scholarly journals Use of phosphatidylcholine in Tris-based extender with or without egg yolk to freeze Bapedi ram semen

2020 ◽  
Vol 50 (3) ◽  
pp. 389-396
Author(s):  
K.S. Mafolo ◽  
C.M. Pilane ◽  
T. Chitura ◽  
T.L. Nedambale
Keyword(s):  
Sperm Cell ◽  
Agricultural Research ◽  
Membrane Integrity ◽  
Germplasm Conservation ◽  
Egg Yolk ◽  
Protective Agent ◽  
Liposome Membrane ◽  
Cell Membrane Integrity ◽  
Agricultural Research Council ◽  
Acrosome Integrity

Traditionally, egg yolk is a protective agent that is used to freeze semen in various species. However, the addition of egg yolk in extender risks the introduction of disease. Therefore, an alternative cryoprotective agent should be found to preserve ram semen. The aim of this study was to evaluate the effect of phosphatidylcholine (PC) as a protective agent in extender with or without egg yolk on semen characteristics and acrosome integrity of frozen then thawed Bapedi ram semen. Semen was collected from four mature Bapedi rams, in the Agricultural Research Council (ARC) Germplasm Conservation Programme, using an artificial vagina. Following collection, semen samples were randomly diluted into Tris-based extender (1: 2), with and without egg yolk, and supplemented with four concentrations of PC liposome (0 mg/ml), 0.25 mg/ml, 0.5 mg/ml and 0.75 mg/ml). Supplementation of PC liposome in extender with or without egg yolk did not improve the semen total motility (TM), progressive motility (PM) and rapid motility (RM) rate. The sperm cell membrane integrity in extender with or without egg yolk was not influenced by the supplementation of PC liposome after thawing (P >0.05). The addition of PC liposome to Tris-based extender with egg yolk had a similar result to control (Tris-based extender with egg yolk) on sperm cell acrosome integrity. In conclusion, supplementation of PC liposome to Tris-based extender without egg yolk had lower sperm cell viability and motility rates compared with the extender with egg yolk, regardless of concentration.Keywords: acrosome, cryoprotectant, liposome, membrane, motility

90 EFFECT OF EGG YOLK ON THE KINEMATICS AND ACROSOME MEMBRANE INTEGRITY OF COOLED-REWARMED CANINE SPERMATOZOA

2010 ◽  
Vol 22 (1) ◽  
pp. 204
Author(s):  
J. Dorado ◽  
M. J. Galvez ◽  
M. R. Murabito ◽  
S. Demyda ◽  
L. J. De Luca ◽  
...  
Keyword(s):  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Mean Values ◽  
Head Displacement ◽  
Healthy Dogs ◽  
Acrosome Integrity ◽  
Digital Manipulation ◽  
Lateral Head

Tris-egg yolk-based diluents provide adequate cryoprotection for the sperm of most species. This study was conducted to compare the ability of Tris-glucose extender containing 2 different concentrations of egg yolk to maintain sperm motility and acrosome integrity of canine spermatozoa during 72 h of preservation. For this purpose, a total of 20 ejaculates from 4 clinically healthy dogs (2 Spanish Greyhound, 1 German Pointer, and 1 Crossbreed) were collected by digital manipulation. The sperm-rich fraction of each ejaculate was divided into 2 aliquots. Then, they were diluted in Tris-based extender and centrifuged at 700g for 8 min. Sperm pellets were resuspended in either Tris buffer added to 20% (EY20) or 10% centrifuged egg yolk (EY10) and cooled to 5°C over 72 h. The effects of these extenders on motility and acrosome integrity were assessed objectively using a computer-aided semen analyzer (Sperm Class Analyzer, Microptic SL, Spain) and Spermac® staining, respectively. Each cooled-rewarmed semen sample was evaluated after 24, 48, and 72 h of preservation. Sperm motion parameters shown by computer-assisted semen analysis (CASA) are progressively motile (PMS) and motile spermatozoa (MS), curvilinear velocity (CLV), average path velocity (APV), progressive speed (SLV), and lateral head displacement (LHD). Data were statistically analysed by ANOVA. Dependent variables expressed as percentages were arsine-transformed before analysis. Differences between mean values were evaluated by the Duncan method. Data were presented as mean ± SEM. Differences were considered significant when P < 0.05. Analyses were performed using the statistical package SPSS 12.0. A total of 98 172 motile sperm trajectories were analyzed by CASA: 52 259 in EY20 and 45 913 in EY10. After 24, 48, and 72 h of preservation, MS and PMS were statistically higher (P < 0.01) in EY20. No significant differences were found for LHD using either extender over a 72-h period. No significant differences were observed for CLV using either extender during the first 2 days. At Day 3, CLV data were significantly higher (P < 0.01) in EY20. Similarly, from Day 2, APV was significantly higher (P < 0.001) in EY20. After 24 h of preservation, SLV was statistically higher (P < 0.001) in EY10, whereas the opposite tendency was found at Day 3. No significant differences were observed for SLV using either extender after 48 h of preservation. During the first 2 days, acrosome integrity was statistically higher (P < 0.001) in EY20. At hour 72, higher acrosome integrity (P < 0.001) was observed in EY10. In conclusion, we have observed that the EY20 extender provided higher motility after 72 h of chilled preservation; however, the acrosome membrane integrity was better preserved in EY10.

scholarly journals Slow freezing versus vitrification for the cryopreservation of zebrafish (Danio rerio) ovarian tissue

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lis S. Marques ◽  
Ana A. N. Fossati ◽  
Rômulo B. Rodrigues ◽  
Helen T. Da Rosa ◽  
Aryele P. Izaguirry ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Ovarian Tissue ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Slow Freezing ◽  
Mitochondrial Activity ◽  
Trypan Blue Exclusion ◽  
Cell Membrane Integrity ◽  
Cell Plasma ◽  
Cryopreservation Method

Abstract The aim of the present study was to compare the efficiency of vitrification and slow freezing techniques for the cryopreservation of zebrafish ovarian tissue containing immature follicles. In Experiment 1, assessment of cell membrane integrity by trypan blue exclusion staining was used to select the best cryoprotectant solution for each cryopreservation method. Primary growth (PG) oocytes showed the best percentage of membrane integrity (63.5 ± 2.99%) when SF4 solution (2 M methanol + 0.1 M trehalose + 10% egg yolk solution) was employed. The vitrification solution, which presented the highest membrane integrity (V2; 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose + 10% egg yolk solution) was selected for Experiment 2. Experiment 2 aimed to compare the vitrification and slow freezing techniques in the following parameters: morphology, oxidative stress, mitochondrial activity, and DNA damage. Frozen ovarian tissue showed higher ROS levels and lower mitochondrial activity than vitrified ovarian tissue. Ultrastructural observations of frozen PG oocytes showed rupture of the plasma membrane, loss of intracellular contents and a large number of damaged mitochondria, while vitrified PG oocytes had intact mitochondria and cell plasma membranes. We conclude that vitrification may be more effective than slow freezing for the cryopreservation of zebrafish ovarian tissue.

Assessment of changes in sperm cell membrane integrity occurring during the storage of semen from genetically different males using two diagnostic methods

2014 ◽  
Vol 94 (4) ◽  
pp. 601-606 ◽  
Author(s):  
Anna Wysokińska ◽  
Stanislaw Kondracki
Keyword(s):  
Cell Membrane ◽  
Sperm Cell ◽  
Cell Membranes ◽  
Intact Cell ◽  
Membrane Integrity ◽  
Diagnostic Methods ◽  
Cell Membrane Integrity ◽  
Staining Methods ◽  
Breed Crosses

Wysokińska, A. and Kondracki, S. 2014. Assessment of changes in sperm cell membrane integrity occurring during the storage of semen from genetically different males using two diagnostic methods. Can. J. Anim. Sci. 94: 601–606. The present study was carried out to assess changes in sperm cell membrane integrity occurring during the storage of semen collected from genetically different domestic male pigs. The study was aimed at assessing differences in the course of changes in the integrity of cell membranes in spermatozoa produced by males with different degrees of genetic diversity (pure-bred males, two-breed hybrids and multi-breed crosses) and testing the usefulness of two methods of sperm cell membrane integrity evaluation, based on material collected from genetically different males. The experiments were conducted on 56 ejaculates collected from 28 domestic male pigs. The examination of sperm cell membrane integrity was performed three times for each ejaculate, i.e., after 1 h, after 24 h and after 48 h from collection. The preparations for analysing cell membrane integrity were made using two methods: the SYBR 14/PI method and the eosin–nigrosin method. It was found that both SYBR 14/PI and eosin–nigrosin staining methods make it possible to successfully assess the integrity of the plasma membrane of domestic pig sperm cells under in vitro conditions. Hybrid pig spermatozoa, especially those from multi-breed crosses, better retain the integrity of their plasmalemmas than the spermatozoa of pure-bred boars. The ejaculates of Hypor cross-breed boars assessed after 1, 24 and 48 h of storage contain more spermatozoa with intact cell membranes than the ejaculates of pure-bred Duroc and Pietrain boars. The ejaculates of Hypor boars also show fewer decaying spermatozoa than those produced by pure-bred boars.

scholarly journals Effect of sunflower lecithin on Kalahari Red goat semen during cryopreservation

2018 ◽  
Vol 51 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Sakirat Opeyemi Adeyanju ◽  
James Olatinbo Daramola ◽  
Jimoh Alao Olanite ◽  
Olufiropo Samson Awokola
Keyword(s):  
Soybean Lecithin ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Domestic Animal ◽  
Arginase Activity ◽  
Semen Parameters ◽  
Phospholipid Content ◽  
Acrosome Integrity ◽  
Semen Extender ◽  
Different Levels

Abstract Soybean lecithin had been used as an alternative to egg yolk in domestic animal semen extender during cryopreservation due to its characteristic phospholipid content which played a major cryoprotective role. This composition of soybean lecithin informed the replacement of soybean with sunflower lecithin (SL) in the extender for the Kalahari Red (KR) buck semen cryopreservation in this study. Effect of different levels of SL on the quality of the KR buck semen during cryopreservation using slow freezing method was evaluated. Semen samples were collected from four KR bucks of between two and two and half of age using artificial vagina, evaluated for motility and then diluted in extenders containing different levels of SL (1.5%, 3.0% and 4.5%) as experimental group and 0% SL or 20% egg yolk as control. Semen parameters including motility, acrosome integrity (AcI), membrane integrity (MI), malondialdehyde (MDA) concentration, cholesterol level and seminal arginase activity were evaluated for. The results showed that motility, acrosome integrity (AI) and membrane integrity were comparable at 0%, (22.00 ± 4.58, 82.00 ± 3.51 and 96.00 ± 2.03); 1.5%, (23.00 ± 2.08, 87.00 ± 3.79 and 89.00 ± 2.08); 3.0%, (13.00 ± 2.52, 81.33 ± 0.41 and 76.67 ± 1.20) and 4.5% (11.00 ± 4.51, 85.33 ± 9.88 and 84.00 ± 8.50), respectively, after thawing. SL at 0% had the highest (P < 0.05) values for MDA, cholesterol and seminal arginase activity (1.10 ± 0.008 nmol/ml, 236.35 ± 4.08 mg/dl and 0.54 ± 3.3 E-3 units/mg protein, respectively). Our data suggest that 1.5% sunflower lecithin can be used in place of soy lecithin as a substitute for egg yolk during the cryopreservation of caprine semen.

scholarly journals Integrity of Sperm Cell Membrane in the Semen of Crossbred and Purebred Boars during Storage at 17 °C: Heterosis Effects

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3373
Author(s):  
Anna Wysokińska ◽  
Dorota Szablicka
Keyword(s):  
Cell Membrane ◽  
Sperm Cell ◽  
Cell Membranes ◽  
Semen Quality ◽  
Intact Cell ◽  
Membrane Integrity ◽  
Storage Conditions ◽  
Cell Membrane Integrity ◽  
Semen Storage ◽  
Sperm Membrane

The aim of the study was to assess changes in the integrity of sperm cell membranes during the storage of semen collected from Duroc × Pietrain crossbred boars and purebred boars of the component breeds. To compare the cell membrane integrity of sperm heads in crossbred and purebred boars, heterosis effects were estimated. The study was conducted on 48 ejaculates collected from Duroc × Pietrain crossbred boars and from purebred Duroc and Pietrain boars used for artificial insemination. Microscope slides were prepared from each ejaculate for the evaluation of the cell membrane integrity of the sperm, at 1, 24, 48, 72, and 96 h after collection of the ejaculate. Diluted ejaculates were stored at 17 °C. Sperm membrane integrity was analysed by two methods: SYBR-14/PI and eosin–nigrosin. Our results showed that the cell membrane integrity of sperm heads changed with storage time, but the extent of the changes varied depending on the genetic group of boars. The semen of Duroc × Pietrain crossbreds was clearly seen to be less sensitive to storage conditions than that of boars of the parent breeds, which was confirmed by the calculated heterosis effects. The percentage of sperm with an intact cell membrane was higher in crossbred boars than in purebred boars (p ≤ 0.05). In addition, significantly fewer moribund sperm spermatozoa and spermatozoa with a damaged cell membrane were observed in crossbred boars (p ≤ 0.05). In the semen of purebred Duroc and Pietrain boars, the cell membrane integrity of the sperm should be assessed more often during storage than in the semen of Duroc × Pietrain crossbred boars. This study provides valuable information for the development and implementation of semen quality monitoring in crossbred boars and boars of the parent breeds during storage at 17 °C with respect to the cell membrane structure of sperm heads. The evaluation methods used effectively identify damage to the cell membranes of the sperm during semen storage.

scholarly journals Sperm Antioxidant Biomarkers and Their Correlation with Clinical Condition and Lifestyle with Regard to Male Reproductive Potential

2020 ◽  
Vol 9 (6) ◽  
pp. 1785
Author(s):  
Wirginia Krzyściak ◽  
Monika Papież ◽  
Ewelina Bąk ◽  
Eva Morava ◽  
Paweł Krzyściak ◽  
...  
Keyword(s):  
Male Infertility ◽  
Clinical Condition ◽  
Sperm Cell ◽  
Eating Habits ◽  
Membrane Integrity ◽  
Reproductive Potential ◽  
Cell Membrane Integrity ◽  
Alternative Approach ◽  
Potential Trap ◽  
Total Sensitivity

Measurement of sperm oxidative-antioxidant indicators is widely used in the assessment and detection of biochemical causes of male infertility. The main purpose of this study was to identify biomarkers that assist in diagnostics and monitoring of male reproductive potential. We performed the assessment of oxidative-antioxidant malondialdehyde (MDA), glutathione (GSH), and total redox antioxidant potential (TRAP) indicators in seminal plasma, seminogram, clinical condition, and lifestyle of people with reproductive problems. The combined assessment of GSH and TRAP as potential biomarkers of male infertility in semen plasma was characterized by the highest total sensitivity and specificity. Furthermore, we provide evidence that male reproductive potential is significantly correlated with basic sperm parameters, sperm cell membrane integrity, their morphology, lifestyle, eating habits, occupation, and mental health. Our results provide evidence on the importance of oxidative stress and defense against free radicals in diagnosing and monitoring men with infertility that are consistent with previously conducted research. We provide an alternative approach on the possibility of interpreting the combination of the biomarkers that can bring benefits to a multi-threaded approach to the diagnosis and treatment of male infertility.

scholarly journals Assessment of genetic variation in Bapedi sheep using microsatellite markers

2020 ◽  
Vol 50 (2) ◽  
pp. 318-324
Author(s):  
A. Maqhashu ◽  
N.O. Mapholi ◽  
H.A. O’Neill ◽  
K.A. Nephawe ◽  
F.V. Ramukhithi ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Genetic Variation ◽  
Microsatellite Markers ◽  
South African ◽  
Agricultural Research ◽  
Germplasm Conservation ◽  
Genetic Distances ◽  
Fixation Index ◽  
Indigenous Sheep ◽  
Agricultural Research Council

This study was conducted to assess genetic variation in Bapedi sheep using 14 microsatellite markers. Blood samples were collected from 174 unrelated Bapedi sheep on six farms in various districts of Limpopo and from the Agricultural Research Council Animal Production Institute (ARC-API) in Gauteng. Genotypes from other South African indigenous sheep, namely Zulu (N = 14), Damara (N = 11), Dorper (N = 8), and Namaqua (N = 11), were included to represent reference populations. The effective number of alleles averaged 5.6 for across the Bapedi flocks and was 4.9 for the reference breeds. Among the Bapedi flocks, the observed heterozygosity (Ho) ranged from 0.56 ± 0.05 to 0.69 ± 0.03 and expected heterozygosity (He) values were between 0.75 ± 0.04 and 0.88 ± 0.01. Thus, there is considerable genetic diversity within the Bapedi sheep populations. However, the fixation index was high, indicating the possibility of inbreeding becoming a problem for these flocks. A neighbour-joining tree was constructed from the estimates of Nei’s genetic distances among flocks. The presence of Bapedi sheep flocks on all of the main branches of the tree along with one of the reference breeds suggests the present-day Bapedi is not an entirely distinct breed and that there are genetic differences between flocks of these South African indigenous sheep. Sustainable breeding and conservation programmes are needed to control inbreeding and to foreclose possible genetic dilution of Bapedi sheep. Keywords: genetic diversity, germplasm conservation, inbreeding, indigenous sheep

41 THE EFFECT OF AMBIENT TEMPERATURE ON SPERM MOTILITY DURING LIQUID STORAGE OF VENDA COCK SEMEN AND INDIVIDUAL DIFFERENCES IN SPERM CRYOTOLERANCE

2012 ◽  
Vol 24 (1) ◽  
pp. 133
Author(s):  
M. L. Mphaphathi ◽  
D. Luseba ◽  
M. B. Masenya ◽  
B. Sutherland ◽  
T. L. Nedambale
Keyword(s):  
Cell Motility ◽  
Sperm Motility ◽  
Sperm Cell ◽  
Agricultural Research ◽  
Germplasm Conservation ◽  
Storage Period ◽  
Liquid Storage ◽  
Frozen Semen ◽  
Significant Difference ◽  
Motility Rate

Improving techniques for liquid storage of cock semen can increase the efficiency of AI programs in the poultry industry. The aims of the present study were (1) to compare storage of cock sperm for 24 h at 5 and 25°C and (2) to test the cryotolerance of sperm cell motility in individual Venda cocks. Semen was collected with the abdominal massage method, from 6 indigenous Venda cocks. Cocks were 26 weeks of age and were kept under the same conditions. After macroscopic analysis, semen was pooled and diluted (1:2) with Kobidil+ extender and divided into 3 equal parts. Part 1 was evaluated immediately (0 h), part 2 was stored at 5°C and part 3 was stored at 25°C and evaluated for sperm motility and velocity parameters at 4, 8, 12 and 24 h of storage. For cryopreservation, semen was diluted (1:2) with modified Kobidil+ extender supplemented with 8% of dimethyl sulfoxide. Individual ejaculates were equilibrated at 5°C for 4 h and then loaded into the programmable freezer. Then, semen straws were thawed at 5°C. Sperm motility and velocity parameters were evaluated using the Sperm Class Analyzer® system. Six replicates were done per trial. Data were analysed using the statistical programme GenStat®. Treatment means were separated using Fisher's protected t-test least significant difference (P < 0.05). Total sperm cell motility rate was 87.5% and decreased significantly during in vitro storage and was <31% after 24 h at 25°C. Semen samples stored at 5°C showed a total sperm cell motility rate of above 50% after 24 h. There was a slight linear decrease in the percentage of sperm with progressive motility and rapid velocity as the storage period increased, irrespective of the storage temperature. The rapid and medium motility percentages were higher in fresh semen and significantly decreased (P < 0.05) during the incubation period. There was variation in the total sperm cell motility of fresh and frozen semen among cocks. There was no significant difference in variation in non-progressive and medium percentage (P > 0.05) motility in diluted fresh or frozen sperm cells or in the percentage of sperm with rapid motility in thawed semen. There was variation in <25% of the cocks in total sperm motility rate. In summary, cryopreservation reduced sperm cell motility and velocity rates in all the cock semen donors. We found that cryotolerance of cock sperm does vary among males. Furthermore, the lower temperature 5°C was suitable for semen storage of Venda cocks. This temperature (5°C) could potentially improve methods of semen equilibration before cryopreservation. The study was supported by an Agricultural Research Council Parliamentary Grant, Department of Agriculture Forestry and Fisheries and National Research Foundation-GUN No RT21 and 24000 (NRF). The Germplasm Conservation & Reproduction Biotechnologies (GCRB) group is thanked for their support.

138 EFFECT OF SEMINAL PLASMA ON CHILLING AND FREEZING OF CANINE SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 187
Author(s):  
I. Yu ◽  
Y. J. Kim ◽  
I. S. Kim ◽  
S. P. Leibo ◽  
N. Songsasen
Keyword(s):  
Pisum Sativum ◽  
Seminal Plasma ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Beneficial Effects ◽  
Progressive Motility ◽  
Healthy Dogs ◽  
Sperm Survival ◽  
Acrosome Integrity ◽  
Group 1

Seminal plasma (SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing (Barrios et al. 2000 Biol. Reprod. 63, 1531–1537; Moore et al. 2005 Theriogenology 63, 2372–2381; Vadnais et al. 2005 Anim. Reprod. Sci. 87, 121–132). The purpose of this study was to determine the effect on sperm survival of adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from 4 healthy dogs (3–4 years old) of various breeds were pooled and centrifuged at 300g for 10 min at 25�C; the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris (EYT) buffer. The study comprised 2 experiments: Exp. 1: Sperm were suspended in EYT extender containing 0%, 20%, 50%, 80%, or 100% SP, and were slowly cooled to 4�C for 2 h or held at 25�C as controls. Exp. 2: Sperm samples, each of which contained 1 � 108 sperm mL-1, were assigned to 5 groups to be frozen. In group 1, sperm in EYT + 20% SP were cooled to 4�C, diluted to contain final concentrations of 5% glycerol + 10% SP in EYT, and then frozen. In the 4 other groups, sperm in EYT alone were first cooled slowly to 4�C, then diluted to contain 5% glycerol plus 0%, 20%, 40%, or 50% SP in EYT, and then frozen. Spermatozoa were frozen at 25�C min in plastic straws that were suspended above liquid nitrogen and thawed in water at 38�C for 30 s. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at 400� magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that 20%, 50%, 80%, or 100% SP did not improve motility, membrane integrity, or acrosome integrity of spermatozoa chilled to 4�C compared to those chilled without SP (P &gt; 0.05). Survival of spermatozoa suspended in EYT + 20% SP and maintained at 25�C was significantly higher than for those that were chilled (P &lt; 0.05). The results of the second experiment showed that spermatozoa suspended in EYT + 20% SP and then diluted at 4�C to contain 5% glycerol + 10% SP exhibited the highest progressive motility and membrane integrity after being frozen and thawed (P &lt; 0.05). In summary, although seminal plasma did not affect spermatozoa that were only chilled, addition of seminal plasma did significantly improve survival of canine spermatozoa that were frozen and thawed.

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.

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