Ovine epididymal spermatozoa preservation in liquid state with or without seminal plasma epididimários ovinos

ABSTRACT: Preservation and use of spermatozoa that have been recovered after death can extend the use of genetically superior animals. The objective of this study was to evaluate the maximum period for which ovine spermatozoa could be successfully stored in refrigerated dilution medium post-mortem, with or without added seminal plasma. Three samples of spermatozoa collected in an artificial vagina from 10 rams, or from the tails of four epididymes from the same rams at the time of death (G0) and six (G6), twelve (G12), twenty-four (G24) and forty-eight (G48) hours after death were used. After recovery, the spermatozoa were refrigerated at 5°C in either control medium (CM) or control medium plus 20%homologous seminal plasma (SP) and evaluated for 72 hours from the start of refrigeration. The G48 samples had a lower(P <0.05) total motility (TM) and plasma membrane integrity in the hyposmotic test (HOST) than the other groups evaluated at all analyzed times. The TM decreased (P <0.05) after 24 hours of cooling in semen collected in AV, at G0 and G24 and after 48 hours of refrigeration in G6 and G12. The TM and HOST integrity and sperm morphology did not differ between samples refrigerated in CM or SP. In conclusion, it is possible to collect epididymal spermatozoa up to 24 hours after death. Sperm viability can be prolonged fora further 48 hours by refrigeration. However, total motility decreases from 24 hours after refrigeration and the supplementation of 20% seminal plasma to the extender has no effect on spermatozoa longevity.

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Relationship between semen quality and seminal plasma cholesterol, triacylglycerols and proteins in the domestic cat

Journal of Feline Medicine and Surgery ◽

10.1177/1098612x19889072 ◽

2019 ◽

Vol 22 (10) ◽

pp. 882-889 ◽

Cited By ~ 1

Author(s):

María F García ◽

Romina Nuñez Favre ◽

María C Stornelli ◽

Ramiro Rearte ◽

María C García Mitacek ◽

...

Keyword(s):

Seminal Plasma ◽

Sperm Morphology ◽

Semen Quality ◽

Sperm Count ◽

Sodium Dodecyl ◽

Sperm Concentration ◽

Membrane Integrity ◽

Domestic Cat ◽

Sds Page ◽

The Relationship

Objectives The current study aimed to evaluate the relationship between specific seminal plasma components – cholesterol (CHOL), triacylglycerols (TAG) and total protein (PROT) concentrations – and semen quality in cats. A further aim was to determine the relationship between specific seminal protein bands and semen quality. Methods Thirteen toms, 2–5 years of age, were included. Semen collection was performed by electroejaculation every 4 weeks. Fifty-eight ejaculates were assessed for motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology. Samples were divided into two groups: good semen quality (GSQ) and poor semen quality (PSQ). After evaluation, seminal plasma was separated from the sperm by centrifugation and stored at −20°C. CHOL, TAG and PROT concentrations were then assessed and seminal plasma protein profile was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results Seminal plasma CHOL and TAG concentrations, motility, velocity, sperm concentration, total sperm count and sperm morphology were significantly higher in GSQ cats compared with PSQ cats ( P <0.01). Moreover, seminal plasma SDS-PAGE analysis showed an identifiable extra band exclusively in the GSQ group. Conclusions and relevance Data obtained in this study showed that seminal plasma CHOL and TAG concentrations and specific protein bands could be used to improve semen evaluation in toms. In this sense, the 14 kDa protein band could be a valuable marker for semen quality in the cat and should be further investigated. However, more studies are necessary to determine its relationship with fertility.

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Sex sorting sperm of sumba ongole bulls by using snakehead fish (Channa striata) albumin extract

Journal of the Indonesian Tropical Animal Agriculture ◽

10.14710/jitaa.44.1.106-113 ◽

2019 ◽

Vol 44 (1) ◽

pp. 106 ◽

Cited By ~ 2

Author(s):

T. Maulana ◽

S. Said ◽

R. I. Arifiantini ◽

M. A. Setiadi

Keyword(s):

Concentration Ratio ◽

Room Temperature ◽

Membrane Integrity ◽

Control Group ◽

Sperm Viability ◽

Freeze Dried ◽

Bottom Fraction ◽

Channa Striata ◽

Artificial Vagina ◽

Better Than

The objective of this research was to investigate the potential of snakehead albumin extract (channalbumin) for sorting X and Y sperm of Sumba Ongole (SO) and its characteristic. Semen was collected from three SO bulls using artificial vagina and the freeze dried channalbumin was extracted from snakehead fish. Channalbumin column was made with different concentration ratio of top and bottom fraction: 2%:4%; 3%:5%; 4%:6% respectively and BSA 5%:10% as control. Semen was put in top fraction then incubated for 30 min at room temperature then each fraction was centrifuged at 1800 rpm for 10 minutes. The pellet was evaluated for motility, abnormality, viability, membrane integrity and head sperm morphometric. The results showed that the channalbumin capable to maintain sperm motility in the top fraction better than the bottom fraction. Sperm viability and membrane integrity in control group were significantly higher (P<0.05) than all channalbumin treatment. BSA 5%:10% has highest proportion of X and Y sperm (69%:76.77%) compared with 2%:4% (42.33%:79.13%), 3%:5% (55.97%:75.73%) and 4%:6% of channalbumin (62.77%:68%). It’s concluded that channalbumin 4%: 6% was effective for separation of XY sperm with higher proportion.

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Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen

Brazilian Journal of Veterinary Research and Animal Science ◽

10.11606/issn.1678-4456.bjvras.2021.174301 ◽

2021 ◽

Vol 58 ◽

pp. e174301

Author(s):

Carolina Natalia Alonso ◽

Catalina Castañeira ◽

Ana Flores Bragulat ◽

Luis Losinno

Keyword(s):

Kinetic Parameters ◽

Seminal Plasma ◽

Membrane Integrity ◽

Milk Proteins ◽

Egg Yolk ◽

Reproductive Efficiency ◽

Sperm Viability ◽

Progressive Motility ◽

Live Sperm

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus(R) extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.

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Viability of ovine spermatozoa collected from epididymides stored at 18°-25°C for 48 hours post mortem

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-10058 ◽

2018 ◽

Vol 70 (4) ◽

pp. 1023-1028

Author(s):

T.G. Bergstein-Galan ◽

R.R. Weiss ◽

T.S.R. Barbosa ◽

L.E. Kozicki ◽

S.D. Bicudo

Keyword(s):

Morphological Changes ◽

Membrane Integrity ◽

Post Mortem ◽

Integrity Test ◽

Vitro Fertilization ◽

Progressive Motility ◽

Viable Cells ◽

Epididymal Spermatozoa ◽

Artificial Vagina

ABSTRACT The objectives of this study were to verify the time during which viable ovine spermatozoa could be recovered from the cauda epididymis kept at ambient temperature (18-25°C). Sperm collected in an artificial vagina (AV) were used as control. Spermatozoa samples were collected with an AV and from epididymis at 0 (G0), 6 (G6), 12 (G12), 24 (G24), and 48 (G48) hours post mortem. Total motility (TM), progressive motility (PM), hypo-osmotic membrane integrity test (HOST) and morphological changes were assessed. TM decreased (P<0.05) from 24 hours post mortem (70.0±1.9%) compared to AV (86.4±1.0%). PM decreased (P<0.05) from 12 hours after death (31.3±4.0%) compared to AV group (73.2±1.4%). The percentage of viable cells in HOST decreased (P<0.05) in the G48 (60.0±8.9%). Spermatozoa recovery was lower (P<0.05) 48 hours after death (2064.2±230.7 x 106 spermatozoa) compared to G0(2623.6±288.4 x 106 spermatozoa). In conclusion, under the conditions of this study, it would be possible to use epididymal spermatozoa recovered up to 24 hours after death for artificial insemination or in vitro fertilization; however, fertility trials are necessary to prove this hypothesis.

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Quality of ram spermatozoa separated with modified swim up method

Bangladesh Veterinarian ◽

10.3329/bvet.v33i2.36459 ◽

2018 ◽

Vol 33 (2) ◽

pp. 62-70 ◽

Cited By ~ 2

Author(s):

A Hossain ◽

MM Islam ◽

F Naznin ◽

RN Ferdousi ◽

FY Bari ◽

...

Keyword(s):

Semen Quality ◽

Membrane Integrity ◽

Quality Parameters ◽

Fluid Medium ◽

Normal Morphology ◽

The Mean ◽

Mass Activity ◽

Fresh Semen ◽

Artificial Vagina

Semen was collected from four rams, using artificial vagina and viability%, motility% and plasma membrane integrity% were measured. Fresh ejaculates (n = 32) were separated by modified swim-up separation using modified human tubal fluid medium. Four fractions of supernatant were collected at 15-minute intervals. The mean volume, mass activity, concentration, motility%, viability%, normal morphology and membrane integrity% (HOST +ve) of fresh semen were 1.0 ± 0.14, 4.1 ± 0.1 × 109 spermatozoa/ml, 85.0 ± 1.3, 89.4 ± 1.0, 85.5 ± 0.7, 84.7 ± 0.5 respectively. There was no significant (P>0.05) difference in fresh semen quality parameters between rams. The motility%, viability% and HOST +ve % of first, second, third and fourth fractions were 53.4 ± 0.5, 68.2 ± 0.3, 74.8 ± 0.3 and 65.5 ± 0.4; 55.5 ± 0.4, 66.2 ± 0.4, 74.5 ± 0.3 and 73.6 ± 0.3 and 66.7 ± 0.5, 66.8 ± 0.5, 65.2 ± 0.4 and 74.7 ± 0.5 respectively. The motility%, viability% and membrane integrity% of separated semen samples differed significantly (P<0.05) between four fractions. The mean motility% and viability% were significantly higher (P<0.05) in third fraction (74.8 ± 0.3%), whereas the mean HOST +ve% was significantly higher (P<0.05) in fourth fraction (74.7 ± 0.5). All quality parameters of separated spermatozoa were significantly (P<0.05) lower than that of fresh semen. The pregnancy rates were higher with fresh semen (71%) in comparison to that of separated sample (57%).Bangl. vet. 2016. Vol. 33, No. 2, 62-70

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Macro- and microelements in serum and seminal plasma as biomarkers for bull sperm cryotolerance

Acta Veterinaria Scandinavica ◽

10.1186/s13028-021-00590-2 ◽

2021 ◽

Vol 63 (1) ◽

Author(s):

Maja Zakošek Pipan ◽

Petra Zrimšek ◽

Breda Jakovac Strajn ◽

Katarina Pavšič Vrtač ◽

Tanja Knific ◽

...

Keyword(s):

Seminal Plasma ◽

Semen Quality ◽

Sperm Quality ◽

Membrane Integrity ◽

Quality Characteristics ◽

Freezing And Thawing ◽

Additional Tool ◽

Bull Semen ◽

Bull Sperm ◽

Fresh Semen

ABSTRACT Background Wide variation in fertility rates is observed when using frozen bull semen, even when the bulls have met quality standards for semen production. Therefore, a simple and reliable test to assess the freezing potential of bull semen based on the analysis of fresh semen or blood would be of great value. Attention is now turning to assessment of seminal plasma components such as proteins and elements. In the present study, the concentrations of macro- and microelements in fresh bull semen plasma and in serum and their correlation with quality characteristics of fresh semen and with semen quality after freezing and thawing were determined. Ejaculates were collected from 30 mature bulls, and semen volume, concentration, sperm motility, morphology, tail membrane integrity, plasma membrane permeability and DNA fragmentation were determined on the day of collection and after freezing and thawing. The concentrations of macroelements (Na, Mg, K and Ca) and microelements (Cu, Fe, Zn and Se) were determined in the seminal plasma and serum. The semen samples were classified into satisfactory and unsatisfactory groups according to the fresh semen quality. Results Zinc and Se levels measured in serum were associated with almost all fresh and frozen-thawed semen quality characteristics, while Fe levels were associated only with acrosomal defects in fresh semen. Zinc and Fe levels in fresh seminal plasma were associated with various quality characteristics of fresh and frozen-thawed semen, while Se level in fresh seminal plasma was not associated with any of the semen quality characteristics. Conclusions Microelements were shown to be useful as biomarkers involved in the analysis of bull sperm quality and could be used as an additional tool to predict bull semen quality after freezing and thawing. Our results confirm that the analysis of Zn and Se levels in serum and Zn, Cu and Fe levels in fresh seminal plasma can provide information to discriminate between bull semen samples with spermatozoa with high or low cryotolerance.

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Role of Glutathione in Male Infertility

Bangladesh Journal of Medical Biochemistry ◽

10.3329/bjmb.v4i2.13772 ◽

2013 ◽

Vol 4 (2) ◽

pp. 20-25

Author(s):

ZU Naher ◽

SK Biswas ◽

FH Mollah ◽

M Ali ◽

MI Arslan

Keyword(s):

Male Infertility ◽

Seminal Plasma ◽

Antioxidant Defense ◽

Sperm Morphology ◽

Sperm Count ◽

Antioxidant Systems ◽

Infertile Male ◽

Semen Volume ◽

Infertile Group ◽

Male Subjects

Infertility is a worldwide problem and in almost 50% of cases infertility results from abnormality of the male partners. Apart from endocrine disorders, definitive cause and mechanism of male infertility is not clear in many cases. Recent evidence indicates that imbalance between pro-oxidant stress and antioxidant defense plays an important role in the pathogenesis of male infertility. Among the endogenous antioxidant systems, reduced glutathione (GSH) plays a significant role in the antioxidant defense of the spermatogenic epithelium, the epididymis and perhaps in the ejaculated spermatozoa. The current study was therefore designed to evaluate any association that may exist between GSH levels and male infertility. Infertile male patients (having female partners with normal fertility parameters; n=31) and age- matched healthy male fertile control subjects (n=30) were included in this study. In addition to medical history, semen analyses including semen volume, sperm count, motility and morphology were done for each subject. As a measure of antioxidant capacity erythrocyte and seminal plasma GSH concentrations were measured by Ellman's method in fertile and infertile male subjects. The infertile subjects were similar to fertile subjects in terms of age. However, semen volume and sperm count was found significantly lower (p<0.001) in infertile males compared with healthy fertile male subjects. Percentage of subjects with abnormal sperm morphology and motility were found higher in infertile group compared with fertile group. The median (range) erythrocyte GSH level did not differ between the two groups (12.62 (0.67-29.82) versus 13.93 (2.10-21.08) mg/gm Hb). However, the seminal plasma GSH level was found markedly suppressed in infertile group (1.64 (0.23-7.50)) compared with fertile group (4.26 (2.32-7.50)) mg/dl (p<0.001). In the present study seminal plasma GSH level was found markedly suppressed along with abnormal values for semen volume, sperm concentration and sperm morphology and motility in infertile subjects compared with fertile subjects. This finding indicates that low level of seminal plasma GSH level may be associated with male infertility. DOI: http://dx.doi.org/10.3329/bjmb.v4i2.13772 Bangladesh J Med Biochem 2011; 4(2): 20-25

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Cooling of equine semen at 16°C for 36 hours with addition of different glutathione concentrations

Semina Ciências Agrárias ◽

10.5433/1679-0359.2015v36n6p3699 ◽

2015 ◽

Vol 36 (6) ◽

pp. 3699

Author(s):

Rodrigo Arruda de Oliveira ◽

Marco Antônio De Oliveira Viu ◽

Maria Lúcia Gambarini

Keyword(s):

Lipid Peroxidation ◽

Sperm Motility ◽

Membrane Integrity ◽

Membrane Lipid ◽

Sperm Cells ◽

Sperm Viability ◽

Membrane Lipid Peroxidation ◽

Acrosomal Membrane ◽

In Vitro Effects

Handling equine semen during the refrigeration process reduces sperm viability, and consequently causes membrane lipid peroxidation, among other challenges. The present study aimed to evaluate the in vitro effects of glutathione (control, 1. 0, 1. 5, and 2. 5 mM) on equine semen in a refrigeration protocol of 16ºC for 36 hours. The following variables were evaluated after 0, 12, 24, and 36 hours refrigeration: total sperm motility, vigor, viability, and plasma and acrosomal membrane integrity. Motility was higher with 2. 5mM of glutathione (57. 8 ± 7. 3) after 12 hours of refrigeration compared to the control (53. 2 ± 8. 3) (P < 0. 05). After 36 hours of refrigeration, motility was higher with 1. 5 mM (43. 4 ± 12. 7) and 2. 5mM glutathione (45. 5 ± 6. 2), than it was with 1mM glutathione (38. 2 ± 9) and the control (35. 5 ± 18. 4) (P < 0. 05), respectively. Vigor was highest with 1. 5mM glutathione (3. 7 ± 0. 3) after 36 hours compared to the control (3. 2 ± 1. 1), (P < 0. 05). Viability differed between control and 1mM treatments (79. 5 ± 1. 8) only after 24 hours (75. 5 ± 9. 7) (P < 0. 05). Throughout the investigation, no significant differences were noted in plasma and acrosomal membrane integrity (P > 0. 05). The 1. 5 and 2. 5mM glutathione levels were more efficient in protecting sperm cells and yielded higher total motility values after 36 hours of refrigeration.

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Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze - thaw damage

Reproduction Fertility and Development ◽

10.1071/rd08238 ◽

2009 ◽

Vol 21 (4) ◽

pp. 571 ◽

Cited By ~ 23

Author(s):

T. Leahy ◽

J. I. Marti ◽

G. Evans ◽

W. M. C. Maxwell

Keyword(s):

Seminal Plasma ◽

High Speed ◽

Sperm Quality ◽

Membrane Integrity ◽

Weight Fraction ◽

Protein Supplementation ◽

Freezing Process ◽

Spermatozoa Motility ◽

Functional Integrity

Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n = four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37°C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze–thawing.

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