Tris-egg Yolk Extender Research Articles

Cryoprotective role of organic Zn and Cu supplementation in goats (Capra hircus) diet

The current study focused on cryopreservation and assessment of characters of post-thaw semen of indigenous Osmanabadi bucks maintained with standard diet, supplemented with different concentrations of organic zinc (Zn), copper (Cu) or in combination, for a period of 180 days. The different doses of organic Zn and Cu were fed per kg DM basis, Zn groups (low: Zn20, medium: Zn40 and high: Zn60), Cu groups: (low: Cu12.5, medium: Cu25 and high: Cu37.5) and combination of Zn + Cu groups (low: Zn20 + Cu12.5, medium: Zn40 + Cu25 and high: Zn60 + Cu37.5) respectively. The control group bucks were maintained mainly on the basal diet without any additional mineral supplementation. Two hundred and forty (240) semen samples were collected from 40 bucks aged 11 months, through electro ejaculator method, processed and analysed for sperm quality parameters both at pre freeze and post-thaw stage. The semen samples were diluted in Tris egg yolk extender, cooled and equilibrated for 4 h at 5 °C, cryopreserved using programmable freezer (PLANER Kryo 360–1.7) and stored at −196 °C. The organic trace minerals (Zn, Cu and Zn + Cu) protected the spermatozoa against the cryoinjury and maintained higher post-thaw semen parameters except in high Zn group. Additional feeding of organic Cu and Zn to bucks had a protective role and resulted in higher sperm liveability, plasma membrane and acrosome integrities, motility and velocity and reduced oxidative stress in supplemented goats (P < 0.05).

Semen Quality of Post-Thawed Local Ram’s in Tris-Egg Yolk Extender with Different Glutathione Level

The aims of this research were to find out the effect of glutathione level on semen quality of local ram and to find out the glutathione level that provide the best quality of local ram semen. This research use Completely Randomized Design (CRD) with five treatments of glutathione level (0 mM, 4 mM, 5 mM, 6 mM and 7mM) and was repeated for 10 times. Data were analyzed using analysis of varians (ANOVA) and differences between treatment was analyzed using Duncan test. The parameter were semen quality consist of motility, intact plasma membrane (IPM), abnormality, and recovery rate. Result of this research showed that glutathione level significantly (p<0.05) affect on motility, IPM, abnormality and recovery rate. Duncan test result showed that recovery rate from treatment of 5 mM glutathione level have significantly higher than other treatment, whereas for motility, IPM and abnormality were not significant different with 4 mM, and 6 mM. It is concluded that semen quality of local ram was affected by glutathione level, and the level of 5 mM in egg yolk tris extender provide the best quality of local ram semen.

Alpha-zearalenol negatively influences ram sperm parameters during liquid storage.

This study was aimed to investigate the effects of 17 𝛽-estradiol (E2) and 𝛼-zearalenol (α-ZOL) on motility parameters, plasma membrane integrity, levels of produced nitric oxide (NO) and total antioxidant capacity of Ghezel ram sperm during the liquid storage at 4 ˚C, for various periods of time. Semen samples were collected from four rams and diluted with Tris–egg yolk extender and supplemented with E2 (100 µmol) or different concentrations of α-ZOL (100 pmol, 100 nmol and 100 µmol) at a final concentration of 200 × 106 sperm per mL. We failed to show any significant effect of E2 at 100 µmol concentration on ram’s sperm parameters while α-ZOL resulted in a significant decrease of plasma membrane integrity at 100 µmol concentration (55.40% for α-ZOL vs 62.20% for control) after 96 hr incubation. Alpha-ZOL had decreasing effect on sperm motility parameters including curvilinear velocity and average path velocity at 100 µmol concentration after 96 hr storage. Although remarkable reduction of total antioxidant capacity at high concentration of α-ZOL and long incubation time was found, however no significant changes were recorded in NO level during storage time. It was concluded that the detrimental effect of α-ZOL on ram sperm might be attributed to its induced oxidative stress and damage to the plasma membrane.

Effects of apple and orange juices on quality of refrigerated goat semen

This study investigated the effects of apple and orange juices on quality of refrigerated spermatozoa of goat bucks. Semen samples from WAD goat bucks were diluted with Tris-egg yolk extenders each supplemented with apple and orange juices at 0, 2.5, 5, 7.5 and 10/100 ml of diluents. The diluted semen samples were assessed for sperm viability and malondialdehyde (MDA) concentration after in vitro storage for 240 hours at 5oC. The ability to maintain sperm motility was higher in the extenders with 7.5% orange juice followed by 10% apple juice compared to other treatments (P&lt;0.05). The extenders supplemented with 2.5%, 5% and 7.5% apple juice, and 5% orange juice had higher intact acrosome compared to other treatments and the control (P&lt;0.05). The 10% orange juice had higher percentage membrane integrity compared to other treatments. Consistent and reduced (P&lt;0.05) MDA levels were observed in the extenders supplemented with fruit juices and lower MDA was observed in the extenders supplemented with 10% apple juice compared to other treatments and the control (P&lt;0.05). The findings reveal that additions of the fruit juices to semen extenders to maintain the viability of refrigerated spermatozoa were best at concentrations of 10 ml/100 ml of apple juice and 7.5 ml/100 ml of orange juice.

A two-step dilution tris-egg yolk extender containing Equex STM significantly improves sperm cryopreservation in the African wild dog (Lycaon pictus)

Conservation management of endangered African wild dogs (AWD; Lycaon pictus) can benefit greatly from development of sperm freezing and artificial insemination. Previous freezing attempts yielded nearly 0% motile sperm within 2 h of thawing. In this study, two canine freezing protocols were tested: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution. Semen was collected by electroejaculation from n = 24 AWDs, of which eight ejaculates of sufficient quality (four good quality with initial sperm motility of 75.0 ± 4.4% and four poor quality; showing rapid decrease in sperm motility to 3.3 ± 3.3% prior to freezing) were frozen. For good quality samples, motility and sperm motility index persisted for up to 8 h for Protocol 2, and was higher between 2 and 6 h after thawing with a decrease from 4 h of incubation. Motility dropped to nearly 0% after 2 h incubation for Protocol 1. Viability was higher for Protocol 2 throughout the 8 h of incubation, with a decrease after 6 h, compared to 4 h for Protocol 1. Acrosome integrity was higher for Protocol 2 throughout post-thaw incubation, with a decrease after 2 h for both protocols. Protocols did not differ in normal sperm morphology or DNA integrity. Poor quality samples yielded similar results, except for acrosome integrity, which declined for Protocol 2. In conclusion, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it suitable for sperm banking and artificial insemination initiatives.

Impact of Eurycoma longifolia extract on DNA integrity, lipid peroxidation, and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.

An endeavor to improve longevity of cryopreserved equine sperm

Exposure of sperm cells to the oxidative stress pending hypothermic storage of semen has been suggested to be responsible, in part, for the decline of their motility and fertility. This study was conducted to evaluate the in-vitro effects of antioxidants (AOs) and / or caffeine on longevity of cryopreserved stallion spermatozoa. Aliquots from the gel-free fraction of semen ejaculates (n=12), collected from 5 Arabian stallions (9-18 years old) of unknown sperm freezability, were mixed 1:1 with a Tris-egg yolk extender (TEYE), centrifuged at 500 χ g for 5 min and sperm cells were frozen in the form of 0.25-ml concentrated pellets after 2-step addition of TEYE supplemented with or without AOs (0.50 mg /ml Na pyruvate, 1 mg / ml Na thiosulfate, 5 mg / ml bovine serum albumin, 0.15 mg / ml zinc chloride and 0.50 mg / ml ferulic acid). The final pre-freeze concentrations of glycerol and sperm cells were 5% and 562-924 χ IO6 / ml, respectively. Frozen pellets from non-AOs and AOs-treated sperm were thawed in a Tris-citric acidglucose solution (40°C) containing 0, 0.49, 0.97 or 1.94 mg / ml caffeine and incubated (140-230 χ IO6 sperm / ml) at 30°C for 3 h. Sperm progressive motility (%) was assessed after centrifugation, before freezing and after 0, 1, 2 and 3 h of thawing. The results revealed significant (P&lt;0.05) effects of sperm treatments only on post-thaw motility. Neither AOs alone nor caffeine alone could significantly ameliorate the maintenance of sperm motility. AOs plus 0.97 or 1.94 mg / ml caffeine were the superior supplements in improving the longevity of stallion spermatozoa.

Effects of supplementation of the Tris-egg yolk extender with n-3 polyunsaturated fatty acids (PUFAs) on frozen-thawed ram semen characteristics

The present study was conducted to evaluate the effects of supplementation of Tris-egg yolk extender with the different concentrations of n-3 PUFAs (DHA+EPA) on the freezability of ram semen. Semen samples were collected from five fertile rams using an artificial vagina, once a week, for 8 weeks in July and September. The selected ejaculates were extended and pooled into Tris-egg yolk extender supplemented with 0 (control), 10, 30 and 50ngml−1 concentrations of n-3 PUFAs. The extended samples were cooled for 3h at 4°C and packed in 0.5ml straws, frozen in liquid nitrogen for a week. The mean percentage of sperm motility in 50ngml−1 (35.6±1.8%) and 30ngml−1 (37.5±1.8%) n-3 FAs supplemented groups was significantly lower than that of control group (43.1±1.8%; P<0.01). The mean percentage of sperm viability in 30ngml−1 (38.9±1.5%) and 50ngml−1 (36.4±1.5%) n-3 FAs supplemented groups was significantly lower than that of control group (43.8±1.5%; P<0.01). The plasma membrane integrity was significantly lower in 50ngml−1 n-3 FAs supplemented group (29.6±1.6%) than that of control group (34.2±1.6%; P<0.05). The mean percentage of sperm tail defects was also significantly higher in group containing the n-3 FAs of 50ngml−1 (P<0.05). In addition, post-thaw sperm motility (P<0.05), viability (P<0.001) and plasma membrane integrity (P<0.05) in n-3 FAs supplemented groups decreased more significantly in September than those in July. In conclusion, the addition of n-3 PUFAs in Tris-egg yolk extender did not improve the freezability of ram semen and had more detrimental effects on sperm cell after freezing-thawing in the breeding season.

Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen-thawed Thai native bull sperm.

This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen-thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen-thawed spermatozoa from all groups were used for invitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post-thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P<0.05). However, iodixanol had no beneficial effect on post-thaw sperm invitro fertilization ability and pregnancy rate after AI (P>0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on invitro fertilization ability and pregnancy rate after AI.

Effect of osmotic pressure on spermatozoa characteristics of cryopreserved buffalo bull (Bubalus bubalis) semen

ABSTRACTThis study was designed to determine the effect of different osmotic pressures on spermatozoa characteristics of cryopreserved buffalo bull semen using tris egg yolk extender (TEYE). Semen of Nili-Ravi buffalo bulls (n = 4) housed at a semen production unit was collected at weekly intervals for 10 weeks. Three solutions of tris-citric acid-fructose with osmotic pressures of 255, 275 and 295 mOsm/kg were used in extender preparation. Semen straws containing 20 × 106 spermatozoa were processed and stored at −196°C in liquid nitrogen. Post-thaw analyses of spermatozoa included motility, viability, acrosomal integrity, plasma membrane integrity, DNA integrity and lipid per-oxidation. Significantly higher (P < .05) sperm motility, acrosomal and DNA integrity were recorded at osmotic pressures of 275 and 295 mOsm/kg compared to 255 mOsm/kg. However, differences in spermatozoa viability, plasma membrane integrity and lipid per-oxidation were non-significant among three osmotic pressures. It is concluded that osmotic pressure of the solution used in extender preparation plays an important role in post-thaw quality of cryopreserved buffalo bull semen.

Preservability of bull spermatozoa in Tris-egg yolk extender enriched with different concentrations of butylated hydroxytoluene

Objective: To explore the effect of BHT on cattle spermatozoa during cooling and cryopreservation. Methods: Pooled bull semen were diluted by Tris-Citrate-Fructose egg yolk (TCFY) diluent considered as control (0 BHT) and different concentrations of BHT (1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mM were prepared in ethanol in prewarmed (37 °C) test tubes. The ethanol was allowed to evaporate so that, a thin crystallized layer of BHT was deposited on the inner surface of the tubes. Then extended semen was added into the tubes and incubated at 37 °C for 5 min to allow uptake of BHT by spermatozoa. The tubes were cooled slowly (approximately for 2 h) up to 5 °C and equilibrated for 4 h. After equilibration, semen freezing process was carried out. Extended semen was subjected to evaluation (motility, alive sperm, intact sperm membrane (HOST) % and acrosome integrity) in both cooled and cryopreserved semen. Results: The result revealed that sperm motility of post-cooled spermatozoa improved (P

The effect of equilibration time on semen freezing of local swamp buffalo (Bubalus bubalis) with combination extender of lactose and glycerol

Eriani K, Sari N, Ihdina M, Rosnizar. 2017. The effect of equilibration time on semen freezing of local swamp buffalo (Bubalus bubalis) with combination extender of lactose and glycerol. Nusantara Bioscience 9: 77-82. This study was to determine the different time of equilibration on semen of swamp buffalo (Bubalus bubalis) which was diluted by adding Tris-Egg Yolk extender of lactose and glycerol cryoprotectant combination. Fresh sperm of the swamp buffalo (B. bubalis) was diluted by using Tris-Egg Yolk extender of lactose cryoprotectants combination 0 mM (L0), 60 mM (L60), 120 mM (L120) and glycerol 3% (G3), 5% (G5), 7% (G7) with the equilibration of 2,5, 3, and 4 hours. The parameter used in this study was the percentage of sperm motility and live treatment. The results of the 2.5-hour equilibration with a combination of cryoprotectants L60G7 showed the best quality in all parameters. The results of the 3-hour equilibration with combination of cryoprotectants L60G7 showed the best quality in all parameters whereas the best quality in all parameters in 4-hour equilibration was the combination of cryoprotectants L120G7.

Pengaruh Pemberian Susu Skim dengan Pengencer Tris Kuning Telur terhadap Daya Tahan Hidup Spermatozoa Sapi pada Suhu Penyimpanan 5ºC

&lt;p&gt;The objective of the study was to determine the effect of skim milk and Tris yolk&lt;br /&gt;extender on the viability bovine spermatozoa at 5 ºC. This study used Simmental bull semen. The design used was Randomized Design Group (RAK) with 5 treatments and 5 groups of ejaculate. The treatments were: P0 = skim milk 100% without Tris yolk extender; P1 = 95% skim milk + 5% Tris yolk extender, P2 = 90% skim milk + Tris yolk extender 10%, P3 = skim milk 85% + Tris yolk extender 15%, P4 = skim milk 80% + Tris yolk extender 20%. The observed variables were the percentage of live spermatozoa and the motility of spermatozoa after storage for 2 days at 5ºC. Skim milk with Tris yolk extender affect the viability and motility of spermatozoa stored at 5ºC (P&amp;lt;0.01). Provision of extender up to 15% gave the best results and suppressed the decline in the viability of spermatozoa storaged for 2 days at 5ºC.&lt;/p&gt;&lt;p&gt;Key words: skim milk, spermatozoa, Tris yolk extender, viability&lt;/p&gt;

Pengaruh Pemberian Susu Skim dengan Pengencer Tris Kuning Telur terhadap Daya Tahan Hidup Spermatozoa Sapi pada Suhu Penyimpanan 5ºC

&lt;p&gt;The objective of the study was to determine the effect of skim milk and Tris yolk&lt;br /&gt;extender on the viability bovine spermatozoa at 5 ºC. This study used Simmental bull semen. The design used was Randomized Design Group (RAK) with 5 treatments and 5 groups of ejaculate. The treatments were: P0 = skim milk 100% without Tris yolk extender; P1 = 95% skim milk + 5% Tris yolk extender, P2 = 90% skim milk + Tris yolk extender 10%, P3 = skim milk 85% + Tris yolk extender 15%, P4 = skim milk 80% + Tris yolk extender 20%. The observed variables were the percentage of live spermatozoa and the motility of spermatozoa after storage for 2 days at 5ºC. Skim milk with Tris yolk extender affect the viability and motility of spermatozoa stored at 5ºC (P&amp;lt;0.01). Provision of extender up to 15% gave the best results and suppressed the decline in the viability of spermatozoa storaged for 2 days at 5ºC.&lt;/p&gt;&lt;p&gt;Key words: skim milk, spermatozoa, Tris yolk extender, viability&lt;/p&gt;

Efectos de la piridoxina en combinación con diferentes antioxidantes sobre parámetros de viabilidad y estrés oxidativo del semen caprino criopreservado

Se estudiaron los efectos de la piridoxina sola, o en combinación con diferentes antioxidantes, sobre los parámetros de viabilidad y estrés oxidativo de los espermatozoides caprinos criopreservados. Muestras combinadas de semen, diluidas con diluyente yema de huevo-tris se complementaron, en el estudio I con 0, 2, 4, 6 y 8 mM de piridoxina; en el estudio II con 2 mM de piridoxina (P), en combinación con diferentes concentraciones (0, 2, 4, 6 y 8 mM) de vitamina E (PE), vitamina C (PC) o melatonina (PM); y en el estudio III con 2 mM de piridoxina suplementada con combinaciones de vitamina C, vitamina E y melatonina (PCEM) a diferentes concentraciones (0, 2, 4, 6 y 8 mM). Los resultados mostraron mayor (p

The Effect of Canthaxanthin on the Quality of Frozen Ram Spermatozoa.

The addition of antioxidants to semen cryopreservation extenders has been employed for combating oxidative damage. This work aimed to evaluate the addition of carotenoid canthaxanthin to a cryopreservation extender of ram semen. Three breeder rams were used and, after semen collection, with 48-hour intervals between collection, the samples were included in the pool formation (n = 6). The experimental groups comprised 0 (control), 0.1, 1, 10, and 25 μM of canthaxanthin. After thawing (37°C/30 s) and incubation at 37°C for 2 hours, semen aliquots from each group were evaluated for sperm kinetics (CASA), the integrity of the plasma and acrosomal membranes (iPAM), intracellular reactive oxygen species (ROS) production, and lipid peroxidation (LPO) by flow cytometry associated with the image. The control group and canthaxanthin 1 μM after incubation at 37°C for 2 hours showed increases of curvilinear velocity and amplitude of lateral head displacement with decreases of linearity, straightness, and wobble (p < 0.05), which were not observed for the canthaxanthin 10 and 25 μM. The supplementation of a Tris-egg yolk extender with canthaxanthin had no effect on the iPAM, intracellular ROS production in viable spermatozoa, or LPO. In conclusion, supplementation with 10 and 25 μM of canthaxanthin in a Tris-egg yolk extender used for ram semen cryopreservation is able to protect ovine sperm from kinetic changes after incubation at 37°C for 2 hours post-thawing.

Protective effects of Opuntia ficus-indica extract on ram sperm quality, lipid peroxidation and DNA fragmentation during liquid storage

The present study aimed to assess the phenolic composition of the acetone extract from Opuntia ficus indica cladodes (ACTEX) and its effects on ram semen variables, lipid peroxidation and DNA fragmentation during liquid storage at 5°C for up to 72h in skim milk and Tris egg yolk extenders. Semen samples from five rams were pooled extended with Tris-egg yolk (TEY) or skim milk (SM) extenders containing ACTEX (0%, 1%, 2%, 4% and 8%) at a final concentration of 0.8×109 sperm/ml and stored for up to 72h at 5°C. The sperm variables were evaluated at different time periods (8, 24, 48 and 72h). Sperm total motility and viability were superior in TEY than in SM whereas the progressive motility, membrane integrity, abnormality and spontaneous lipid peroxidation were greater in SM compared to TEY (P<0.05). The results also indicated that the inclusion of 1% ACTEX in the SM or TEY extender increased the sperm motility, viability, membrane integrity, and decreased the abnormality, lipids peroxidation up to 72h in storage compared to control group. Similarly, even at 72h of storage, 1% ACTEX can efficiently decrease the negative effects of liquid storage on sperm DNA fragmentation (P<0.05). In conclusion, SM and TEY supplemented with 1% of ACTEX can improve the quality of ram semen. Further studies are required to identify the active components in ACTEX involved in its effect on ram sperm preservation.

Semen supplementation with palmitoleic acid promotes kinematics, microscopic and antioxidative parameters of ram spermatozoa during liquid storage.

The purpose of the present experiment was to investigate the protective effects of palmitoleate on the quality of ram semen during low temperature liquid storage. Ejaculates were collected using the artificial vagina from four Qezel rams twice a week. Ejaculates were pooled, diluted with Tris-egg yolk extender without palmitoleate (control) or supplemented with 0.125 (P 0.125), 0.25 (P 0.25), 0.5 (P 0.5) and 1 (P 1) mM palmitoleate at a final concentration of 500×106 spermatozoa/ml. Total motility and forward progressive motility (FPM) as well as other spermatozoa kinematics were evaluated by computer-assisted sperm analysis. Moreover, viability and membrane functionality were determined in the spermatozoa. Additionally, amounts of malondialdehyde (MDA), total antioxidant activity (AOA), nitric oxide (NO) and superoxide dismutase (SOD) activities were evaluated in the medium and spermatozoa at 0, 24, 48 and 72hr of storage. The palmitoleate supplementation resulted in a significant (p<.05) increase in total motility and FPM with the highest increase at 0.5 mM concentration for 72hr. P 0.5 group also resulted in the highest percentage of membrane-intact spermatozoa (76.60±1.95%) and viability (75.81±1.34%) at 72hr (p<.05). The amounts of MDA and NO were lower in P 0.125, P 0.25 and P 0.5 groups compared to control at 48hr and 72hr (p<.05). Higher amounts of AOA were obtained in palmitoleate-treated groups in medium and spermatozoa during storage time (p<.05). Furthermore, palmitoleate supplementation increased the SOD activities in spermatozoa compared to the control (p<.05). The results of the present experiment reveal that supplementation with 0.5 mM palmitoleate improves ram spermatozoa motion characteristics, AOA levels and SOD activities during liquid storage. Then, palmitoleate could be used as an antioxidant source during liquid storage of ram semen.

IGF1 stabilizes sperm membrane proteins to reduce cryoinjury and maintain post-thaw sperm motility in buffalo (Bubalus bubalis) spermatozoa

Insulin like growth factor 1 (IGF1) in the seminal plasma is reported to improve sperm motility by reducing oxidative stress. The present study was conducted to assess the effect of addition of IGF1 on sperm function and protein composition during cryopreservation process. Semen samples were collected from six Murrah buffaloes (2 ejaculates from each animal) and diluted (80 million/ml) in tris egg yolk extender and divided into control, T1, T2 and T3, groups supplemented with 0, 50, 100 and 150 ng of IGF1/mL, respectively. The semen was filled in straws (250 μL) and straws from each group were divided into two batches. One batch was processed for freezing and another batch was incubated at 4 °C for 4 h. The sperm kinematic and functional parameters were studied in both the batches. A significant (P < 0.05) positive effect of IGF1 was observed on functional membrane integrity (%) during incubation at 4 °C for 4 h in T3 as compared to control group. The spermatozoa (%) positive for structural membrane integrity, mitochondrial membrane potential and the metabolic activity in post-thaw semen were significantly (P < 0.05) high in T3 than the control group. The acrosomal integrity was significantly (P < 0.05) higher in T2 group as compared to control. The proteins (kDa) of 17.3 with pI 4.2 (calmodulin), 11.3 with pI 6.5 (dermcidin) and 18.1 with pI 5.5 (sperm acrosome membrane associated protein3) were protected in IGF1 group. The study suggests that IGF1 can be added to the extender for improving cryosurvial of buffalo spermatozoa.

Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein.

Aim:This study was aimed at evaluating the anti-apoptotic effects of Bcl-2 protein in cryopreserved buffalo bull sperm.Materials and Methods:A total 10 ejaculates from two buffalo bulls (5 each) were collected using artificial vagina method, and semen was evaluated using a standard protocol. Semen was extended by Tris egg yolk extender supplemented with Bcl-2 protein at 5, 10, and 15 µM. Semen was cryopreserved at ultra-low temperature using traditional vapor freezing method. Pre-freeze and post-thaw semen samples were evaluated for percent motility, viability, hypo-osmotic swelling test (HOST) reactive sperms; status of mitochondrial membrane activity and status of sperm phospholipase A1 and phospholipase A2 activity.Results:There were no significant effects of Bcl-2 protein supplementation on pre-freeze sperm quality. Percent motility and active mitochondria in post-thaw Bcl-2 supplemented and control groups were also similar. However, viable sperms were significantly (p<0.05) higher (74.29±4.23%) in Bcl-2 supplemented group (5 µM) as compared to control (51.6±5.77%). The proportion of HOST reactive sperms was also higher (63.1±6.73%) in Bcl-2 supplemented (5 µM) group as compared to control (50.7±6.98%). The sperm with low PLA activity (non-apoptotic) was significantly (p<0.05) higher in all the supplemented doses of Bcl-2 protein, i.e., at 5 µM (73.42±5.79%), 10 µM (75.51±6.22%), and 15 µM (74.78±5.89%) as compared to control (60.23±4.45%). We found that Bcl-2 protein supplementation at 5 µM dose improved the post-thaw semen quality indicated by higher viability, HOST reactive sperms, and sperm with low PLA activity (non-apoptotic sperms).Conclusion:Bcl-2 protein supplementation exerts its protective effect on spermatozoa against apoptosis-like changes developed during cryopreservation.