scholarly journals A Comparison of Freezing Methods and Diluents Types on Post-Thaw Sperm Quality of Ram Sperm

2020 ◽  
Vol 7 (2) ◽  
pp. 235-241
Author(s):  
Pankaj Kumar Jha ◽  
M Golam Shahi Alam ◽  
Farida Yeasmin Bari
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Sperm Parameters ◽  
Plasma Membrane Integrity ◽  
One Step ◽  
Acrosome Integrity ◽  
Ram Sperm

The effect of freezing methods and diluents types on post-thaw sperm quality of Bangladeshi ram semen was studied. Two freezing methods and three diluents was tested as pooling effects (freezing methods or diluents) on post-thaw sperm parameters; sperm motility (SM), viability (SV), plasma membrane integrity (SPMI) and acrosome integrity (SAI), respectively. From selected ten rams, eight ejaculates were used for each freezing group (freezing methods × diluents). Semen samples were diluted by using two-steps for hand-made tris-based diluents (20% egg yolk): D1 (7% glycerol) and D2 (5% glycerol), and one-step dilution for commercial diluents: D3 (Triladyl®) at 35°C. After 4h of equilibration of temperature at 5°C, diluted semen samples was aspirated into 0.25 mL straws, and sealed. Straws were frozen in liquid nitrogen (LN2) vapour using two methods: F1 (manually in Styrofoam box, using three-steps method; +5°C to -80°C at -11.33°C/min, -80°C to -120°C at -26.66°C/min, and -120°C to -140°C at - 13.33°C/min) and F2 (programmable bio-freezer, using two-steps method; +5°C to -100°C at - 20°C/min and -100°C to -140°C at -10°C/min). Two semen straws from each batch were evaluated (37°C for 20 sec) for sperm parameters. In pool effects between freezing methods; SAI differed significantly (P < 0.001). The SM (56%) and SV (72%) were observed competitive. However, SPMI (67.58 ± 2.02%) and SAI (76.13 ± 1.42%) were higher in F1. Among diluents, SM (P < 0.006), SV (P < 0.008), SPMI (P < 0.012) and SAI (P < 0.019) differed significantly. The SM (61.25 ± 1.80%), SV (77.13 ± 1.47%), SPMI (68.31 ± 1.91%) and SAI (74.75 ± 1.64%) were highest in D3. In conclusion, the combination of manual freezing (three-steps) and handmade tris-based diluents (20% egg yolk, 5% glycerol) is suitable and sustainable method for cryopreservation of ram semen. Res. Agric., Livest. Fish.7(2): 235-241,  August 2020

scholarly journals QUALITY OF CHILLED CANINE SEMEN IN TRIS-EGG YOLK EXTENDER SUPPLEMENTED WITH SERICIN

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Kim Chwin Khye ◽  
Tuty Laswardi Yusuf ◽  
Faisal Amri Satrio ◽  
Ni Wayan Kurniani Karja
Keyword(s):  
Plasma Membrane ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Plasma Membrane Integrity ◽  
Spermatozoa Motility ◽  
Acrosome Integrity

                                                                 ABSTRACTThe objective of this research was to evaluate the quality of chilled canine semen in Tris-egg yolk (TEY) extenders containing different concentrations of sericin. Semen were collected from four dogs by massage method. Canine semen was collected using sterile urine pots and evaluated. Sperm-rich fractions were pooled and divided into four equal aliquots, which were then diluted with TEY extenders supplemented with different concentrations of sericin (0%, 0.1%, 0.25%, 0.5%). The diluted semen aliquots were preserved at 4 ℃ in sterile centrifuge tubes and were then evaluated for spermatozoa motility, viability, plasma membrane integrity and acrosome integrity every 12 hours up to 72 h. The TEY extenders supplemented with 0.25% and 0.5% sericin resulted in higher spermatozoa motility and viability at 72 h compared to other TEY extenders (P0.05). The integrity of plasma membrane and acrosome of spermatozoa showed no significant differences among the groups extenders at 72 h. In conclusion, sericin in concentration of 0.25% and 0.5% were able to prevent the motility and viability of canine spermatozoa after storage for 72 h.

scholarly journals Efficiency of Commercial Egg Yolk-Free and Egg Yolk-Supplemented Tris-Based Extenders for Dromedary Camel Semen Cryopreservation

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 999 ◽  
Author(s):  
Ayman Abdel-Aziz Swelum ◽  
Islam M. Saadeldin ◽  
Hani Ba-Awadh ◽  
Mohsen G. Al-Mutary ◽  
Abdullah F. Moumen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Dromedary Camel ◽  
Plasma Membrane Integrity ◽  
Semen Cryopreservation

This study compared the efficiency of commercial egg yolk-free (AndroMed, OPTIXcell) and egg yolk-supplemented (Triladyl, Steridyl) Tris-based extenders for semen cryopreservation in seven adult dromedary camels. The camel-specific extender SHOTOR was used as control. The collected semen samples were evaluated and diluted with SHOTOR, Triladyl, Steridyl, AndroMed, or OPTIXcell. The diluted semen was gradually cooled and equilibrated for two hours before liquid nitrogen freezing. Semen was evaluated prior to freezing and after freeze-thawing cycles for motility, kinetics, vitality, abnormality, plasma membrane integrity, and DNA fragmentation using computer-assisted sperm analysis. In pre-freezing evaluation, progressive sperm motility was higher in SHOTOR-diluted samples (21.54 ± 1.83) than in samples diluted with Steridyl, OPTIXcell, or AndroMed (15.76 ± 1.80, 17.43 ± 1.10, and 13.27 ± 1.07, respectively). Moreover, Triladyl and SHOTOR resulted in significantly (p < 0.05) better sperm vitality and DNA integrity than all other diluents, but Triladyl resulted in a significantly (p < 0.05) better plasma membrane integrity (87.77 ± 0.31) than SHOTOR (85.48 ± 0.58). In the post-thawing evaluation, Triladyl led to significantly (p < 0.05) higher sperm motility (38.63 ± 0.81%; p < 0.05) when compared to SHOTOR, Steridyl or AndroMed (35.09 ± 1.341%, 34.4 ± 0.84%, and 31.99 ± 1.48%, respectively), with OPTIXcell being the least efficient (28.39 ± 0.86%). Progressive sperm motility was the highest when using Triladyl. Post-thawing curvilinear, straight line and average path sperm velocities were highest with Triladyl and lowest with AndroMed. Triladyl led to the highest linearity coefficient and straightness sperm coefficient, while SHOTOR to the highest DNA and plasma membrane integrity. OPTIXcell and AndroMed resulted in poor post-thawing sperm vitality, while Steridyl was less efficient than Triladyl. The highest rate of sperm abnormalities was recorded with OPTIXcell and the lowest with SHOTOR or Triladyl. In conclusion, SHOTOR, Triladyl, Steridyl, AndroMed, and OPTIXcell can all be used for camel semen cryopreservation; however, SHOTOR and Triladyl provided the best post-thawing sperm quality. Based on our findings, Triladyl is the best commercially available extender for dromedary camel semen cryopreservation to date.

scholarly journals Butaphosphan and Cyanocobalamin Supplementation in Semen Extender on Chilled Boar Sperm Quality and Life Span

2020 ◽  
Vol 7 ◽  
Author(s):  
J. Suwimonteerabutr ◽  
S. Chumsri ◽  
P. Tummaruk ◽  
Morakot Nuntapaitoon
Keyword(s):  
Plasma Membrane ◽  
Life Span ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Control Group ◽  
Plasma Membrane Integrity ◽  
Progressive Motility ◽  
Boar Sperm ◽  
Acrosome Integrity ◽  
Semen Extender

The objective of the present study was to determine the effect of butaphosphan and cyanocobalamin supplementation in semen extender on chilled boar sperm quality and life span. A total of 35 ejaculates of boar semen were included. The semen was diluted with Beltsville thawing solution extender supplemented with different concentrations of butaphosphan and cyanocobalamin [0 (control), 0.1, 0.2, 0.3, 0.4, and 0.5%] in the diluted semen. The semen samples were evaluated using a computer-assisted sperm analysis system to determine sperm motility and sperm kinetic parameters (i.e., the curvilinear velocity, VCL; straight line velocity, VSL; average path velocity, VAP; linearity, LIN; straightness, STR; amplitude of lateral head, ALH; wobble, WOB; and beat cross frequency, BCF). Additionally, sperm viability, acrosome integrity, mitochondrial activity, and plasma membrane integrity were evaluated after 4 (day 0), 72 (day 3), 120 (day 5), and 168 (day 7) h of storage using SYBR-14–ethidium homodimer-1 (EthD-1), EthD-1, JC-1, and the short hypo-osmotic swelling test, respectively. The analyses were carried out by using the general linear mixed model (MIXED) procedure of SAS. The statistical models for each data set included group, day after storage, and interaction between group and day after storage. The boar was included as a random effect. On day 0 after storage, progressive motility, VCL, VSL, VAP, and plasma membrane integrity of boar sperm in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the 0.4 and 0.5% groups (P &lt; 0.05). On day 3 after storage, total motility and progressive motility, VCL, VSL, VAP, LIN, WOB, BCF, and plasma membrane integrity in 0.3% of butaphosphan and cyanocobalamin supplementation were significantly greater than those in the control group (P &lt; 0.05). The total motility and progressive motility, VAP, and WOB in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 5 after storage (P &lt; 0.05). No effects of butaphosphan and cyanocobalamin supplementation on acrosome integrity and mitochondria activity were found on days 3, 5, and 7 after storage. However, the motility and progressive motility and the values for all sperm kinetic parameters except ALH in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 7 after storage (P &lt; 0.05). In conclusion, 0.3% of butaphosphan and cyanocobalamin supplementation in semen extender improved sperm motility, sperm activity, morphology, and life span in chilled boar sperm.

Cryoprotective Effect of Glycerol Concentrations on Indian Red Jungle Fowl (Gallus Gallus Murghi) Spermatozoa

2018 ◽  
Vol 11 (2) ◽  
pp. 80-88 ◽  
Author(s):  
Bushra Allah Rakha ◽  
Muhammad Sajjad Ansari ◽  
Shamim Akhter ◽  
Elisabeth Blesbois
Keyword(s):  
Plasma Membrane ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Gallus Gallus ◽  
Recovery Rates ◽  
Plasma Membrane Integrity ◽  
Red Jungle Fowl ◽  
Frozen Semen ◽  
Acrosome Integrity

Semen cryopreservation protocols for wild avian species need to be optimised in order to achieve optimum post-thaw sperm quality and fertility. The present study was designed to evaluate the cryoprotective effect of different glycerol concentrations (11%, 15% and 20%) on post-thaw quality, recovery rates, absolute livability index and fertility of Indian Red Jungle Fowl (Gallus gallus murghi) semen. Semen was collected from eight mature cocks and cryopreserved for storage at −196 °C. Frozen semen was thawed at 37 °C for 30 s and assessed for motility, plasma membrane integrity, viability and acrosome integrity at 0, 2 and 4 h incubation at 37 °C. Percentages of motility, plasma membrane integrity, viability and acrosome integrity were recorded higher (P<0.05) post-thaw at 0, 2 and 4 h at 37 °C with 20% glycerol compared to 15% and 11% glycerol. Likewise, recovery rates (%) of aforementioned parameters after cryopreservation and absolute livability index were observed highest (P<0.05) with 20% glycerol. By comparing values of R2 after multivariate regression analysis, least negative effects of hours of incubation were observed on semen quality in extenders with 20% glycerol followed by 15% and 11% glycerol. The fertility outcomes (number of fertile eggs, fertility [%], number of hatched chicks, percent hatch and hatchability of fertilised eggs) were recorded higher (P<0.05) with 20% glycerol followed by 15% and 11% glycerol. It is concluded that the concentration of 20% glycerol gives the best cryoprotection for quality and fertility of Indian Red Jungle Fowl semen.

scholarly journals Quality of Sexed Sperm of Bali Bull in Regional Artificial Insemination Center of Riau Province

2019 ◽  
Vol 43 (2) ◽  
Author(s):  
Yendraliza Yendraliza ◽  
Anwar Efendi Harap ◽  
July Handoko ◽  
Muhammad Rodiallah
Keyword(s):  
Plasma Membrane ◽  
Pregnancy Rate ◽  
Artificial Insemination ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Female Offspring ◽  
Plasma Membrane Integrity ◽  
Frozen Semen ◽  
Calving Rate

This study aimed to evaluate the quality of frozen semen of Bali bull resulted from sexing procedure on calf or offspring production with desired sex. The tested sperm of Bali bull were collected from Bali bull raised at Regional Artificial Insemination Center of Riau Province (BIBD Riau). The study was carried out in 2 stages. The first stage was X and Y chromosome separation by albumin method. The extender used in the sexing procedure is trice citrate fructose and egg yolk. The second stage was mainly testing the sexed sperm collected in 60 Bali cow in Langkat Village, Bengkalis Regency. To determine the quality of post thawing frozen semen collected from the sexing procedure, the study evaluated motility, viability, mortality, abnormality and plasma membrane integrity of the spermatozoa. The pregnancy rate, calving rate, and birth accuracy of inseminated sexed sperm to offspring’ sex were also evaluated. The evaluation resulted in motility (66.3-75.3%), viability (70-78.5%), plasma membrane integrity (60-65.8%), abnormality (6.05-8.05%), mortality (20.05-30.05%), and pregnancy rate (83.33-90%). The calving rate on this study was 100% with the birth accuracy of 81.8% for male offspring and 40% for female offspring. As conclusion, the sexed sperm evaluated on this study have fairly good fertility.

scholarly journals Changes in motility, morphology, plasma membrane and acrosome integrity during stages of cryopreservation of buck sperm

2014 ◽  
Vol 85 (1) ◽  
Author(s):  
Mushtaq Ahmad ◽  
Rashad Nasrullah ◽  
Hasan Riaz ◽  
Abdul Sattar ◽  
Nasim Ahmad
Keyword(s):  
Plasma Membrane ◽  
Sperm Morphology ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Structure And Function ◽  
Freezing And Thawing ◽  
Plasma Membrane Integrity ◽  
Acrosome Integrity ◽  
Live Sperm ◽  
And Function

Changes in sperm structure and function occur during the processing of semen. The present study was designed to investigate the effect on buck sperm during different stages of semen preparation including dilution, cooling, equilibration and freeze-thawing. Semen ejaculates from three mature bucks (replicates = 5) were diluted with tris-citric acid egg yolk glycerol extender at 37 ºC, cooled to 4 ºC over 90 min, equilibrated at 4 ºC for 2 h, transferred to 0.5 mL straws, placed in nitrogen vapour, frozen and thawed and then analysed. Sperm samples were assessed for percentage motility, acrosomal and plasma membrane integrity, live sperm, and morphology after dilution, cooling, equilibration and thawing. Mean percentage motility after dilution (86.0 ± 1.4%) was reduced significantly (p < 0.05) due to cooling and equilibration (77.6 ± 1.3% and 74.6 ± 1.4% respectively); furthermore, it decreased significantly (p < 0.05) after freezing and thawing (42.3 ± 2.5%). Mean percentage of live sperm was higher (p < 0.05) after dilution (89.3 ± 1.4%)compared with cooling (84.8 ± 1.8%) and equilibration (80.2 ± 2.5%) and further reduced (p < 0.05) after freezing and thawing (56.0 ± 3.4%). Sperm morphology dropped significantly (p < 0.05) from 96.4 ± 0.3% after dilution to 88.8 ± 1.3% at cooling and further decreased (p < 0.05) after freezing and thawing (81 ± 1.9%). Mean percentage of sperm with normal plasma membrane after dilution (82.2 ± 1.1%) was significantly reduced (p < 0.05) at cooling or equilibration (73.8 ± 1.8) and further decreased (p < 0.05) after freezing and thawing (50.1 ± 2.9%). The percentage of sperm with normal acrosomes did not differ significantly due to dilution, cooling or equilibration (85.8 ± 1.7%, 83.2 ± 1.6%, 81.7 ± 1.8%) but was significantly reduced after freezing and thawing (45.2 ± 2.8%). In conclusion, frozen thawed sperm showed maximum damage to motility, morphology, plasma membrane and acrosome integrity following cooling.

scholarly journals Effect of Incubation Time During Sperm Sexing Process on Sperm Quality of Pasundan Bull

2020 ◽  
Vol 25 (3) ◽  
pp. 112
Author(s):  
Siti Darodjah Rasad ◽  
Nurcholidah Solihati ◽  
Kikin Winangun ◽  
Annisa Yusrina ◽  
Fahmy Avicenna
Keyword(s):  
Plasma Membrane ◽  
Incubation Time ◽  
Lower Layer ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Dna Integrity ◽  
Test Parameter ◽  
Plasma Membrane Integrity ◽  
Randomized Design

The research was conducted to evaluate the effect of incubation time on viability, plasma membrane integrity, abnormality, and DNA integrity of sexed Pasundan’s bulls sperm. The sperm sexing used 5% and 10% concentrations of Bovine Serum Albumin (BSA).  A completely randomized design with three treatments and six replications was used in this study. The data were analyzed using variance analysis followed by Duncan’s multiple distance test.  Parameter evaluated were sperm longevity, plasma membrane integrity (PMI), abnormality, and DNA integrity of sexed Pasundan bulls sperm. Results showed that incubation time gave significant effect (P&lt;0.05) on the longevity of sperm, but not on the PMI of Pasundan bulls sexed sperm.  The incubation time of 45 minutes gave the highest value of longevity sperm on the upper layer (4.33 days) and the lower layer (4.17 days). Furthermore, the abnormality of sperm X in the upper layer was 4.00%-4.20% and the lower layer was 4.10%- 4.40%.  Meanwhile, the DNA integrity of an upper layer was 98.16%-98.66%, and the lower layer was 97.83%-98.58%.  It is concluded that 45 minutes of incubation time significantly affected the longevity of sperm, but not plasma membrane integrity, abnormality, and DNA integrity of Pasundan bulls sexed sperm.

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

2020 ◽  
Vol 32 (2) ◽  
pp. 145
Author(s):  
C. Souza ◽  
F. Brandão ◽  
J. Santos ◽  
V. Alfradique ◽  
V. Brair ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Young Scientist ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Intact Cells ◽  
Plasma Membrane Integrity ◽  
Head Displacement ◽  
Thawing Process ◽  
Semen Extender ◽  
Ram Sperm

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

Effect of different concentrations of trehalose and glycerol on the freezability of ram semen using soybean lecithin-based diluents

2015 ◽  
Vol 55 (5) ◽  
pp. 666 ◽  
Author(s):  
Zh. Bohlool ◽  
M. Mohammadi ◽  
M. Roostaei-Ali Mehr ◽  
N. Ghavi Hossein-Zadeh
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Soybean Lecithin ◽  
Membrane Integrity ◽  
Sperm Viability ◽  
Plasma Membrane Integrity ◽  
Main Effect ◽  
Acrosome Integrity ◽  
Ram Sperm ◽  
Desirable Effect

This study was conducted to determine the effect of different levels of trehalose and glycerol on ram sperm cryosurvival using lecithin-based diluents. Ejaculates were collected from four rams, pooled after initial evaluation, diluted with Tris-soybean lecithin extender and split into nine equal parts. A total of 0 (T0), 50 (T50) or 100 (T100) mM of trehalose and 3% (G3), 5% (G5) or 7% (G7) of glycerol were added to each part. Sperm motility, viability, plasma membrane integrity and acrosome integrity were evaluated immediately after thawing (0 h), and subsequently after 3 h and 6 h post-thawing incubation at 37°C. Results indicated that there was interaction between trehalose and glycerol on sperm motility. In addition, interaction of trehalose and glycerol with incubation time on sperm motility, viability, plasma membrane integrity and acrosome integrity was not significant (P > 0.05). Sperm motility was greatest in the sperm treated with 100 mM trehalose and 7% glycerol (27%; P < 0.05). The effect of trehalose was significant on viability and plasma membrane integrity of ram spermatozoa (P < 0.05). The main effect of trehalose showed that sperm viability was higher in T100 (47.06%) than T50 (53.96%; P < 0.05). The highest membrane integrity was observed in T100 (47.04%; P < 0.05). Membrane integrity was higher (P < 0.05) in G5 (49.97%) than G3 (41.56%) and there was no difference between G7 (46.86%) and G3 (41.56%; P > 0.05). The best sperm viability and plasma membrane integrity was observed at 0 h (65.75% and 51.58%, respectively). It was concluded that simultaneous use of 7% glycerol and 100 mM trehalose had a desirable effect on motility of ram frozen–thawed sperm.

208EFFECT OF REFRIGERATION AND CRYOPRESERVATION ON THE QUALITY OF LION EPIDIDYMAL SPERMATOZOA

2004 ◽  
Vol 16 (2) ◽  
pp. 225 ◽  
Author(s):  
A.F. Malo ◽  
F. Martinez-Pastor ◽  
F. Olivier ◽  
T. Spies ◽  
E.R.S. Roldan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Osmotic Shock ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Storage Period ◽  
Egg Yolk ◽  
Plasma Membrane Integrity ◽  
Sperm Plasma Membrane ◽  
Epididymal Spermatozoa

Epididymal spermatozoa from harvested wild animals is potentially useful for conservation purposes, as it can be used for subsequent artificial insemination or stored in Biological Resource Banks for future use. The potential of sperm banking is of particular interest for use in lion (Panthera leo) populations maintained in small National Parks, as translocation of males to effect gene-flow is often problematic, resulting in the translocated lion being killed by resident pride males. We measured the change in sperm quality over time during cool storage (at 4°C) and after thawing of samples cryopreserved at −196°C. Also, we present a correlation between sperm plasma membrane integrity and mitochondrial activity as measured by fluorescent analysis. The testes from a pride lion were removed and transported to the laboratory (at 4°C) within 6h. The epididymides were removed and both cauda epididymides were flushed with 1mL of Tris-citrate egg yolk extender (Fraction A, Biladyl, Minitub, Germany). The sample containing 2930×106 cells mL−1 was washed (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH;; 400mOsm/kg, pH 7; Sigma, South Africa) and after centrifugation (5min. at 600g), the pellet was resuspended in 0.5mL of washing solution (with 197mM NaCl instead of sucrose). One aliquot of spermatozoa was kept at 4°C and evaluated at 24h intervals for 7 days. A second aliquot of the sperm sample was extended in Tris-citrate egg yolk extender with glycerol (Fraction B, Biladyl), frozen in liquid nitrogen (LN) vapor and stored in LN. The frozen sample was later thawed and evaluated as for the cooled samples. Percentages of motile (MS) and progressive (PS) spermatozoa were assessed using a phase contrast microscope (×200; stage at 37°C). Sperm plasma membrane damage was assessed by determining the percentage of cells exhibiting red fluoresence after staining with propidium iodide (PI, 50ng/mL; 10min RT). Spermatozoa that did not stain red in PI were classified as plasma membrane intact (PMI). Resilience to hypo-osmotic shock and plasma membrane integrity were evaluated by incubating a portion of the sample in a 100mOsm/kg solution (10nM glucose, 20nM HEPES, 30nM NaCl) containing PI for 15min at room temperature. The percentage of sperm cells with active mitochondria (MIT) was determined by counting spermatozoa showing orange fluoresence over the mid-piece after staining with JC-1(7.5 uM Sigma) for 30min at 37°C. At collection, MS was 15% and did not show a significant decrease during the 7-day storage period. Initially, PS was 10% and dropped to 5% after 7 days, with values fluctuating during the storage period. Both PMI and HOSPMI were 80% on Day 1, gradually decreasing to 75% on Day 7 of storage. PMI and MIT showed a highly significant correlation (r=0.88; P=0.003; n=8). In frozen-thawed sperm samples, MS fell from a pre-freeze value of 15% to 5% after thawing. Similarly, PS fell from 10% in pre-freeze to 3% in frozen-thawed samples. Likewise, PMI, HOSPMI and MIT values were 80% and 45%, 87% and 45% and 89% and 49%, respectively. Our study showed that lion sperm PMI and MIT remained high after 7 days at 4°C. MS and PS, although low, did not vary during this same period. PI and JC-1 showed a significant correlation, suggesting that both might be affected by the same deleterious factors. Although PMI, HOSPMI and MIT values decreased approximately 40% after freezing, we feel that such sperm samples could be used for in vitro embryo production, if not by IVF, by ICSI. Of course, additional studies are needed to validate our suggestion.

Sign in / Sign up

Export Citation Format

Share Document