Examination of Some Processing Methods for Freezing Boar Semen

Five experiments were conducted to examine the effect of processing methods and diluents on survival and morphology of boar spermatozoa after freezing. Post-thawing survival of spermatozoa was better for Beltsville-F3 (BF3) than for tris-fructoseEDT A freezing diluent when the seminal plasma and glycerol were removed prior to freezing (method A). Both freezing diluents yielded similar viability results when the spermatozoa were frozen in the presence of seminal plasma and glycerol (method B). Viability of spermatozoa after thawing was better when glycerol concentration in the prefreezing diluent (method A) or in the freezing medium (method B) was 2� 5 and 5� 0 rather than 7� 5 %. Cooling of diluted semen to 5�C beyond 4 h decreased the post-thawing survival of spermatozoa. The proportion of spermatozoa with undamaged acrosomes after processing and thawing by different methods was indistinguishable and relatively low. When the semen was frozen at cell concentrations ranging from 0�25 to 2�0 x 109/mI, the viability of spermatozoa declined with increasing concentration following freezing in BF3 and S-1 diluents. Viability results were very similar for all cell concentrations examined when tris-fructose-EDTA diluent was used, indicating the possibility of freezing boar semen in a concentrated state.

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INFLUENCE OF GLYCEROL CONCENTRATION AND FREEZING TO −79 C ON OXYGEN UPTAKE AND LIVABILITY OF BOAR AND BULL SPERMATOZOA

Canadian Journal of Animal Science ◽

10.4141/cjas72-007 ◽

1972 ◽

Vol 52 (1) ◽

pp. 65-72 ◽

Cited By ~ 2

Author(s):

L. M. SANFORD ◽

G. J. KING ◽

J. W. MACPHERSON

Keyword(s):

Oxygen Uptake ◽

Incubation Period ◽

Skim Milk ◽

Egg Yolk ◽

Freezing Process ◽

Glycerol Concentration ◽

Motile Cells ◽

Boar Semen ◽

Boar Spermatozoa

Boar and bull spermatozoa were diluted in a skim milk–egg yolk–glucose extender containing 0, 7.5, or 15% glycerol (v/v) and incubated aerobically for 6 hr at 37 C. Other partially diluted boar semen samples were cooled to 5 C. Glycerol was added to a final concentration of 0, 7.5, and 15%. Samples were frozen to −79 C, rewarmed, and incubated for 3 hr at 37 C. The presence of glycerol in the extender depressed (P < 0.01) the oxygen uptake by nonfrozen boar and bull spermatozoa during the 6-hr incubation period. The reduction of oxygen uptake by semen samples increased as the level of glycerol in the extender increased. There was a corresponding decrease (P < 0.01) in the number of motile cells at the conclusion of the incubation period. Glycerol appeared to have more of a detrimental effect on boar spermatozoan oxygen uptake. The rate of oxygen uptake by boar semen samples postfreezing was extremely depressed, suggesting that spermatozoa surviving the freezing process metabolize at a much lower rate than normal. Active progressive motility of most of the surviving boar spermatozoa ceased within 1–2 hr of incubation under the in vitro conditions of this experiment.

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Deep Freezing of Boar Semen II. Effects of Method of Dilution, Glycerol Concentration, and Time of Semen-Glycerol Contact on Survival of Spermatozoa

Australian Journal of Biological Sciences ◽

10.1071/bi9730231 ◽

1973 ◽

Vol 26 (1) ◽

pp. 231 ◽

Cited By ~ 10

Author(s):

I Wilmut ◽

S Salamon ◽

C Polge

Keyword(s):

Glycerol Concentration ◽

Factorial Experiments ◽

Deep Freezing ◽

Boar Semen ◽

Boar Spermatozoa

Five factorial experiments were conducted to examine the effects of glycerol concentration and processing procedures prior to freezing on the revival of boar spermatozoa upon thawing.

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Seminal plasma affects sperm sex sorting in boars

Reproduction Fertility and Development ◽

10.1071/rd14088 ◽

2016 ◽

Vol 28 (5) ◽

pp. 556 ◽

Cited By ~ 6

Author(s):

Diego V. Alkmin ◽

Inmaculada Parrilla ◽

Tatiana Tarantini ◽

David del Olmo ◽

Juan M. Vazquez ◽

...

Keyword(s):

Seminal Plasma ◽

Holding Time ◽

Oxygen Species ◽

Plasma Membrane Fluidity ◽

Progressive Motility ◽

Liquid Storage ◽

Boar Semen ◽

Sorting Efficiency ◽

Boar Spermatozoa

Two experiments were conducted in boar semen samples to evaluate how both holding time (24 h) and the presence of seminal plasma (SP) before sorting affect sperm sortability and the ability of sex-sorted spermatozoa to tolerate liquid storage. Whole ejaculate samples were divided into three aliquots immediately after collection: one was diluted (1 : 1, v/v) in Beltsville thawing solution (BTS; 50% SP); the SP of the other two aliquots was removed and the sperm pellets were diluted with BTS + 10% of their own SP (10% SP) or BTS alone (0% SP). The three aliquots of each ejaculate were divided into two portions, one that was processed immediately for sorting and a second that was sorted after 24 h storage at 15–17°C. In the first experiment, the ability to exhibit well-defined X- and Y-chromosome-bearing sperm peaks (split) in the cytometry histogram and the subsequent sorting efficiency were assessed (20 ejaculates). In contrast with holding time, the SP proportion influenced the parameters examined, as evidenced by the higher number of ejaculates exhibiting split and better sorting efficiency (P < 0.05) in semen samples with 0–10% SP compared with those with 50% SP. In a second experiment, the quality (viability, total and progressive motility) and functionality (plasma membrane fluidity and intracellular generation of reactive oxygen species) of sex-sorted spermatozoa were evaluated after 0, 72 and 120 h storage at 15–17°C (10 ejaculates). Holding time and SP proportion did not influence the quality or functionality of stored sex-sorted spermatozoa. In conclusion, a holding time as long as 24 h before sorting did not negatively affect sex sorting efficiency or the ability of sorted boar spermatozoa to tolerate long-term liquid storage. A high proportion of SP (50%) in the semen samples before sorting reduced the number of ejaculates to be sorted and negatively influenced the sorting efficiency, but did not affect the ability of sex-sorted spermatozoa to tolerate liquid storage.

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Fractionated Seminal Plasma of Boar Ejaculates Analyzed by LC–MS/MS: Its Effects on Post-Thaw Semen Quality

Genes ◽

10.3390/genes12101574 ◽

2021 ◽

Vol 12 (10) ◽

pp. 1574

Author(s):

Leyland Fraser ◽

Karolina Wasilewska-Sakowska ◽

Łukasz Zasiadczyk ◽

Elżbieta Piątkowska ◽

Krzysztof Karpiesiuk

Keyword(s):

Seminal Plasma ◽

Semen Quality ◽

Membrane Integrity ◽

Gel Filtration ◽

Protein Composition ◽

Liquid Chromatography Mass Spectrometry ◽

Beneficial Effects ◽

Sperm Membrane ◽

Boar Semen ◽

Boar Spermatozoa

This study aimed to characterize the protein composition of fractionated seminal plasma (SP) by liquid chromatography mass spectrometry (LC–MS/MS) analysis and investigate its effects on survival of frozen-thaw (FT) boar spermatozoa following storage. Seminal plasma (SP) was fractionated by gel filtration chromatography to give two fractions, SP1 with more than 40 kDa (>40 kDa) and SP2 with less than 40 kDa (<40 kDa). SP1 and SP2 were subjected to LC–MS/MS and bioinformatics analysis. Following cryopreservation, FT boar semen (n = 7) was thawed in Beltsville Thawing Solution (BTS), BTS + SP1 or BTS + SP2, stored at different periods and subjected to post-thaw (PT) quality assessment. A total of 52 and 22 abundant proteins were detected in SP1 and SP2, respectively. FN1, ANGPTL1, and KIF15 proteins were more abundance in SP1, whereas a high abundance of spermadhesins (PSP-I and PSP-II) was detected in SP2. Proteins of the fractionated SP were involved in various biological processes, such as cell motility and signal transduction. The dominant pathway of SP1 proteins was the apelin signaling pathway (GNA13, MEF2D, SPHK2, and MEF2C), whereas a pathway related to lysosome (CTSH, CTSB, and NPC2) was mainly represented by SP2 proteins. In most of the boars, significantly higher motility characteristics, membrane integrity, and viability were observed in FT spermatozoa exposed to SP1 or SP2 compared with BTS. The results of our study confirm that a combination of several proteins from the fractionated SP exerted beneficial effects on the sperm membrane, resulting in improved quality characteristics following PT storage.

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Fertility Test of Frozen Boar Semen

Australian Journal of Biological Sciences ◽

10.1071/bi9760335 ◽

1976 ◽

Vol 29 (4) ◽

pp. 335 ◽

Cited By ~ 3

Author(s):

O Osinowo ◽

S Salamon

Keyword(s):

Seminal Plasma ◽

Sperm Concentration ◽

Cryoprotective Agent ◽

Freezing Medium ◽

Boar Semen ◽

The Mean

The fertility results of two experiments are presented. In experiment 1, the semen was frozen in tris-fructose-EDTA or BF3 diluents at 0'25xl09/mI sperm concentration and extended after thawing with either seminal plasma (SP) or the freezing medium (FM) containing no cryoprotective agent. In the second experiment the semen was glycerolated by two methods, frozen at 1� 0 x 109/ml sperm concentration, and extended with FM before insemination. Fertility after double insemination within one oestrus with semen frozen in tris-fructose-EDTA or BF 3 diluents varied depending on the medium used for extension of thawed semen. The farrowing rates for semen frozen in the former diluent with FM and SP post-thawing media were 4/8 and 1/8 respectively, and for semen frozen in BF3 diluent with FM and SP post-thawing extenders 1/8 and 5/8 resp_ectively. The mean farrowing for the 32 animals inseminated was 34�4 %. Pregnancies for semen frozen in tris-fructose-EDTA and glycerolated at 30 or 5�e were 5/12 and 4/12 respectively, and for single and double inseminations 6/12 and 3/12 respectively. Of 24 animals inseminated 37� 5 % farrowed.

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Difference of seminal plasma and sperm proteins in good and poor freezability boar ejaculates

Veterinarska stanica ◽

10.46419/vs.53.2.8 ◽

2021 ◽

Vol 53 (2) ◽

Author(s):

Janyaporn Rungruangsak ◽

Junpen Suwimonteerabutr ◽

Kakanang Buranaamnuay ◽

Sariya Asawakarn ◽

Naphat Chantavisoote ◽

...

Keyword(s):

Sperm Motility ◽

Seminal Plasma ◽

Plasma Proteins ◽

Triosephosphate Isomerase ◽

Progressive Motility ◽

Sperm Proteins ◽

Seminal Plasma Proteins ◽

Boar Semen ◽

High Level ◽

Boar Spermatozoa

The present study was performed to compare the expression of sperm proteins, i.e. triosephosphate isomerase (TPI) and acrosin binding protein (ACRBP) and seminal plasma proteins, i.e. glutathione peroxidase 5 (GPX5) and fibronectin 1 (FN1), in boar semen with good, moderate and poor freezability. The study was conducted by determining the protein contents in 32 sperm samples and 38 seminal plasma samples of semen. The ejaculated semen was divided into two portions: the first portion was centrifuged to separate the pellet of sperm from the seminal plasma and the second portion was cryopreserved. After thawing, the ejaculates were classified into three groups according to their post-thawed sperm motility: good (60.2 ± 1.7%), moderate (29.3 ± 2.0%) and poor (16.6 ± 2.2%) freezabilities. The expressions of GPX5 and FN1 in seminal plasma and TPI and ACRBP in sperm were determined using Western blot analysis. It was found that, for sperm proteins, the level of TPI was negatively correlated with the post-thawed total sperm motility (r = -0.38, P = 0.029). For seminal plasma proteins, the level of FN1 in the seminal plasma was positively correlated with the post-thawed total sperm motility (r = 0.37, P = 0.021) and progressive motility (r = 0.39, P = 0.016). The expression of GPX5 was not correlated with any of the frozen–thawed sperm qualities (P > 0.05). In conclusions, boar semen containing a high level of FN1 in seminal plasma has better freezability. Frozen–thawed sperm motility was positively correlated with the level of FN1 in boar seminal plasma and negatively correlated with TPI in boar spermatozoa.

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Thawing boar semen in the presence of seminal plasma improves motility, modifies subpopulation patterns and reduces chromatin alterations

Reproduction Fertility and Development ◽

10.1071/rd15530 ◽

2017 ◽

Vol 29 (8) ◽

pp. 1576 ◽

Cited By ~ 8

Author(s):

Rocío Fernández-Gago ◽

Manuel Álvarez-Rodríguez ◽

Marta E. Alonso ◽

J. Ramiro González ◽

Beatriz Alegre ◽

...

Keyword(s):

Seminal Plasma ◽

Chromatin Structure ◽

Sperm Chromatin ◽

Chromatin Compaction ◽

Sperm Analysis ◽

Positive Effects ◽

Fragmentation Index ◽

Non Linear ◽

Boar Semen ◽

Dna Fragmentation Index

Seminal plasma could have positive effects on boar semen after thawing. In the present study we investigated changes in the motility and chromatin structure in spermatozoa over 4 h incubation (37°C) of boar semen thawed in the presence of 0%, 10% or 50% seminal plasma from good-fertility boars. Cryopreserved doses were used from seven males, three of which were identified as susceptible to post-thawing chromatin alterations. Motility was analysed by computer-aided sperm analysis every hour, and data were used in a two-step clustering, yielding three subpopulations of spermatozoa (slow non-linear, fast non-linear, fast linear). Chromatin structure was analysed using a sperm chromatin structure assay and flow cytometry to determine the DNA fragmentation index (%DFI) as a percentage, the standard deviation of the DFI (SD-DFI) and the percentage of high DNA stainability (%HDS), indicating chromatin compaction. Thawing without seminal plasma resulted in a rapid loss of motility, whereas seminal plasma helped maintain motility throughout the incubation period and preserved the subpopulation comprising fast and linear spermatozoa. The incidence of chromatin alterations was very low in samples from non-susceptible males, whereas samples from males susceptible to post-thawing chromatin alterations exhibited marked alterations in %DFI and %HDS. Seminal plasma partly prevented these alterations in samples from susceptible males. Overall, 50% seminal plasma was the most efficient concentration to protect motility and chromatin. Some changes were concomitant with physiological events reported previously (e.g., semen thawed with 50% seminal plasma increased the production of reactive oxygen species and yielded higher fertility after AI). Thawing in the presence of seminal plasma could be particularly useful in the case of samples susceptible to post-thawing chromatin damage.

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