Effect Of Different Extenders Research Articles

Effect of different extenders and cryoprotectants on sperm viability of mud crab, Scylla tranquebarica for short term storage

The aim of the present study was to determine the extenders such as calcium-free saline, Ringer’s solution and phosphate buffer and to determine the effect of different concentrations of cryoprotectants with different duration of exposure for short term storage of mud crab, Scylla tranquebarica sperm cells. A total of 30 mud crabs were used in the present study. In the present study, three treatments with three replicates were tested to determine the suitable extender. Besides, three treatments (dimethyl sulfoxide, glycine and glycerol) were tested to determine the suitable cryoprotectants. Eight treatments (25, 20, 16, 4, 2, -4, -20 and -80 °C) were assessed to determine the optimal cooling rate. The result showed the phosphate buffer solution produced the highest sperm viability (82.36 ± 5.84 %) after 60 min compared the Calcium free saline and Ringer’s solution. The results for sperm viability at -4 °C, -20 C and -80 °C after 24 hours were 95.63 0.13 % (5 % glycine), 91.51 ± 1.91 % (5 % DMSO) and 93.97 ± 0.43 % (5 % glycine). By using the 5 % glycine as the best cryoprotectant, the results for the sperm viability in 6 h, 12 h, 24 h, day 7, day 14 and day 21 at 80 °C were 95.17 ± 0.92 %, 94.11 ± 0.64 %, 93.31 ± 0.51 %, 90.84 ± 2.05 % and 82.82 ± 0.41 %. In a nutshell, the phosphate buffer and 5 % glycine were the best extender and cryoprotectant in the present study.

Effects of different extenders on epigenetic patterns and functional parameters of bull sperm during cryopreservation process.

The cryopreservation process induces alterations in cellular parameters and epigenetic patterns in bull sperm, which can be prevented by adding cryoprotectants in the freezing extenders. The purpose of this study was to compare the protective effects of two extenders based on soybean lecithin (SLE) and egg yolk (EYE) on epigenetic patterns and quality parameters of sperm such as motility parameters, mitochondrial membrane integrity, DNA fragmentation, viability, and apoptotic-like changes of bull sperm after cryopreservation. Results demonstrated that cryopreservation significantly (p < .05) reduced the level of DNA global methylation, H3K9 histone acetylation, and H3K4 histone methylation in both frozen groups compared to the fresh sperm. Also, the level of H3K9 acetylation was lower in the frozen SLE group (21.2 ± 1.86) compared to EYE group (15.2 ± 1.86). In addition, the SLE frozen group had a higher percentage of viability, progressive motility, and linearity (LIN) in SLE frozen group compared to EYE frozen group. However, no difference was observed in mitochondrial membrane integrity and DNA fragmentation between SLE and EYE frozen groups. While soybean-lecithin-based extender showed some initial positive impacts of epigenetics and semen parameters, further investigations can provide useful information for better freezing.

Post-thaw quality of boar spermatozoa is affected by ejaculate fractions and extenders.

The aim of this study was to investigate the effect of different extenders on the post-thaw (PT) quality of sperm originating from the sperm-rich fraction (SRF) and post-sperm-rich fraction (PSRF) of boar ejaculate. Motility and velocity parameters, analyzed using a computer-assisted semen analysis (CASA) system, and membrane integrity parameters were markedly higher in frozen-thawed (FT) spermatozoa of the SRF in both the Belstville Thawing Solution (BTS) and Androhep Plus (AHP) extenders, irrespective of the post-thaw (PT) storage time. Furthermore, reduced cryo-survival was more marked in FT spermatozoa of the PSRF in both extenders following storage for 60 min. It was found that the SRF-stored samples in the AHP extender for 60 min exhibited significantly higher percentages of spermatozoa with total motility, mitochondrial function and acrosome integrity than those stored in the BTS extender. The findings of this study confirm that components of the ejaculate fractions and extender have varying effects on the cryo-survival of boar spermatozoa.

Effects of different extenders, storage temperatures, and antioxidant supplementation on chilled semen quality: a review.

The successful preservation of ram semen is essential to promote genetic variability, ensure semen transportation, and inseminate multiple ewes. Currently, either animal or plant-based lipoprotein-based extenders are used for semen preservation. Animal product-based extenders include milk and egg yolk, while soybean lecithin is a plant-based extender. Although extenders containing products of animal origin better preserve the quality of chilled semen, the in vivo efficacy after 24h of storage is still of great concern. Storage temperature is another important and effective factor in preserving sperm quality, whereby different storage temperatures are adopted to enhance the storage life of sperm. Low temperatures (4-5°C) better preserve sperm quality for a longer duration than high temperatures (15, 20, and 25°C). Moreover, antioxidant supplementation has a positive impact on sperm quality during liquid storage. The current review summarizes the outcomes of various extenders, different storage temperatures, and antioxidant supplementation on the liquid storage of ram sperm.

Effect of extenders, cryoprotectants and dilution ratios on the spermatological properties of cryopreserved milt of cobia Rachycentron canadum

Cobia (Rachycentron canadum) culture depends on the availability of seeds which isaffected by asynchronous spawning and brooders availability. The possible solution forthese problems is the use of cryopreserved, high quality gametes from selected individuals.Therefore, in the present study, the effect of different extenders, different concentrationsof cryoprotectants and dilution ratios on the spermatological properties of cryopreservedcobia milt was analysed. Milt was collected from R. canadum brooders and diluted withMarine Fish Ringer’s Solution (MFRS) and 0.85% physiological saline (PS) as extendersand dimethyl sulphoxide (DMSO) as cryoprotectant at three different concentrations(5, 10 and 15%) at three dilution ratios such as 1:5, 1:10 and 1:15. Motility duration, motilitypattern, motility score and percentage of live and dead cells were analysed. The milt wasrapidly frozen employing liquid nitrogen vapour for 10 min. The milt sample diluted with PSand 10% DMSO at 1:5 dilution ratio exhibited highest post-thaw motility parameters. Keywords:Cryoprotectants, Cryopreservation, Dilution ratios, Extenders, Spermatological properties, Rachycentron canadum

Africanized honeybee (Apis mellifera) semen freezing using Tris-based and Collins extenders.

This study is aimed at evaluating the effect of different extenders on the cryopreservation of semen from Africanized honeybees (A. mellifera). Semen from honeybee drones from 10 different colonies was obtained by endophallus exposure technique and immediately evaluated for motility, viability using fluorescent probes, functional membrane integrity using the water test, and morphology. Samples from each colony were divided in three aliquots and subjected to a dilution ratio of 12:1 (diluent: semen) using Tris, Tris + egg yolk (Tris+EY), and Collins extender. Samples were cryopreserved and stored in liquid nitrogen for one week and then rewarmed and reevaluated. Immediate dilution provoked no significant effect on sperm motility and functional membrane integrity, regardless of the extender used; however, the greatest values (P < 0.05) for normal sperm morphology were found at the use of isolate Tris (69.3 ± 1.9%). After thawing, there were no significant differences among extenders with relation to the preservation of sperm motility, viability, and functional membrane integrity, but the Tris extender provided the highest post-thawing values (P < 0.05) for sperm normal morphology (49.2 ± 4.9%) while the Collins extender provoked the highest amounts (P < 0.05) of curled tail defects (67.5 ± 3.2%). Moreover, the Tris was the only extender at preserving the proportion of normal sperm after thawing similar to what was verified for fresh samples. In summary, we suggest the use of a Tris-based extender for the cryopreservation of Africanized honeybee semen.

Effects of extender type and storage time on sperm quality parameters of Kail ram semen stored at 5 °C

The study was designed to evaluate the effect of different extenders and storage times on sperm quality parameters of extended Kail ram semen. Semen was collected from five adult Kail rams using an artificial vagina. Semen samples with >70% total sperm motility were pooled, diluted with Tris (TR), sodium citrate (SC), and skim milk (SM)-based extenders, and stored at 5 °C. Sperm motility and kinematics, viability, and plasma and acrosomal membrane integrity were assessed every 24 hrs for 120 hrs. Sperm longevity was estimated by placing semen in a water bath at 37°C to assess sperm kinematics. Storage time as the main effect had a negative (P < 0.05) impact on sperm quality parameters. The percentages of total motile (TM), progressive motile (PM), rapid, and medium progressive (MP) motile sperm were similar at 24 hrs of storage for the three extenders. However, semen extended in TR had a higher (P < 0.05) percentage of TM, PM, rapid, and MP sperm compared to SM and SC extenders at 48 hrs of storage. The sperm kinematics (VCL, VSL, VAP, and ALH) of progressive sperm were similar for all three extenders up to 72 hrs of storage. In addition, semen extended in TR had a higher (P < 0.05) percentage of sperm with intact plasma and acrosomal membranes at a storage time of 48 hrs. At 37 °C, the percentage of TM and PM sperm was higher (P < 0.05) in the TR extender than in the SM and SC extenders at 60 minutes and beyond. In conclusion, the Kail ram sperm extended in TR and stored at 5 °C maintained better quality after 48 hrs storage than sperm extended in SM and SC extenders. At 37 °C, sperm extended in TR also retained better quality parameters at 60 min and afterward than sperm extended in SM and SC.

Ide (Leuciscus idus) sperm short-term storage: Effects of different extenders and dilution ratios

In cyprinids, Tyrode’s (TLP) and Volckaert’s (VRT) solutions are the most frequently used extenders for short-term sperm storage. The effectiveness of TLP and VRT on ide (Leuciscus idus) sperm short-term storage was analyzed using a × 4 (sperm:extender) dilution ratio over 48 h. A × 4 (1:3) dilution ratio was compared to a × 10 (1:9) dilution ratio for ide sperm storage using TLP supplemented with antibiotics and was tested for a 14-day period. Sperm motility (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity of sperm (VCL, µm s-1), movement linearity (LIN, %), beat cross frequency (BCF, Hz), and the amplitude of lateral head displacement (ALH, µm) were verified using a CASA system. After 48 h, most CASA parameters were significantly higher in TLP compared to VRT. The dilution ratio also had a significant impact (P < 0.0001) on the efficiency of ide sperm short-term storage over 14 d in comparison to undiluted sperm (control). Ide sperm diluted with TLP supplemented with penicillin/streptomycin and stored short term, regardless of the dilution ratio used, retained motility and fertilization capacity over 14 d at 4 °C. The highest embryo survival rates of 70% and 73% were noted using sperm diluted with TLP at × 4 and × 10 dilution ratios compared to 48% for raw sperm that was not stored short term.

The Effect of Different Extenders on the Quality Characteristics of European Red Deer Epididymal Sperm Stored at 5 °C.

Simple SummaryThe use of commercial extenders for storing cervid epididymal spermatozoa in a liquid state can contribute to popularizing this method of semen preservation in cervid farms. The aim of this study was to assess the effect of various extenders (Bovidyl®, BoviFree®, and BioXcell®) on the quality of European red deer epididymal spermatozoa stored at 5 °C. The analyses demonstrated that the tested extenders and storage time significantly affected sperm motility and functionality of different sperm structures. The Bovidyl® extender containing chicken egg yolk exerted the most beneficial influence on the quality of stored sperm. Spermatozoa stored in the Bovidyl® extender were characterized by satisfactory motility (above 60% on storage day 9) and the functionality over a long period of time. In turn, extenders containing plant-based ingredients and glycerol (BioXcell® and BoviFree®) significantly decreased membrane integrity and mitochondrial activity of spermatozoa already on the first day of storage. These results indicate that European red deer epididymal spermatozoa can be stored in commercial extenders, but their viability and functionality are significantly influenced by the composition of the applied extenders. The extender containing chicken egg yolk without glycerol produced the most satisfactory results. The aim of this study was to evaluate the effect of different extenders on the quality of European red deer epididymal sperm stored at 5 °C. Epididymal spermatozoa were collected post mortem from 10 stags and diluted with three extenders (Bovidyl®, BoviFree®, and BioXcell®) and stored at 5 °C. Sperm motility (TMOT), motility parameters (system CASA), plasma membrane integrity (SYBR-14+/PI−), acrosomal membrane integrity (FITC-PNA−/PI−), mitochondrial activity (JC-1/PI), viability, and apoptotic-like changes (YOPRO/PI) were evaluated. The analyses were conducted on the first and successive days of storage (D1–D7). The applied extender, storage time, and the interactions between these factors significantly (p < 0.001) affected most of the analyzed parameters whose values were highest in sperm samples stored in Bovidyl®, regardless of storage time. In Bovidyl®, BoviFree®, and BioXcell® extenders, TMOT values were estimated at 83%, 63%, and 59%, respectively, on D3. The extenders significantly influenced DNA integrity on D7. The percentage of dead sperm increased from D3. The quality of stored sperm cells was significantly influenced by the extenders’ biochemical composition. BoviFree® and BioXcell® contain glycerol which could contribute to deteriorating the quality of spermatozoa stored at 5 °C. Sperm cells stored in the egg yolk-based extender (Bovidyl®) were characterized by the highest viability and functionality.

Optimization of a sperm cryopreservation protocol for giant grouper (Epinephelus lanceolatus)

Characterized by the extremely fast growth performance, giant grouper (Epinephelus lanceolatus) is one of the most promising marine culture species in South East Asia. Motivated by the rapid expansion in grouper aquaculture, giant grouper semen remains in increasing demand for massive seed production, but which is often constrained by the shortage of broodstock and asynchronous availability of gametes for the bulk of farmers. Sperm cryopreservation has a great potential for increased flexibility and efficiency for artificial seed production in aquaculture. To develop an efficient and standardized cryopreservation protocol for giant grouper semen, some key factors were investigated in this study, including the effects of different extenders (MPRS, E2, ELRS3), freezing heights (3, 5, 7 and 9 cm) above the liquid nitrogen surface corresponding to different freezing rates, final sperm concentration in the straw (1–10 × 109 spermatozoa/mL), equilibration (0–180 min) and pre-freezing storage time (0–60 h). Moreover, the fertilizing ability of cryopreserved semen was tested at sperm:egg ratios ranging from 100:1 to 1× 106:1. With 10% DMSO as cryoprotectant, the glucose-based extender ELRS3 at freezing height 5–7 cm produced higher post-thaw sperm motility. The optimal sperm concentrations in straws ranged from 2 to 8 × 109 spermatozoa/mL. Besides, sperm motility after thawing was not affected by a 120-min equilibration, but a significant decrease was detected after 180 min of equilibration. Concerning pre-freezing storage, semen stored within 24 h after collection could be successfully cryopreserved, while extended storage time would compromise the cryopreserved sperm motility. Similar fertilization results were obtained at the range of 1000 to 1 × 106 spermatozoa per egg for cryopreserved semen. Compared with fresh semen, the fertilization capacity of thawed sperm was not affected by 120 min of post-thaw storage, although with reduced motility parameters. This standardized procedure will be useful for the implementation of cryopreserved giant grouper semen in hatchery practice.

Effects of different extenders on preservation of Jamunapari buck semen

The aim of this study was to investigate the effects of different extenders on the quality of Jamunapari buck semen. Semen was collected from three adult Jamunapari bucks by Artificial Vagina (AV) method once a week. Immediate after collection, fresh semen was evaluated for recording its quality including volume, color, density, concentration, mass motility, pH and membrane potentiality. Each semen sample was divided into two groups; one for chill and another for cryo-preservation. Three different types of semen extenders; Tris (T), Skim milk (S) and Skim milk D-glucose (SD) were added in each group of semen dividing into three parts and then preserved accordingly. Chilled semen was evaluated on day 2 and day 4 of chilling at refrigerator (4ºC) and frozen semen on day 5 and day 12 of cryopreservation at liquid nitrogen (LN2). Preserved semen was evaluated for motility, viability, membrane potentiality (HOS test) and morphology. Average volume, mass motility, and sperm concentration were 1.22±0.26 ml, 4.01±0.67and 3.13±0.27×109 /ml, respectively. The motility, viability, normal morphology and membrane potentiality and pH were 86.67.5±3.00, 82.13±2.18, and 96.52±1.69, 83.2±3.20, and 6.74±0.28, respectively. The quality of chill semen was the best in Trisbased extender (P

Effect of Different Extenders on Sperm Motility and Vitality in Goose Semen Cryopreservation

This study aimed to investigate the usability of different diluents containing 6% Dimethylformamide (DMF) for cryo-preservation of the semen of geese (Anser cygnoides). The diluents of Glucose (G), Tris-Glucose (T), Lactated Ringer’s-Glucose (LG), and Lactated Ringer’s (L), all of which contained 6% DMF, were used as cryoprotectants. The researchers collected semen samples from four geese, twice a week over a four-week period, by means of abdominal massage; they then calculated how much sperm each goose ejaculated. Next, the semen samples were pooled and their spermatological parameters were determined. Their volume (4x (mL)), concentration (×108/mL), pH, motility (%), and vitality (%) rates were 0.31±0.01, 3.49±0.32, 7.13±1.06, 67.75±1.28, and 70.00±2.03, respectively. Then, these pooled semen samples were equally divided into four groups. Once they were frozen and thawed, the researchers discovered that the diluent L had the highest motility rate: 40.12% ± 1.35. The motility rates of the other diluents were as follows: LG (28.25%± 1.48), G (21.50% ± 1.41), and T (5.12% ± 0.83). Likewise, the vitality rates (%) of the diluents were as follows: L (41.93% ±1.87), LG (31.50%±1.88), G (29.43% ±1.45), and T (10.56%±1.34), respectively. Freezing and thawing appeared to lower each diluent’s vitality and motility rates. However, for the Lactated Ringer’s (L), this decrease was predictable. Therefore, Lactated Ringer’s diluent containing 6% DMF can be used in cryo-preservation of goose semen.

Cryopreservation of Spix's yellow-toothed cavy epididymal sperm using Tris- and coconut water-based extenders supplemented with egg yolk or Aloe vera

Addressing the establishment of biobanks for the conservation of wild hystricomorph rodents' germplasm, we verified the effects of different extenders and distinct concentrations of non-permeant cryoprotectants on the sperm parameters of Spix's yellow-toothed cavies. Nine testis-epididymis complexes were used for sperm collection by retrograde washing using Tris or a powdered coconut water extender (ACP®-116c). Spermatozoa were diluted and frozen with the same extenders supplemented with egg yolk or Aloe vera at a 10% or 20% concentration. After recovery and cryopreservation, all samples were evaluated for sperm kinetic parameters, morphology, membrane integrity, osmotic response, and sperm-binding capability using an egg yolk perivitelline membrane assay. After recovery, no differences were observed between Tris and ACP®-116c that provided 515.4 × 106 sperm/mL and 561.6 × 106 sperm/mL, presenting >65% motile sperm, respectively. After cryopreservation, most effective preservation of sperm kinetic parameters (68.1 ± 5.9% motile sperm) and membrane integrity (48.2 ± 7.4%) was provided by Tris extender supplemented with 10% egg yolk. However, both extenders supplemented with any concentration of egg yolk or Aloe vera presented similar preservation of osmotic response and sperm-binding ability after cryopreservation. In summary, we suggest the use of a Tris extender supplemented of 10% egg yolk for cryopreservation of Spix's yellow-toothed cavy epidydimal sperm.

Protective Effect of Vitamin C (Ascorbic Acid) Supplementation on Post-Thaw Motility and Fertility of Cryopreserved Rainbow Trout (Oncorhynchus mykiss) Sperm

Ascorbic acid (Vitamin C) is one of the important antioxidants, which naturally present in seminal plasma of fish. On the other hand, whether its effect may improve sperm quality following cryopreservation process still remains its uncertainty. Thus, the present study aimed to analyse the effect of different extenders supplemented with different ascorbic acid concentrations on post-thaw motility and fertility of frozen-thawed rainbow trout (Oncorhynchus mykiss) sperm. Selected sperm samples were pooled and diluted at 1:3 ratios with two different extenders (E) composing such as (E-1) 300 mM glucose, 10% egg yolk and 10% DMSO and (E-2) 0.6 mM sucrose and 10% DMSO. Each extender was supplemented with vitamin C at 1, 5 and 10 mM concentrations. Following dilution, the sperm was loaded into 0.25 ml straws and frozen in liquid nitrogen vapour. The straws were then plunged into liquid nitrogen for storage. Fertilization was carried out using the dry fertilization technique. Highest post-thaw motility (50±5.77) and fertilization (56±1.00) results were obtained with the extender-1 (E-1) containing 10% DMSO concentration. In conclusion, the present study indicated that addition of ascorbic acid to the extenders improved rainbow trout sperm motility resulting higher fertilization of the eggs.

The Effect of Different Extenders on Some Fertility Properties of Roosters Semen

The Effect of Different Extenders on Some Fertility Properties of Roosters Semen

Effect of three commercial extenders on sperm motility and fertility in liquid ram semen stored at 15 °C or 5 °C

The effect of different extenders on sperm motility and fertility was evaluated during liquid storage of ram semen at 5 °C and 15 °C. The semen was collected, pooled and diluted in three commercial extenders: Inra 96® (INRA) based on skimmed milk, Biladyl® A fraction (BIL) based on egg yolk, and Ovixcell® (OVIX) based on soybean lecithin. Then, sperm motility was evaluated at 0, 6, 24, 48, 72 and 96 h. In order to evaluate fertility, samples stored at 15 °C were used after dilution in INRA and OVIX. Results showed that progressive motility was significantly higher up to 72 h of storage in sperm samples maintained at 5 °C in comparison with 15 °C, similarly for each tested diluent. When samples were stored at 5 °C in OVIX, kinematic parameters such as velocity (except curvilinear velocity, VCL), trajectory [linearity (LIN), straightness (STR), wobble (WOB)], amplitude of lateral head displacement (ALH) and beat/cross frequency (BCF) were higher than in INRA and BIL. No significant differences in pregnancy rate were detected between INRA (62.6%) and OVIX (58.9%). In conclusion, liquid storage at 5 °C with OVIX extender is an interesting option since non-animal components are used, and this extender offers similar in vitro and in vivo efficacy as other extenders containing animal components.

Effects of different extenders and storage temperatures on longevity of small East African goat (Capra hircus) semen

Goat production is central to resource-poor rural livelihoods in the tropics. However, it is constrained by high inbreeding levels and poor access to good quality breeding bucks. In this study, semen extenders differing in egg yolk and energy level were compared in their ability to maintain liquid semen from a tropical goat breed, when stored at physiological (37 °C) or refrigerator (4 °C) temperature. Semen was collected weekly for nine weeks from seven bucks of the Small East African goat breed, diluted in three extenders and incubated at 37 °C or 4 °C for 24 h. The semen parameters: viability, motility and morphology were better preserved when semen was incubated at 4 °C compared to 37 °C across all extenders. Sperm motility after 24 h at 4 °C was 50.4 ± 5.5%, 50.4 ± 6.2% and 54.3 ± 5.4% for Extender 1 (high glucose, 18% egg yolk), Extender 2 (low glucose, 2.5% egg yolk) and Extender 3 (low fructose, no egg yolk), respectively. Extender 2 maintained significantly higher viability values (67.7 ± 2.5%) at 24 h compared to Extenders 3 (59.3 ± 2.1%) and 1 (46.7 ± 4.2%). Normal morphology after 24 h (88.1 ± 3.1% at 4 °C and 85.6 ± 3.1% at 37 °C) fell within the acceptable range for good quality semen. Acceptable quality was maintained within 8 h and up to 24 h from collection for non-frozen goat semen stored at 37 °C and 4 °C temperature, respectively. These results imply that Small East African goat semen diluted in low or non-egg yolk based extenders and stored at refrigerator temperature can be valuable for artificial reproduction of native goats on low resource capacity farms in developing countries.

Effect of Different Extenders and Dilution Ratios on the Spermatological and Fertilization Parameters of Cyprinus carpio During Cryopreservation.

Production of quality seeds is a critical need, and cryopreservation serves as an important biotechnological tool in achieving that goal. In the present study, the effects of different extenders and dilution ratios on the spermatological and fertilization parameters of cryopreserved milt were evaluated in common carp brooders. Milt was collected from Cyprinus carpio brooders and diluted and cryopreserved at three dilution ratios such as 1:40, 1:80, and 1:120, using Freshwater Fish Saline (FWFS) and Modified Fish Ringer as extenders and dimethyl sulfoxide as cryoprotectant (90:10). Motility duration, fertilization, and hatching rates were studied after different treatments. Rapid freezing of straws was done for 20 minutes at a freezing rate of -78°C/min. It can be concluded that C. carpio milt can be cryopreserved with FWFS at a dilution ratio of 1:40 to obtain the highest post-thaw motility duration (60.33 ± 1.52 seconds), fertilization rate (87.6% ± 1.52%), and hatching rate (57.1% ± 4.16%). The difference in the values between extenders, dilution ratios, and cryopreserved and fresh milt were statistically significant (p < 0.05) when analyzed using ANOVA.

Effects of different extenders and additives on liquid storage of Awassi ram semen

The aim of this study was to evaluate the effects of different extenders and additives on ram semen during liquid storage and to demonstrate the role of oxidative stress parameters on this process. In the present study, ejaculates taken by artificial vagina twice a week from 4 rams during the breeding season were used. They were mixed and used if motility and viability were above 70% and there was a 95% intact acrosome. The semen specimens were diluted by Tris-citrate-glucose (TRIS), Tris-TES (TEST), HEPES-buffered Tyrode lactate (TL-HEPES), and Dulbecco?s phosphate-buffered saline (PBS) extenders supplemented with different additives [centrifuged egg yolk, Equex-STM, bovine serum albumin (BSA), and ethylenediaminetetraacetic acid (EDTA)]. The specimens were stored at 4 °C for up to 96 h and evaluated for motility, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), and oxidative stress parameters. At the end of the 96-h storage, the highest sperm motility was 64.2 ± 3.7% (P < 0.05) and significant loss of sperm motility and membrane integrity were not detected in extender TEST-3, but the MMP rate significantly declined. Acrosome integrity was not affected by storage time or extender types. BSA and EDTA decreased lipid peroxidation (LPO) and total oxidant status (TOS), but did not positively affect motility or membrane integrity. As a result, TRIS, TEST, and TL-HEPES-3 were observed to provide better protection for ram semen during liquid storage at 4 °C than other extenders. The role of oxidative stress and MMP are considerable in liquid storage.

Effect of different extenders for donkey sperm vitrification in straws.

Aseptic vitrification of semen samples packed in straws has been successfully developed in human but not in donkeys. The aim of this study was to compare the effect of two extenders for donkey sperm vitrification using straws. Ejaculates from four Andalusian donkeys were collected, and samples were extended in INRA-96 (I) or Gent (G) supplemented with sucrose 0.25M and 1% bovine serum albumin (BSA). Extended samples were cooled for one hour at 5°C. For vitrification, samples were filled in covered 0.25ml straws and then plunged directly into liquid nitrogen. For warming, straws were immersed in INRA-96 at 43°C. Results showed no significant differences between I and G treatments for TM (34.2%±8.7 vs. 30.7%±9.6) and PM (26.8%±7.3 vs. 24.6%±7.9), respectively. In conclusion, donkey sperm could be vitrified in straws either with INRA-96 or with Gent in combination with sucrose and BSA.