Tris-egg Yolk Extender Research Articles

Relationship of extender and packaging system an the length of preservation and the quality of chilled semen of Boer goat

<p class="abstrak2">The aim of this research was to compare the effectiveness of different extender (either Triladyl or Tris Egg Yolk extender) and different packaging method (pool and straw) of chilled semen an the length of preservation and the quality of chilled semen of Boer goat. Semen was collected using an artificial vagina from 3 two years old Boer bucks with body weight of 50-55 kg. It was evaluated under a microscope, then each was diluted either in Tris egg yolk extender (TEY) or Triladyl. Those diluted sperms were then packed either in pool or straw and preserved at 5⁰C refrigerator. Sperm motility, viability and membrane integrity of each group were evaluated every 24 h for up to 5 days. Results showed that sperm motility in Triladyl of pool packaging system up to 3 days was higher than straw packaging system or TEY in pool or straw packaging system which were 45.8%, 26.1%, 32.1% and 9.1%, respectively (P<0.05). Percentage of sperm membrane integrity showed the same pattern to Triladyl both in pool and straw packaging system which was higher than TEY group (75.2% and 77,2%; P<0.05). Sperm viability in Triladyl both in pool or straw packaging system decreased (P<0.05) after 3 days of preservation (77.1% and 76.2%) but TEY significanly decreased after 4 days of preservation either in pool or straw packaging system (73.2% and 58.0%; P<0.05). It was concluded that sperm quality decreased with increasing of the length of preservation while Triladyl extender in pool packaging system showed the best quality.</p><strong>Key Words: </strong>Chilled Semen, Boer, Triladyl, Tris Egg Yolk, Straw

Assessment of motion and kinematic characteristics of frozen-thawed Sirohi goat semen using computer-assisted semen analysis.

Aim:The aim was to determine the motion and kinematics characteristic of frozen-thawed spermatozoa in Sirohi goat using computer-assisted semen analysis.Materials and Methods:A study was carried out in Sirohi buck. Semen collection was made biweekly from each buck with the help of artificial vagina. A total of 12 ejaculates were collected from two bucks (six ejaculates from each buck). Freshly collected semen was pooled and later evaluated. The pooled semen sample was extended with standard glycerolated egg yolk tris extender and later subjected to a process of cryopreservation. The motion and kinematic characteristics of spermatozoa were studied during freez-thawing process.Results:Significantly (p<0.01) higher value of live percent, hypo-osmotic swelling test, and acrosomal integrity were recorded in neat semen followed by diluted and frozen thaw semen. The proportion of spermatozoa showing slow progression were the highest in the neat and diluted semen followed by rapid and non-progressively motile, while a reverse pattern was observed in the frozen thaw semen where the proportion of non-progressively motile spermatozoa were significantly (p<0.01) higher followed by slow and rapid progression.Conclusion:This study showed that the best results for motion, vitality, plasma membrane integrity, and acrosome status were obtained in the neat semen followed by diluted and frozen thaw semen. Further, the process of cryopreservation results in a shift of motility from slow to non-progressive in the post-thaw semen with a significant decrease in the path velocities when compared to neat and diluted semen. Hence, it can be concluded that freezing-thawing process reduces the motility and kinematic characters spermatozoa and may be an important factor affecting the fertilizing ability of spermatozoa resulting in poor conception rate after insemination in goats.

Influence of catalase and pre-freezing equilibration on post-thaw semen quality and conception rate in ewes laparoscopically inseminated

The aim of this study was to investigate the effects of catalase and pre-freezing equilibration during ram sperm cryopreservation on motility and membrane and acrosomal integrity of frozen-thawed semen, as well as conception rate following laparoscopic timed-insemination. Semen was collected from four mature Dorper rams, pooled and diluted in Tris egg-yolk extender basic solution (CON), or this solution supplemented with catalase (CAT; 20 U/100 × 106 sperm). Extended semen was packaged in 0.25 ml mini straws (25 × 106 sperm/straw), chilled (to 5°C), and then either frozen immediately (CON and CAT) or maintained at 5°C for 12 h of pre-freezing equilibration (CON12 and CAT12). Immediately after thawing and at 1 h after incubation at 37°C, kinematic parameters (CASA), plasma membrane integrity (PI-FITC), and acrosomal status (FITC-PNA) of sperm were assessed. There were no significant differences among the four groups on sperm traits evaluated immediately post-thaw. However, after 1 h of incubation, total motility (46.7 and 25.0%) and plasma membrane integrity (38.7 and 25.7%) were higher (P < 0.05) in CAT12 than CON. When these two treatments were used for laparoscopic timed artificial insemination of ewes (with synchronized ovulation), conception rate was similar for CAT12 and CON (32.8%, n = 61 vs. 27.3%, n = 55). In conclusion, the combination of catalase and pre-freezing equilibration resulted in significantly improved quality of post-thawed ram semen without affecting conception rate in fixed-time laparoscopically intrauterine inseminated ewes.

Different extenders in the cryopreservation of bovine epididymal spermatozoa

The objective of this study was to evaluate the effects of two different egg yolk extenders incubated with or without Sperm Talp on the motility and plasma membrane integrity of cryopreserved bovine epididymal spermatozoa after freezing. Twenty-five testicles with epididymides from mature bulls were collected at the abattoir. Epididymal sperm recovery was performed by retrograde flushing using a skim milk-extender (Botu-Semen™). After recovery, sperm were incubated either without or with Sperm Talp and then submitted to centrifugation. For the freezing process, half of the testes were processed with Tris egg yolk extender, and half were processed with Botu-Bov™ egg yolk extender. Samples incubated in Sperm Talp exhibited better results than epididymal spermatozoa that were incubated without Sperm Talp (p<0.05). Both Botu-Bov™ and Tris could be utilised to freeze sperm from the bovine epididymides if the sperm were previously incubated with Sperm Talp. The extenders examined in this work did not differ in their effect on plasma membrane integrity after freezing.

Effects of pyridoxine supplementation or in combination with other antioxidants on motility, in vitro capacitation and acrosome reaction of goat buck spermatozoa during cryopreservation

Effects of semen extenders supplemented with pyridoxine or in combination with other antioxidants on motility, in vitro capacitation and acrosome reaction of spermatozoa obtained from West African Dwarf (WAD) goat bucks during cryopreservation were studied. Tris-based extenders supplemented with 2, 4, 6 and 8mM of pyridoxine in study I, and 2mM of pyridoxine (P) supplementation combined with 2mM of vitamin E, vitamin C and melatonin (PE, PC, PM and PCEM) each in study II, were diluted with pooled semen samples. Sperm motility, capacitation and acrosome reaction were assessed following cryopreservation. The results showed higher (P<0.05) sperm motility and acrosome reaction in extenders supplemented with pyridoxine alone or combined with vitamin C, vitamin E and melatonin compared to the control. The improvement in sperm motility was optimal in extenders supplemented with 6mM and PC while the highest percentage of capacitation was observed in extenders supplemented with 4mM, 6mM, PE and PCEM compared to other treatments and the control (P<0.05). The findings revealed that pyridoxine alone at 6mM was consistently effective for improving motility, capacitation and acrosome reaction of cryopreserved spermatozoa in Tris-egg yolk extender of WAD goat bucks during cryopreservation.

Effect of supplementation of butylated hydroxytoluene on post-thaw sperm viability, motility and membrane integrity of Hariana bulls.

Aim:This study was aimed to see the beneficial effect of butylated hydroxytoluene (BHT) as a semen additive of Hariana bull semen.Materials and Methods:The study was carried out in Hariana bulls. Twenty-four ejaculates from two bulls were used for this study. Each ejaculate was extended with standard glycerolated egg yolk tris extender and supplemented with BHT at two concentrations as 0.5 mM (T1) and 1.0 mM (T2). After dilution, equilibration and 24 h of cryopreservation, the samples were analyzed for progressive motility, sperm viability and membrane integrity.Results:Progressive motility, sperm viability and sperm membrane integrity were significantly (p<0.05) increased in the samples fortified with BHT as compared to the control during the process of cryopreservation and thawing. The BHT concentration of 1 mM revealed better results as compared to 0.5 mM.Conclusion:Addition of 1.0 mM BHT was found better in cryopreservation of Hariana bull semen compared to 0.5 mM BHT and control samples. The addition of BHT has improved the sperm quality by acting as an antioxidant thereby reducing the lipid peroxidation of the sperms.

Effect of extracellular adenosine 5′-triphosphate on cryopreserved epididymal cat sperm intracellular ATP concentration, sperm quality, and in vitro fertilizing ability

Intracellular adenosine 5′-triphosphate (ATP) is essential for supporting sperm function in the fertilization process. During cryopreservation, damage of sperm mitochondrial membrane usually leads to compromised production of intracellular ATP. Recently, extracellular ATP (ATPe) was introduced as a potent activator of sperm motility and fertilizing ability. This study aimed to evaluate (1) levels of intracellular ATP in frozen–thawed epididymal cat sperm after incubation with ATPe and (2) effects of ATPe on epididymal cat sperm parameters after freezing and thawing. Eighteen male cats were included. For each replicate, epididymal sperm from two cats were pooled to one sample (N = 9). Each pooled sample was cryopreserved with the Tris–egg yolk extender into three straws. After thawing, the first and second straws were incubated with 0-, 1.0-, or 2.5-mM ATPe for 10 minutes and evaluated for sperm quality at 10 minutes, 1, 3, and 6 hours after thawing and fertilizing ability. The third straw was evaluated for intracellular ATP concentration in control and with 2.5-mM ATPe treatment. Higher concentration of intracellular sperm ATP was observed in the samples treated with 2.5-mM ATPe compared to the controls (0.339 ± 0.06 μg/2 × 106 sperm vs. 0.002 ± 0.003 μg/2 × 106 sperm, P ≤ 0.05). In addition, incubation with 2.5-mM ATPe for 10 minutes promoted sperm motility (56.7 ± 5.0 vs. 53.3 ± 4.4%, P ≤ 0.05) and progressive motility (3.1 ± 0.2 vs. 2.8 ± 0.4, P ≤ 0.05), mitochondrial membrane potential (36.4 ± 5.5 vs. 28.7 ± 4.8%, P ≤ 0.05), and blastocyst rate (36.1 ± 7.0 and 28.8 ± 7.4%, P ≤ 0.05) compared with the controls. In contrast, ATPe remarkably interfered acrosome integrity after 6 hours of postthawed incubation. In sum, the present finding that optimal incubation time of postthaw epididymal cat sperm under proper ATPe condition might constitute a rationale for the studies on other endangered wild felids regarding sperm quality and embryo development.

EFFECT OF MELATONIN ON BUFFALO BULL SPERM FREEZABILITY, ULTRASTRUCTURE CHANGES AND FERTILIZING POTENTIALS

Cryopreservation induces sublethal damage to the spermatozoa, which leads to reduce their fertile life. The objective of the present study was to investigate the effect of melatonin on freezability, ultrastructure and fertilizing potentials of the buffalo spermatozoa. Buffalo spermatozoa were cryopresreved with Tris egg yolk extender containing 7% glycerol supplemented with (0.100, 0.250, 0.500, 0.750 and 1 mM melatonin) or Tris-based extender only (control). Cryopresreved spermatozoa were assessed for post-thawing sperm motility, viability and acrosomal integrity, ultrastructure changes and fertilizing potentials. The current results clearly indicated that adding 0.1 mM melatonin to Tris extender significantly improved (P<0.05) post-thawing sperm motility and viability index following cryopreservation (60.00±2.9% and 157.5±11.8, respectively) compared with the control spermatozoa (45.00±2.90% and 97.00±9.09, respectively). The current results illustrated that addition of 0.1mM melatonin protected the plasma membrane, acrosomal region and mitochondria and maintained the ultrastructure integrity of the cryopresreved spermatozoa compared with the control spermatozoa. The present data indicated that addition of melatonin at a dose of 0.1 and 0.25 mM to the semen extender significant increased (P< 0.05) the positive HOST percent (83.66 and 81.33%, respectively) compared with the control semen (43.33 %). Additionally, the current results revealed that addition of 0.1 mM and 0.25 mM melatonin to the semen extender highly significant increased (P< 0.01) the pregnancy rate (66.67 and 60.43%, respectively) compared with the control semen (45.65%). Therefore, the present results revealed that addition of melatonin to the freezing extender might improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.

Cryopreservation of Semen of Mithun and Siri Bulls

The objective of the study was to assess the semen characteristics of Jersey, Mithun and Siri breeds of bulls and to compare the suitability of Tris Eggyolk Citrate and commercial soya lecithin based BIOXcell extenders for cryopreservation of semen from the these breeds of bulls. The mean semen volume was 4.55± 1.57, 4.86±1.33 and 4.83±1.72 ml in Jersey, Mithun and Siri bulls, respectively. Mean mass activity of 3, mean initial motility of 78.0±4.22, 80±4.47 and 78.33±4.08%, mean sperm concentration of 1416±484.19, 1530.91±517.41, 1122.83±293.68 x106 and mean livability of 69.20±6.81, 70.45±8.42, 76.67±8.17% was found in Jersey, Mithun and Siri bulls, respectively. There is no significant difference in all above mentioned variables among the breeds. The post-thaw motility of semen cryopreserved in Tris Eggyolk Citrate extender was found to be 45.50 ± 4.38, 46.82 ± 6.81 and 47.50 ± 8.22% in Jersey, Mithun and Siri bulls, respectively. Whereas in BIOXcell extender, it was 45.50 ± 5.99, 46.36 ± 5.05 and 49.17 ± 5.85 % in Jersey, Mithun and Siri bulls, respectively. Similarly, the plasma membrane integrity of thawed spermatozoa in Tris eggyolk extender was 17.50±2.32, 21.00±3.12 and 21.58±3.83% in Jersey, Mithun and Siri bulls, respectively. Whereas in BIOXcell extender, it was found to be 20.45±2.86, 20.86±2.281 and 22.33±3.24% in Jersey, Mithun and Siri bulls, respectively. No significant difference (p&gt;0.05) was observed in post-thaw motility as well as in plasma membrane integrity between extenders and between breeds, and there was no significant effect (p&gt;0.05) of breed x extender interaction. It can be concluded that the semen characteristics of Siri and Mithun bulls fall with the range established for other breeds of bulls. Furthermore, both Tris eggyolk and BIOXcell extenders are equally suitable for the cryopreservation of semen from these three breeds of Bulls.DOI: http://dx.doi.org/10.3329/bjvm.v12i2.21277 Bangl. J. Vet. Med. (2014). 12 (2): 143-153

Cryopreservation of semen from domestic livestock.

In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation secures future reproduction, and insemination doses can be easily shipped. Processing of semen for cryopreservation can be done with minimal efforts and relatively low costs. In this chapter we describe the entire cryopreservation process for stallion and bull sperm including dilution of sperm in primary and freezing extender, cooling and packaging in straws, freezing in liquid nitrogen vapor, cryogenic storage, and thawing. Special emphasis is given on preparation of commonly used primary and freezing extenders (skim milk extender "INRA-82", TRIS-egg yolk extender "TEY") used in a two-step dilution approach. Furthermore the different cooling rates needed in different temperature regimes during the freezing process are being described. Cryopreservation procedures are described in case of using both specialized automated equipment and simple equipment.

'Soya milk Tris-based phytoextender reduces apoptosis in cryopreserved buffalo (Bubalus bubalis) spermatozoa'.

The objective of this study was to investigate the effect of newly developed soya milk Tris (SMT)-based phytoextender as an alternative to egg yolk Tris (EYT) extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of apoptotic proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS-PAGE followed by immunoblotting against caspase-8, caspase-9, caspase-3, poly(ADP-ribose)polymerase (PARP), cytochrome c and apoptosis inducing factor (AIF). In addition, fluorescence microscopy was used for the detection of mitochondrial membrane potential (JC-1 assay) and apoptotic cells (annexin V-FITC/PI assay). The results obtained clearly indicate the significant (p<0.05) reduction in the expression of caspase-3 (27kDa), caspase-8 (53kDa), caspase-9 (50kDa) precursor and cytochrome c (17kDa) in semen cryopreserved in SMT extender in comparison with EYT extender. A non-significant (p>0.05) reduction in expression of PARP-DNA-binding subunit (24kDa) was observed in SMT extender. No expression of AIF was found in cryopreserved semen samples. A significant (p<0.05) increase in the mean percentage of cells having high mitochondrial membrane potential and a non-significant (p>0.05) decrease in late apoptotic cells (AN+/PI+) was observed in SMT extender when compared to EYT extender. The results demonstrated that cryopreservation of buffalo semen in SMT-based phytoextender can replace the traditional egg yolk extenders as it reduces the expression of apoptotic proteins maintaining high mitochondrial membrane potential and gives better protection to sperms in terms of its non-animal origin.

EFFECT OF TREHALOSE, CYSTEINE AND HYPOTAURINE ON BUFFALO BULL SPERM FREEZABILITY, ULTRASTRUCTURE CHANGES AND FERTILIZING POTENTIALS

Cryopreservation induces sublethal damage to the spermatozoa, which leads to reduce their fertile life. The objective of the present study was to investigate the effect of trehalose, cysteine and hypotaurine on freezability, ultrastructure and in vitro fertilizing potentials of the buffalo spermatozoa. Buffalo spermatozoa were cryopresreved with Tris egg yolk extender containing 7% glycerol suplemmented with100 mM trehalose, 100 mM trehalose + 5 mM cysteine, 100 mM trehalose + 25 mM hypotaurine, 100 mM trehalose + 5 mM cysteine + 25 mM hypotaurine or Tris-based extender only (control). Cryopresreved spermatozoa were assessed for post-thawing sperm motility; viability and acrosomal integrity, ultrastructure changes, biochemical activity and in vitro fertilizing potentials. The current results clearly indicated that adding a mixture of 100 mM trehalose , 5 mM cysteine and 25 mM hypotaurine to Tris extender significantly improved (P<0.05) post-thawing sperm motility, viability index and maintained acrosomal integrity following cryopreservation (63.33±7.27%, 155.83±21.06and 12.33±2.73%, respectively) compared with the control spermatozoa (38.33±4.41%, 94.17±12.28 and 25.33±3.49%, respectively). The current results illustrated that addition of combination of additives protected the plasma membrane, acrosomal region and mitochondria and maintained the ultrastructure integrity of the cryopresreved spermatozoa compared with the control spermatozoa. Additionally, the current results revealed that combination of trehalose, cysteine and hypotaurine significantly increased the total antioxidant capacity level (TAC), superoxide dismutase activity (SOD) and glutathione reductase activity (GSH) and reduced significantly the lipid peroxidation rate (0.60±0.89 mµ/ml, 70.00 ±5.78 U/L, 81.66±4.41 U/L and 7.33±1.86 nmol/ml, respectively) compared with the control extender (0.23±0.04 mµ/ml, 34.67±6.74U/L, 51.67±14.82 U/Land 20.67±3.84 nmol/ml, respectively). Furthermore, a mixture of trehalose, cysteine, hypotaurine significantly improved (P<0.05) the rate of in vitro fertilization, cleavage and embryo development to morula and blastocyst stages (62.66±6.28, 53.54±3.89, 23.87±6.28 and17.94±2.57%) compared with the control extender (31.19±4.42, 18.47±5.04, 8.36±1.91 and .42±1.71%, respectively). Therefore, the present results revealed that addition of a mixture of trehalose, cysteine and hypotaurine to the freezing extender could improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.

Effect of rosemary (Rosmarinus officinalis) extracts and glutathione antioxidants on bull semen quality after cryopreservation

The present study determined the effects of the addition of rosemary extract (ROM), glutathione (GSH), and their combination (ROM + GSH) to freezing extender on the quality of bull semen after cryopreservation. Before cryoperservation, the samples were diluted in a tris-egg yolk (TEY) extender containing 5 mM GSH (treatment I), 5 or 10 g L–1 ROM (treatments II and III), and ROM with GSH (5 mM GSH with 5 or 10 g L–1 of ROM) (treatments IV and V). An extender containing no antioxidants (non-ROM/GSH-treated) served as control group. Kinematic parameters were evaluated by means of a computer-assisted semen analysis (CASA). The viability and membrane integrity of the sperm were assessed using eosin-nigrosin stain and the hypo-osmotic swelling test (HOST) at 0 and 2 h after freezethawing. Lipooxidative parameters, superoxide dismutase, and glutathione peroxidase (GPx) activity were assessed after thawing. Treatment III showed positive effects for total motility (TM) (p &lt; 0.01), average path velocity (VAP) (p &lt; 0.001), viability (p &lt; 0.01) and HOST (p &lt; 0.01); however, lipid peroxidation (LPO) decreased (p &lt; 0.05) and GPx activity increased (p &lt; 0.05) immediately after thawing compared to the control. The TM (p &lt; 0.01), VAP (p &lt; 0.01), viability (p &lt; 0.01), HOST (p &lt; 0.01) decreased in LPO (p &lt; 0.01) and GPx activity (p &lt; 0.05) for treatment V and the viability and GPx activity (p &lt; 0.05) for treatment I were significantly higher than for the control group at 2 h after thawing. It was concluded that the inclusion of ROM and its combination with GSH improves the post-thaw quality of bull semen.

The effect of Equex STM® in freezing media on post thaw motility, viability and DNA integrity of frozen-thawed ram spermatozoa.

In this study we investigated the effect of Equex STM® on quality and in-vitro survival of ram spermatozoa frozen in Tris egg yolk based extender. Ejaculates from 6 crossbreed rams were frozen according to the standard procedure after two step dilution with Tris-egg yolk extender (1). The second extender, added to the semen at 5°C, contained 14% of glycerol and was supplemented with detergent 0.75% Equex STM® (group OEP) or contained no detergent (control group). After thawing the samples were incubated in a water bath at 37°C and analysis were performed 10 minutes, 6, 12 and 24 hours later. Motility and the viability (Viadent®) of the semen were analysed with Hamilton Thorne Biosciences, Version 12.3 and membrane integrity with HOS (hypoosmotic swelling test). DNA fragmentation (DFI %) of F/T spermatozoa was analyzed 10 minutes and 3 hours after thawing using sperm chromatin structure assay (SCSATM). The sperm membrane integrity was analysed 15 minutes and 3 hours after thawing by Sybr-14/PI test. Percentage of motile spermatozoa was significantly higher in OEP group in comparison to control group at 0, 6, 12 and 24 h (P<0.001). Viability of spermatozoa was significantly higher (P<0.001) in OEP compared to control group in all analysed times after thawing. Percentage of HOS positive spermatozoa was significantly higher in OEP compared to control group respectively for 0 (P=0.001), 6 (P=<0.001), 12 and 24 h (P=0.002) after thawing.

Cryo-survival, cryo-capacitation and oxidative stress assessment of buffalo spermatozoa cryopreserved in new soya milk extender

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 (100mM) or taurine (50mM) 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.

Post-thaw quality of buck semen samples cooled at 5 °C up to 2 days before cryopreservation

This study aimed to assess the influence of cool storage (5°C) prior cryopreservation over the post-thaw quality of buck semen samples. Semen of six Majorera bucks (n=18 ejaculates) was collected, pooled and diluted in a Tris-yolk extender. Then, diluted semen was divided into six aliquots; the first aliquot (group C) was processed and frozen in liquid nitrogen (final concentration of 400×106spermatozoa/mL, 12% egg-yolk and 4% glycerol). The remaining aliquots (diluted with Tris–glucose, 12% egg yolk) were hold for 1–48h at 4°C: R1, the semen was cooled for 1h; R6, the semen was cooled for 6h; R12, the semen was cooled for 12h; R24, the semen was cooled for 24h and R48, the semen was cooled for 48h. After each cooling period, a second extender was added to reach a final composition (400×106spermatozoa/mL, 12% egg-yolk and 4% glycerol) similar to group C; finally, semen was packed and frozen in liquid nitrogen. After freezing–thawing, the sperm motility, acrosome integrity and the percentage of abnormal spermatozoa were assessed. No differences (P>0.05) were detected in progressive sperm motility (mean range: 35.4–39.9%) and damage acrosomes percentages (mean: 10.8–15.5%) among the control group and the cooled semen samples (R1, R6, R12, and R24) for up to 24h; however, R48 samples showed a lower (21.6%, P<0.01) progressive fast spermatozoa percentage and a higher percentage of damage acrosome (38.3%, P<0.01) than those observed in the control group and in R1, R6, R12 and R24 samples. The present study confirmed that buck semen could be preserved at 5°C for up to 24h before freezing; however, after 2 days of chilling, semen quality experienced a notable decrease and its utility could be lower.

제주흑우 정액 동결을 위한 AndreoMed와 Triladyl 희석제의 동결성 비교

This study was carried out to investigate synthetic extender for semen cryopreservation of Jeju Native Black Bull. The semen was collected using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by Tris-Egg yolk extender and contrifuged in 1,500 rpm for 15 minutes. The supernatant was removed. The pellect was diluted to final sperm concentration of 2×108/ml by doubling in every 30 minutes at 4℃ cold chamber. The semen was equilibrated for 4 hours at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm for 10 minutes. The height and duration affect the freezing speed by temperature. The frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at 37℃ water bath. Frozen-thawed sperm were evaluated sperm viability, membrane integrity and acrosome integrity. Post-thawed sperm viability has been significantly higher (p<0.05) in fresh sperm (93.27±1.62%) than frozen-thawed sperm (73.34±3.27%). However, there were no significant differences between fresh and frozen-thawed dead cell rate (7.35±2.63 vs, 13.71±2.85). In sperm motility, between Triladyl and AndroMed Extender, there was no significant different (72.86±2.83 vs, 81.47±2.48), similarly, the dead cell rates was similar (18.41±3.42% and 17.26±4.25). The results of our study suggest that AndroMed to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Jeju Native Black bull semen.

CHARACTERIZATION AND CRYOPRESERVATION OF SEMEN FROM ENDANGERED MARKHOR GOATS (CAPRA FALCONERI HEPTNERI) WITH EVALUATION OF REPRODUCTIVE SEASONALITY

The aims of this study were to determine the reproductive seasonality of four captive markhor goats (Capra falconeri heptneri), to characterize semen collected by electroejaculation, and to compare extenders and processing techniques for semen cryopreservation. Over the course of 1 yr, mean monthly scrotal circumference, serum testosterone, and fecal testosterone were measured and found to be inversely associated with day length. Maximum scrotal circumference (25.2 +/- 0.9 cm), serum testosterone (521.0 +/- 103.4 ng/dl), and fecal testosterone (382.5 +/- 90.3 ng/g) occurred in November, when day length was short (9.7 +/- 0.1 hr). Once a month for 3 mo (December, January, and February), bucks were anesthetized for electroejaculation and semen evaluation. Semen samples were divided into six aliquots for extension and cryopreservation in soy-based Bioxcell or Tris-based extender with 5 or 15% egg yolk, with and without centrifugation. Samples were then thawed for repeat evaluation 1-3 mo later. Postthaw evaluation revealed no significant differences between centrifuged and noncentrifuged samples. Sperm in Tris 5% and 15% egg yolk displayed higher total motility at 0, 3, and 6 hr postthaw and higher progressive motility postthaw compared with sperm in Bioxcell (P < 0.05). Sperm in Bioxcell displayed higher viability than sperm in both Tris-egg yolk extenders (P < 0.01), more intact acrosomes than sperm in Tris-15% egg yolk (P < 0.05), and a tendency for more intact acrosomes than sperm in Tris-5% egg yolk (P < 0.10). Sperm in Tris-5% egg yolk tended to have a higher percentage of morphologically normal sperm compared with Bioxcell (P < 0.10). This study provides evidence that markhor goats exhibit seasonality in scrotal circumference and testosterone levels and that centrifugation may be eliminated from the processing of markhor semen.

Effect of cysteine and glutamine added to extender on post-thaw sperm functional parameters of buffalo bull

Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris-egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post-thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen-thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen-thawed spermatozoa.

Effects of green tea polyphenol on the quality of canine semen after long-term storage at 5 °C

The aim of the present study was to determine the effects of green tea polyphenol on the quality of canine semen after long-term storage at 5°C. The supplementation of a Tris-egg yolk extender with polyphenol (0.5, 0.75, or 1mg/mL) increased the motility and viability of sperm preserved for four weeks at 5°C.