Kolbroek boar semen revealed positive correlations of 0.842, 0.601, 0.164 and 0.202, respectively while large white boars semen showed correlations of -0.909, -0.144, -0.210, and 0.089, respectively. Only BTS and Kobidil showed high negative correlations between osteopontin and viability in Kolbroek (-0.975) and Large white (-0.746) boar semen, respectively. No significant correlations with osteopontin were observed when Tris extender was used in both breeds. In conclusion, Kolbroek boar semen requires osteopontin in Kobodil extender while large white boar semen requires osteopontin in BTS extender.

Effect of Mifepristone, Sericin and Taurine in Tris Extender on Oxidative Markers and Quality of Fresh and Frozen-Thawed Bovine Spermatozoa

Effect of Mifepristone, Sericin and Taurine in Tris Extender on Oxidative Markers and Quality of Fresh and Frozen-Thawed Bovine Spermatozoa

Effect of adding astaxanthin and taurine to the Tris extender on the semen quality of Awassi rams during cooling storage

Effect of adding astaxanthin and taurine to the Tris extender on the semen quality of Awassi rams during cooling storage

38 Ram sperm longevity after cryopreservation in extender containing L-carnitine

The cryopreservation process causes oxidative stress to the sperm cell, and the addition of antioxidants to the extender for semen freezing helps sperm protection. This study assessed the effect of L-carnitine (LC) concentrations (0, 5, or 10mM LC) on two ram semen extenders (Tris-egg yolk or the commercial optiXcell IMV medium (IMV Technologies)) for semen cryopreservation. Four Santa Inês rams were used during the breeding season. After semen collection, macroscopic and microscopic evaluations were performed, and a pool of semen was formed. The semen was diluted, and the final concentration was 100×106 per 0.25-mL straw. Cryopreservation was performed with a cooling rate of 0.25°C min−1 until 5°C, and the freezing rate used was 20°C min−1 from 5 to −120°C. After the freezing-thawing process and throughout incubation (38°C in 5% CO2) in Fert-Tyrode's albumin lactate pyruvate medium, every 1h for up to 3h, several parameters were evaluated: sperm kinetics, hypo-osmotic test, plasma membrane integrity, capacitation status, and lipid peroxidation level. We did not find any protective effect of LC on plasma membrane integrity, hypo-osmotic test, and capacitation status. The sperm kinetics values throughout incubation showed that Tris extender promoted better indices of staight-line velocity, linearity, wobble, and straightness than IMV extender along incubation, regardless of the presence of LC. There were no benefits of the LC addition throughout the incubation, and 10mM was deleterious to few parameters (amplitude of lateral head displacement, linearity, and wobble) compared with the control (0mM) in the Tris extender group. The plasma membrane integrity analysis revealed no differences (P&amp;gt;0.05) among the groups. The average number of intact cells (hypo-osmotic) was higher in Tris extender groups supplemented with 10mM LC at 1h and 5mM LC at 2h compared to the respective extender IMV groups. Regarding capacitation status, the Tris 5mM LC group had more acrosome-reacted cells when compared with the IMV 5mM LC group at 2h. At 3h, the percentage of acrosome-reacted cells was higher in the Tris 0-mM group when compared with the IMV 0-mM group. Regardless the presence of LC, IMV had higher (P&amp;lt;0.05) lipoperoxidation than the Tris treatments. In conclusion, LC supplementation in semen extender had no beneficial effect on freezing-thawing ram sperm and throughout incubation for up to 3h, with no difference in each time point evaluated. Under the conditions of this study, the use of Tris extender was superior to IMV extender for ram sperm. Financial support for this work came from the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (Young Scientist Program of Our State; E-26/203.168/2017).

Recovery and cryopreservation of epididymal sperm from domestic cat using powdered coconut water (ACP-117c) and TRIS extenders

Cryopreservation of cats epididymal spermatozoa allows the conservation of the genetic material and the study of the cryogenic effect applied to the gametes of other felines. However, this biotechnique still presents variable results, being necessary the investigation of alternative extenders. Powdered coconut water (ACP-117c) has been efficient in the sperm freezing of several species and in the cat sperm refrigeration. Therefore, we aimed to evaluate the effect of the freezing stages and the quality of the cats’ epididymal spermatozoa after thawing, using ACP-117c. Epididymides (n = 36) from 18 cats were processed using TRIS (n = 18) or ACP-117c (n = 18) for sperm recovery. The sperm were immediately evaluated. Then, this was cooled, glycerolized, frozen and thawed, and re-evaluated at each stage for sperm kinetics by Computer Assisted Semen Analysis, viability, functionality (HOST), mitochondrial activity (DAB) and morphology. There was a reduction in total motility and progressive motility after thawing in both groups, and TRIS was superior to ACP-117c. The curvilinear velocity reduced after thawing with ACP-117c. Viability decreased after glycerolization in TRIS. Although it also reduced after thawing in both groups, it was higher in TRIS. There was no change on HOST. Mitochondrial activity decreased during the cryopreservation steps for both extenders. Nevertheless, TRIS presented a higher percentage of spermatozoa from DAB class I and II after thawing. Morphology did not differ between extenders. Therefore, ACP-117c is an alternative for the recovery of cat epididymal spermatozoa; however, it is not efficient for freezing. Glycerolization and thawing are the most critical stages, regardless of the extender.

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca)

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ± 7.7%) and membrane functionality (60.5 ± 4.2%) and higher mitochondrial activity (21.5 ± 3.7%) than those preserved in ACP-117c (20.9 ± 5.4% motile sperm; 47.1 ± 2.5% functional membrane; 11.8 ± 1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ± 3.3%) in comparison to samples diluted in ACP-117c (18.6 ± 1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.

Morphological and morphometric characterization of domestic cat epididymal sperm.

Sperm morphometry is the tool that confers objectivity to the morphological evaluation by accurately measuring the dimensions of the gamete and its structures. Thus, the aim of the study was to perform a morphometric characterization of the domestic cat sperm. Therefore, sperm samples were collected from twenty pairs of epididymis in a TRIS extender at 37ºC. An aliquot of the sample was used to make a smear with Rose Bengal solution, and afterwards, the morphology and morphometry were analysed. In the morphology, were quantified the percentage of normal sperm cells, morphological changes of head, midpiece and tail. In morphometry, each normal sperm cell was measured for length, width, area and perimeter of head and midpiece, tail length and total length. The parameters ellipticity, elongation, regularity and rugosity were also determined. The percentage of normal sperm was 67.21%. Of the abnormalities, the curled/folded tail, followed by the curved midpiece, abnormal shaped head and detached head were the most quantified. The sperm head presented 5.56±0.01μm and 3.10±0.01μm of length and width, respectively. The head area was 16.94±0.05μm2 , while the perimeter was 16.16±0.03μm. In the derived parameters, the values were as follows: ellipticity of 1.81±0.00; elongation of 21.39±0.12; regularity of 0.81±0.00; and rugosity of 0.14±0.00. The midpiece presented length and width of 7.96±0.01μm and 0.76±0.01μm, respectively. The mean length of the sperm tail was 45.12±0.06μm, and the total cell size was 58.67±0.06μm. Thus, it was concluded that the cat sperm is an elongated cell, with high rugosity and regularity. The spermatic tail represents more than ¾ of the total length of the cell and the midpiece exceeds the length of the head.

Brèves: Diabète de type 1 et pression artérielle : du nouveau !

Egg yolk is most commonly used in semen extenders for cryopreservation but, wide variability in composition and potential risk of xenobiotic contamination has raised questions upon the use of egg yolk. Therefore, the present study was designed to develop a soya milk based phytoextender for buffalo semen cryopreservation. Soya milk was prepared from fresh soya beans in laboratory. Soya milk (25% v/v) and glycerol (6.4% v/v) was added in Tris citrate buffer to prepare Soya Milk Tris (SMT) extender. To improve post-thaw sperm quality, cryo-protectants like trehalose (100 mM) or taurine (50 mM) was supplemented in SMT extender before semen cryopreservation. Post-thaw quality parameters, degree of cryocapacitation and oxidative stress (lipid peroxidation) of spermatozoa cryopreserved in SMT with or without additives were assessed and compared with Egg Yolk Tris (EYT) extender. SMT extender showed no significant (P>0.05) differences in sperm motility, viability, membrane integrity and acrosome integrity as compared to EYT extender. The degree of cryocapacitation and oxidative damage were found significantly (P<0.05) lower in spermatozoa cryopreserved in SMT extender as compared to EYT extender. Supplementation of additives (trehalose or taurine) in SMT extender significantly (P<0.05) reduced oxidative damage. Thus, soya milk extender (25% soya milk v/v; 6.4% Glycerol in tris citrate buffer) with additives (trehalose or taurine) may substitute conventional egg yolk extender (20% egg yolk v/v; 7% Glycerol in tris citrate buffer) for better cryopreservation of buffalo semen.

Idebenone improves quality of ram sperm by mitigating oxidative stress during cryopreservation

The present study was designed to test the effect of different levels of idebenone, a potent antioxidant on the quality of ram semen at post thaw. Eighteen (18) ejaculates were collected and extended with tris extender supplemented with no antioxidant (CON), with 2 μM idebenone (Id2), 5 μM idebenone (Id5), 7.5 μM idebenone (Id7.5) and 10 μM idebenone (Id10). The sperm quality was determined in terms of percent sperm motility, live sperm percentage, percent hypoosmotic swelling test (HOST) positive spermatozoa and percent intact acrosome (PIA). Moreover, malondialdehyde (MDA) level, an end product of lipid peroxidation (LPO) was also measured at post thaw both in seminal plasma and sperm cell. At post thaw, the percent sperm motility was significantly higher (p < 0.05) for Id10 as compared to Id2, Id5, Id7.5 and control. The live sperm percentage was non-significantly (p > 0.05) higher for Id10 as compared to control, Id5 and Id7.5 but significantly higher than Id2. The percent HOST positive spermatozoa was significantly higher (p < 0.05) for Id10 than control, Id2 and Id5. The MDA level in seminal plasma was significantly lower (p < 0.05) for Id10 than control and Id2. The MDA level in spermatozoa did show similar trend as in seminal plasma. Further, all the sperm parameters at all idebenone levels declined significantly from pre freeze to post thaw. In conclusion, idebenone at 10 μM level improved post thaw sperm quality by mitigating peroxidative stress, hence could be considered as a promising antioxidant additive for cryopreservation of ram semen.

Use of polyvinyl alcohol as a chemically defined compound in egg yolk-free extender for dog sperm cryopreservation.

The objectives of this study were to investigate the effects of polyvinyl alcohol (PVA) as a chemically defined compound in egg yolk (EY)-free extender by determining the appropriate concentration of PVA and the effect of pH adjustment in EY-free PVA extenders on dog spermatozoa. Spermatozoa (1×108 cells/ml) were frozen with EY-free extenders supplemented with 0 (control), 0.025, 0.05, 0.1, 0.2 or 0.3g/100ml PVA. Sperm progressive motility (PM) was assessed immediately after thawing (IAT) and post-thaw incubation (PTI), while viability, acrosome integrity and reactive oxygen species (ROS) levels were evaluated after PTI. Additionally, spermatozoa were frozen using EY-free PVA extenders before pH adjustment (6.45) and after adjustment of pH (6.85). Viability, PM, ROS and gene expression (BCL2 and SMCP) were assessed. Supplementation with 0.05g/100ml or more PVA significantly increased PM compared to the control group in the IAT and PTI. Post-thaw incubation significantly increased sperm motility in all groups. The acrosome integrity in all PVA groups was higher (p<.05) than the control without an effect on ROS and viability. Adjustment of the pH to 6.85 improved (p<.05) sperm PM compared to the non-adjusted groups without affecting viability, ROS or expression of BCL2 and SMCP. We suggest that PVA supplementation in EY-free Tris extenders can effectively protect dog spermatozoa during freezing and can maintain higher motility and acrosome integrity. Adjustment of pH in EY-free PVA extenders can improve post-thaw sperm motility. Therefore, PVA can be used as a compound in EY-free extender for the cryopreservation of dog spermatozoa.

Influence of Antioxidant Sericin in Tris Extender on Oxidative Markers during Cryopreservation (–196ºC) of Bovine Semen

This investigation was carried out during winter season on the semen of three mature, healthy breeding bulls each of Gir cattle and Murrah buffalo breeds. The aim was to assess the effect of different concentration of antioxidant Sericin (0.0, 0.1, 0.25, 0.50 and 1.0%, w/v) in standard Tris fructose egg yolk glycerol (TFYG) extender on cryopreservability of bovine semen based on sperm motility and oxidative markers in seminal plasma of freshly diluted and cryopreserved semen. The mean sperm motility observed in freshly diluted and frozen-thawed Gir bull semen, irrespective of Sericin levels, were 76.93±0.39 and 43.47 ± 0.58 % and in Murrah bulls 78.20±0.38 and 44.10 ± 0.48 %, respectively. The values of malondialdehyde (MDA, μmol/ml) in seminal plasma of freshly diluted and frozen-thawed semen of Gir bulls, irrespective of Sericin levels, were 21.68±0.38 and 24.99 ± 0.56, and in Murrah bulls 21.49±0.57 and 25.60±0.94, respectively. The corresponding values of superoxide dismutase (SOD, U/ml) were 1.77 ± 0.06 and 1.37 ± 0.05 in Gir and 1.18 ± 0.06 and 0.85 ± 0.04 in Murrah bulls, and those of glutathione peroxidase (GPx, nmol/min/ml) 417.10 ± 12.00 and 349.76 ±11.92 in Gir and 385.71±9.21 and 320.02±9.49 in Murrah bull semen. Sperm motility and activities of all three enzymes differed highly significantly (plessthan00.01) between stages. SOD was significantly (plessthan00.05) lower in buffalo than cattle semen. Inclusion of 0.5% and/or 0.25% Sericin in TFYG extender gave better protection to spermatozoa over other levels against ROS mediated injuries as the MDA production was significantly reduced with increased sperm motility and higher levels of SOD and GPx enzymes in the seminal plasma.