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Cryopreservation of boar semen in 0.5mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability

A manutenção da viabilidade do sêmen suíno criopreservado em palhetas de 0,5mL em baixas concentrações espermáticas é melhor do que em altas concentrações

ABSTRACT:

To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.

INDEX TERMS:
Cryopreservation; boar semen; spermatozoa concentration; sperm viability; cryoinjury; cryocapacitation; swine

RESUMO:

Até o momento, não foram realizados estudos que avaliassem o efeito da concentração de espermatozoides/mL em palhetas (0,5mL) para a criopreservação, levando-nos a analisar esta questão. Cada fração-rica do ejaculado (n=25) foi diluída em cinco diferentes concentrações de espermatozoides (100, 200, 300, 600 e 800x106 espermatozoides/mL), envasadas em palhetas de 0,5mL e posteriormente congeladas. Após a descongelação, os espermatozoides de todos os tratamentos foram avaliados a fim de determinar as características de motilidade usando um sistema de análise computadorizada dos espermatozoides (SCA-CASA). A integridade das membranas plasmática e acrosomal, o potencial de membrana mitocondrial, a peroxidação lipídica e a fluidez da membrana foram analisadas por citometria de fluxo. O aumento na concentração de espermatozoides acima de 300x106 espermatozoides/mL diminuiu (p<0,05) a motilidade total e progressiva, velocidade curvilínea, velocidade linear, linearidade e frequência de batimento. No entanto, a integridade da membrana plasmática e acrosomal, potencial de membrana mitocondrial, peroxidação lipídica e fluidez de membrana não foram influenciados (p>0,05) por altas concentrações de espermatozoides durante a criopreservação. Portanto, a fim de melhorar a sobrevivência dos espermatozoides suínos e a motilidade total e progressiva após a descongelação, os espermatozoides suínos devem ser congelados a concentrações não superiores a 300x106 espermatozoides/mL.

TERMOS DE INDEXAÇÃO:
Sêmen suíno; criopreservão; crioinjúria; concentração espermática; criocapacitação; suíno

Introduction

Frozen-thawed boar semen exhibits fertility rates lower than that of cooled semen because cryopreservation leads to significant loss of spermatozoa viability (Hernández et al. 2007Hernández M., Ekwall H., Roca J., Vazquez J.M., Martinez E. & Rodríguez-Martínez H. 2007. Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars. Cryobiology 54(1):63-70. <http://dx.doi.org/10.1016/j.cryobiol.2006.11.004> <PMid:17222399>
https://doi.org/10.1016/j.cryobiol.2006....
). Thus, an excess of spermatozoa (3 to 5 billion sperm) are used to perform artificial insemination (AI) with frozen-thawed boar semen to guarantee a higher live cell proportion (Ekwall et al. 2007Ekwall H., Hernández M., Saravia F. & Rodríguez-Martínez H. 2007. Cryo-scanning electron microscopy (Cryo-SEM) of boar semen frozen in medium-straws and MiniFlatPacks. Theriogenology 67(9):1463-1472. <http://dx.doi.org/10.1016/j.theriogenology.2007.03.004> <PMid:17448530>
https://doi.org/10.1016/j.theriogenology...
), which requires more thawed straws when performing a single insemination. However, cryopreservation of a high concentration of spermatozoa with good integrity and functionality would allow the use of a smaller number of straws when performing AI facilitating its implementation on swine industry.

On the other hand, the cryopreservation process leads to the formation of extracellular ice crystals and channels of unfrozen medium, as demonstrated by Mazur (1984)Mazur P. 1984. Freezing of living cells: mechanisms and implications. I. Am. J. Physiol. 247(3):C125-C142. <http://dx.doi.org/10.1152/ajpcell.1984.247.3.C125> <PMid:6383068>
https://doi.org/10.1152/ajpcell.1984.247...
and Rodríguez-Martínez & Wallgren (2011)Rodriguez-Martinez H. & Wallgren M. 2011. Advances in boar semen cryopreservation. Vet. Med. Int. 2011:1-5. <http://dx.doi.org/10.4061/2011/396181> <PMid:20871820>
https://doi.org/10.4061/2011/396181...
. Some extracellular water (approx. 15%) is present in the channels of unfrozen medium (Amann & Pickett 1987Amann R.P. & Pickett B.W. 1987. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J. Eq. Vet. Sci. 7(3):145-173. <http://dx.doi.org/10.1016/S0737-0806(87)80025-4>
https://doi.org/10.1016/S0737-0806(87)80...
) such that during freezing, the spermatozoa are encapsulated in these channels, increasing post-thawed cell survival (Mazur 1984Mazur P. 1984. Freezing of living cells: mechanisms and implications. I. Am. J. Physiol. 247(3):C125-C142. <http://dx.doi.org/10.1152/ajpcell.1984.247.3.C125> <PMid:6383068>
https://doi.org/10.1152/ajpcell.1984.247...
, Ekwall 2009Ekwall H. 2009. Cryo-scanning electron microscopy discloses differences in dehydration of frozen boar semen stored in large containers. Reprod. Dom. Anim. 44(1):62-68. <http://dx.doi.org/10.1111/j.1439-0531.2007.00994.x> <PMid:18673328>
https://doi.org/10.1111/j.1439-0531.2007...
). Furthermore, an increase in spermatozoa concentration up to the limit of channels of unfrozen medium could reduce the possibity of spermatozoa be encapsulated in these channels. Thus, an ideal spermatozoa concentration to cryopreservation is crucial to ensure spermatozoa encapsulation on channels of unfrozen medium increasing post-thawed cell survival.

Thus, we sought to determine the effects of different spermatozoa concentration of frozen-thawed boar semen in 0.5mL straws on the kinetic parameters, plasma and acrosomal membrane integrity, mitochondrial potential, membrane lipid disorder and peroxidation in cryopreserved boar.

Materials and Methods

Ethics statement. The experiment was conducted at Swine Research Center, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), in compliance with Ethics Principles in Animal Experimentation, being approved by the the Ethics Committee for the use of Animals in the School of Veterinary Medicine and Animal Science, USP, under protocol 3066/2013.

Reagents and chemicals. Semen extender medium (Botu-Sui®) was purchased from Biotech-Botucatu- Ltd⁄ME (Botucatu, SP, Brazil). The fluorescent probes Hoechst 33342, C11-BODIPY581/591, Yo-Pro-1 and Merocyanine 540 were purchased from Molecular Probes (Eugene, OR, USA). Unless otherwise stated, all other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA).

Semen collection, raw semen evaluation, freezing and semen thawing. Five whole sperm-rich fractions were obtained from five cross-bred boars (n=25) using the gloved-hand technique. Only ejaculates with more than 70% of total motility and 80% of normal spermatozoa were used. After the initial raw semen analysis, the semen was diluted (Botu-Sui®) at five different concentrations using a Neubauer Hemocytometer (100x106 spermatozoa/mL, 200x106 spermatozoa/mL, 300x106 spermatozoa/mL, 600x106 spermatozoa/mL and 800x 106 spermatozoa/mL) and stored in 0.5mL straws (IMV, Laigle, France). The straws were cryopreserved in an automatic machine (TK 3000, TK Tecnologia em Congelação Ltda, Uberaba, Brazil) and cooled at a rate was -0.5°C/min from 25°C to 5°C. The freezing rate was -20°C/min from 5 to -120°C. Subsequently, the straws were immersed in liquid nitrogen at -196°C and stored in goblets within cryogenic tanks (Torres et al. 2016aTorres M.A., Ravagnani G.M., Leal D.F., Martins S.M., Muro B.B., Meirelles F.V., Papa F.O., Dell’aqua J.A., Alvarenga M.A., Moretti A.S. & Andrade A.F. 2016a. Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing. J. Anim. Sci. 94(5):1906-1912. <http://dx.doi.org/10.2527/jas.2016-0293> <PMid:27285688>
https://doi.org/10.2527/jas.2016-0293...
). Two straws per ejaculate and treatment were thawed in a water bath at 37°C for 30 seconds and subsequently pooled. The thawed semen was diluted to a final concentration of 25x106 spermatozoa/mL in a freezing extender.

Computer-assisted sperm analysis (CASA). A sample aliquot (5µL) was placed on a pre-warmed (37°C) cover slide and evaluated by phase contrast microscopy (Nikon, Model Eclipse 80i) at 100x magnification. The SCA (Microptics® - Barcelona/Spain) was pre-adjusted to analyze swine semen, and five fields were analyzed to evaluate the total (MOT) and progressive motility (MOP), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF).

Flow cytometry analysis. The samples for staining and flow cytometry analysis were diluted (5x106 spermatozoa/mL) in TALP medium and stained with Hoechst 33342 (H342, 33µg/mL in 150μL, PMT-photomultiplier tubes with a 450±10nm band pass filter) for 10 min at 37°C, this probe was used to exclude particles with similar scatter properties as spermatozoa from analysis (DeAndrade et al. 2012Andrade A.F., Zaffalon F.G., Celeghini E.C., Nascimento J., Bressan F.F., Martins S.M. & Arruda R.P. 2012. Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation. Theriogenology 77(9):1866-1872.e1. <http://dx.doi.org/10.1016/j.theriogenology.2012.01.003> <PMid:22444550>
https://doi.org/10.1016/j.theriogenology...
). Samples were analyzed in a BD FACSAria flow cytometer (Becton Dickinson, San Jose, CA, USA) controlled by BD FACSDiva 6.0 software (Becton Dickinson).

Frozen-thawed boar semen was analyzed to simultaneous integrity of plasma, acrosomal membranes and mitochondrial potential by flow cytometry (Torres et al. 2016bTorres M.A., Díaz R., Boguen R., Martins S.M., Ravagnani G.M., Leal D.F., Oliveira Mde.L., Muro B.B., Parra B.M., Meirelles F.V., Papa F.O., Dell’Aqua Junior J.A., Alvarenga M.A., Moretti Ade.S., Sepúlveda N. & de Andrade A.F. 2016b. Novel flow cytometry analyses of boar sperm viability : Can the addition of whole sperm-rich fraction seminal plasma to frozen-thawed boar sperm affect it? PlosOne 11(8):e0160988. <http://dx.doi.org/10.1371/journal.pone.0160988> <PMid:27529819>
https://doi.org/10.1371/journal.pone.016...
). For this propose, following probes association was used: Hoechst 33342 (H342), propidium iodide (PI -10µg/mL), Pisum sativum agglutinin conjugated to FITC (PSA-13.4µg/mL) and JC-1 (4.08µM). The plasma (IP) and acrosomal membrane (IA) integrity and mitochondrial membrane potential (HM) were also assessed individually.

Peroxidation of plasma membrane lipids was performed with the same protocol described by De Andrade et al. (2012)Andrade A.F., Zaffalon F.G., Celeghini E.C., Nascimento J., Bressan F.F., Martins S.M. & Arruda R.P. 2012. Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation. Theriogenology 77(9):1866-1872.e1. <http://dx.doi.org/10.1016/j.theriogenology.2012.01.003> <PMid:22444550>
https://doi.org/10.1016/j.theriogenology...
by the association of H342, C11- BODIPY581/591 probe (3.3µg/mL) and PI (10µg/mL).

Plasma membrane fluidity was performed by the association of H342, Yo-Pro-1 (25nM) and Merocyanine 540 (2,7µM) and evaluated by flow crytometry cytometry (Rathi et al. 2001Rathi R., Colenbrander B., Bevers M.M. & Gadella B.M. 2001. Evaluation of in vitro capacitation of stallion spermatozoa. Biol. Reprod. 65(2):462-470. <http://dx.doi.org/10.1095/biolreprod65.2.462> <PMid:11466214>
https://doi.org/10.1095/biolreprod65.2.4...
).

Statistical analysis. The data were analyzed using the MIXED procedure in SAS software (Statistical Analisys System 2002Statistical Analysis System 2002. SAS User’s Guide: statistical analysis system. Release 9.3, SAS Institute, Cary, NC.) according to a randomized block that contained the treatments as the main factor. Each boar (n=5) was considered one block, and the experimental unit was 1/5 of the ejaculate. The effects of the treatments were evaluated using the PDDIF test. The effects were considered significant when p<0.05. The results are expressed as the means ± SD.

Results

Concentrations greater than 300x106 spermatozoa/mL were detrimental (p<0.05) to total and progressive motility. Concentrations of up to 600x106 spermatozoa/mL were deleterious (p< 0.05) to VSL, VAP and BCF. The LIN and STR remained stable at concentrations of up to 200x106 spermatozoa/mL (p>0.05) and decreased (p<0.05) at 300 x106 spermatozoa/mL and higher (Table 1).

Table 1.
Mean ± SD of kinematics sperm of frozen-thawed boar semen cryopreserved at five spermatozoa concentrations in 0.5mL straws

None of the concentrations of boar semen used for cryopreservation were harmful (p>0.05) to IPIAH (spermatozoa with simultaneous plasma, acrosomal membranes integrity and high mitochondrial potential), IP, IA or HM spermatozoa population (Table 2).

Table 2.
Mean ± SD of spermatozoa membranes integrity of frozen-thawed boar semen cryopreserved at five spermatozoa concentrations in 0.5mL straws

Lower (p<0.05) levels of lipid peroxidation were observed in the cryopreserved samples at 100x106 spermatozoa/mL, and the levels remained unchanged (p>0.05) at concentrations up to 200x106 spermatozoa/mL. The spermatozoa frozen at 600x106 spermatozoa/mL led to higher (p<0.05) levels of lipid peroxidation (Table 3).

Table 3.
Mean ± SD of median of fluorescence intensity of C11- BODIPY581/591 and Merocyanine 540 in frozen-thawed boar semen cryopreserved in 0.5mL straws

The plasma membrane remained stable (p>0.05) regardless of the spermatozoa concentration and was unaffected at concentrations up to 800x106 spermatozoa/mL (Table 3).

Discussion

The cryopreservation of spermatozoa still remains a challenge, as all of the spermatozoa compartments are subjected to harmful freezing effects and only approximately half of spermatozoa survive after thawing (Vadnais & Althouse 2011Vadnais M.L. & Althouse G.C. 2011. Characterization of capacitation, cryoinjury, and the role of seminal plasma in porcine sperm. Theriogenology 76(8):1508-1516. <http://dx.doi.org/10.1016/j.theriogenology.2011.06.021> <PMid:21803410>
https://doi.org/10.1016/j.theriogenology...
, De Andrade et al. 2012Andrade A.F., Zaffalon F.G., Celeghini E.C., Nascimento J., Bressan F.F., Martins S.M. & Arruda R.P. 2012. Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation. Theriogenology 77(9):1866-1872.e1. <http://dx.doi.org/10.1016/j.theriogenology.2012.01.003> <PMid:22444550>
https://doi.org/10.1016/j.theriogenology...
). Thus, the cryopreservation of high concentrations of spermatozoa might be a useful method to increase the absolute number of viable spermatozoa when inseminating frozen-thawed semen samples (Alvarez et al. 2012Alvarez M., Tamayo-Canul J., Anel E., Boixo J.C., Mata-Campuzano M., Martinez-Pastor F., Anel L. & de Paz P. 2012. Sperm concentration at freezing affects post-thaw quality and fertility of ram semen. Theriogenology 77(6):1111-1118. <http://dx.doi.org/10.1016/j.theriogenology.2011.10.013> <PMid:22192392>
https://doi.org/10.1016/j.theriogenology...
).

Cryopreservation of high concentrations of boar semen was harmful to sperm kinematic parameters, and both the total and progressive motility were decreased at concentrations higher than 300x106 spermatozoa/mL, whereas the VSL, VAP and BCF were only impaired at 800x106 spermatozoa/mL. In rams, the total spermatozoa motility appears to be less affected by high concentrations and did not exhibit additional harmful effects at 800x106 spermatozoa/mL, unlike the progressive motility, in which a deleterious effect was observed at concentration as low as 400x106 spermatozoa/mL (Alvarez et al. 2012Alvarez M., Tamayo-Canul J., Anel E., Boixo J.C., Mata-Campuzano M., Martinez-Pastor F., Anel L. & de Paz P. 2012. Sperm concentration at freezing affects post-thaw quality and fertility of ram semen. Theriogenology 77(6):1111-1118. <http://dx.doi.org/10.1016/j.theriogenology.2011.10.013> <PMid:22192392>
https://doi.org/10.1016/j.theriogenology...
). As previously reported, in jacks, high spermatozoa concentrations at freezing negatively impact sperm kinetics; furthermore, the total and progressive motility appear to be more sensitive to an increase in spermatozoa concentration at freezing compared with other sperm kinetics parameters (Contri et al. 2012Contri A., Gloria A., Robbe D., Sfirro M.P. & Carluccio A. 2012. Effect of sperm concentration on characteristics of frozen-thawed semen in donkeys. Anim. Reprod. Sci. 136(1/2):74-80. <http://dx.doi.org/10.1016/j.anireprosci.2012.10.022> <PMid:23182476>
https://doi.org/10.1016/j.anireprosci.20...
). The cryopreservation of boar semen with spermatozoa concentration up to the limit of unfrozen channels (300x106 spermatozoa/mL, as the evidence of present results) perhaps reduces encapsulated spermatozoa leading to damage in the spermatozoal tails. Besides that, spermatozoa frozen up to 300x106 spermatozoa/mL had a higher ratio of cryoprotectant agents per cell than samples at a higher sperm concentration. The higher the amount of cryoprotectant per sperm cell, possibly the higher the percentage of unfrozen water channels (Nascimento et al. 2008Nascimento J., Raphael C.F., Andrade A.F.C., Alonso M.A., Celeghini E.C.C. & Arruda R.P. 2008. Effects of sperm concentration and straw volume on motion characteristics and plasma, acrosomal, and mitochondrial membranes of equine cryopreserved spermatozoa. J. Eq. Vet. Sci. 28(6):351-358. <http://dx.doi.org/10.1016/j.jevs.2008.04.010>
https://doi.org/10.1016/j.jevs.2008.04.0...
).

The integrity of post-thawed spermatozoa compartments ensures the maintenance of sperm fertility (Rodríguez-Martínez 2007Rodriguez-Martinez H. 2007. Role of the oviduct in sperm capacitation. Theriogenology 68(Suppl.1):138-146. <http://dx.doi.org/10.1016/j.theriogenology.2007.03.018> <PMid:17452049>
https://doi.org/10.1016/j.theriogenology...
). Damage can be caused by physical (crystal ice) and biochemical (osmotic) changes (Watson 1995Watson P.F. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fert. Develop. 7(4):871-891. <http://dx.doi.org/10.1071/RD9950871> <PMid:8711221>
https://doi.org/10.1071/RD9950871...
). We hypothesized that the increase in spermatozoa concentration in the 0.5 straws at freezing would reduce the chances that the spermatozoa would be encapsulated in the channels not frozen. However, our results did not demonstrate that the increase in spermatozoa concentrations at freezing (up to 800x106 spermatozoa/mL) had a harmful effect on sperm membrane integrity or mitochondrial membrane potential, and it is possible that spermatozoa encapsulation in the channels not frozen was not prevented in this study. Although the plasma and acrosomal membranes are the most sensitive sperm compartments to cold shock (Salamon & Maxwell 1995Salamon S. & Maxwell W.M. 1995. Frozen storage of ram semen II causes of low fertility after cervical insemination and methods of improvement. Anim. Reprod. Sci. 38(1/2):1-36. <http://dx.doi.org/10.1016/0378-4320(94)01328-J>
https://doi.org/10.1016/0378-4320(94)013...
), the acrosome seems to be more resistant to the additional damage caused by an increase in spermatozoa concentration at freezing, similar to the results described in jacks (Contri et al. 2012Contri A., Gloria A., Robbe D., Sfirro M.P. & Carluccio A. 2012. Effect of sperm concentration on characteristics of frozen-thawed semen in donkeys. Anim. Reprod. Sci. 136(1/2):74-80. <http://dx.doi.org/10.1016/j.anireprosci.2012.10.022> <PMid:23182476>
https://doi.org/10.1016/j.anireprosci.20...
), stallions (Nascimento et al. 2008Nascimento J., Raphael C.F., Andrade A.F.C., Alonso M.A., Celeghini E.C.C. & Arruda R.P. 2008. Effects of sperm concentration and straw volume on motion characteristics and plasma, acrosomal, and mitochondrial membranes of equine cryopreserved spermatozoa. J. Eq. Vet. Sci. 28(6):351-358. <http://dx.doi.org/10.1016/j.jevs.2008.04.010>
https://doi.org/10.1016/j.jevs.2008.04.0...
), rams (Alvarez et al. 2012Alvarez M., Tamayo-Canul J., Anel E., Boixo J.C., Mata-Campuzano M., Martinez-Pastor F., Anel L. & de Paz P. 2012. Sperm concentration at freezing affects post-thaw quality and fertility of ram semen. Theriogenology 77(6):1111-1118. <http://dx.doi.org/10.1016/j.theriogenology.2011.10.013> <PMid:22192392>
https://doi.org/10.1016/j.theriogenology...
) and dogs (Okano et al. 2004Okano T., Murase T., Asano M. & Tsubota T. 2004. Effects of final dilution rate, sperm concentration and times for cooling and glycerol equilibration on post-thaw characteristics of canine spermatozoa. Theriogenology 66(11):1359-1364. <PMid:15585949>).

Furthermore, regarding mitochondrial membrane potential, equine spermatozoa are less sensitive (impaired at concentrations up to 400x106 spermatozoa/mL) to the increase in spermatozoa concentration at freezing than the plasma membrane (Nascimento et al. 2008Nascimento J., Raphael C.F., Andrade A.F.C., Alonso M.A., Celeghini E.C.C. & Arruda R.P. 2008. Effects of sperm concentration and straw volume on motion characteristics and plasma, acrosomal, and mitochondrial membranes of equine cryopreserved spermatozoa. J. Eq. Vet. Sci. 28(6):351-358. <http://dx.doi.org/10.1016/j.jevs.2008.04.010>
https://doi.org/10.1016/j.jevs.2008.04.0...
). However, our results were not able to demonstrate similar results, and it is possible that boar spermatozoa are not as greatly influenced by the concentration at freezing as equine spermatozoa.

The increase in spermatozoa death after cryopreservation increases free radical generation (Aitken et al. 1989Aitken R.J., Clarkson J.S. & Fishel S. 1989. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod. 41(1):183-197. <http://dx.doi.org/10.1095/biolreprod41.1.183> <PMid:2553141>
https://doi.org/10.1095/biolreprod41.1.1...
), leading us to believe that lipid peroxidation would increase upon freezing at high spermatozoa concentrations. Indeed, the increase in spermatozoa concentration at freezing increased lipid peroxidation of the sperm membranes, and concentration of 600x106 spermatozoa/mL at freezing were the most detrimental. To the best of our knowledge, the effect of increasing spermatozoa concentrations at freezing on the peroxidation of plasma membrane lipids has rarely been studied. Nevertheless, human spermatozoa are more resistant to lipid peroxidation when increased spermatozoa concentrations (5-40x106 spermatozoa/mL) are used at freezing (Wang et al. 1997Wang Y., Sharma R.K. & Agarwal A. 1997. Effect of cryopreservation and sperm concentration on lipid peroxidation in human semen. Urology 50(3):409-413. <http://dx.doi.org/10.1016/S0090-4295(97)00219-7> <PMid:9301706>
https://doi.org/10.1016/S0090-4295(97)00...
).

Capacitation-like changes are commonly observed in frozen-thawed semen samples (Neild et al. 2003Neild D.M., Gadella B.M., Chaves M.G., Miragaya M.H., Colenbrander B. & Agüero A. 2003. Membrane changes during different stages of a freeze-thaw protocol for equine semen cryopreservation. Theriogenology 59(8):1693-1705. <http://dx.doi.org/10.1016/S0093-691X(02)01231-1> <PMid:12566145>
https://doi.org/10.1016/S0093-691X(02)01...
). Ram spermatozoa have been proven to be very sensitive to an increase in concentration at freezing with regard to the early membrane changes (measured by PI/YO-PRO-1) when cryopreserved at 800x106 spermatozoa/mL (Alvarez et al. 2012Alvarez M., Tamayo-Canul J., Anel E., Boixo J.C., Mata-Campuzano M., Martinez-Pastor F., Anel L. & de Paz P. 2012. Sperm concentration at freezing affects post-thaw quality and fertility of ram semen. Theriogenology 77(6):1111-1118. <http://dx.doi.org/10.1016/j.theriogenology.2011.10.013> <PMid:22192392>
https://doi.org/10.1016/j.theriogenology...
). This result differs from boar spermatozoa, in which membrane fluidity was not influenced by the increase in spermatozoa concentration at freezing, as verified in our study.

Conclusions

To achieve high viability after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL, which would allow the majority of the spermatozoa to be encapsulated in the channels not frozen.

The concentration of boar spermatozoa at freezing must be carefully assessed.

More studies are required to define the ideal value because there is a large range of concentrations from 300 to 600 million spermatozoa/mL.

Acknowledgements

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grant number 2011/23484-8).

References

  • Aitken R.J., Clarkson J.S. & Fishel S. 1989. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod. 41(1):183-197. <http://dx.doi.org/10.1095/biolreprod41.1.183> <PMid:2553141>
    » https://doi.org/10.1095/biolreprod41.1.183
  • Alvarez M., Tamayo-Canul J., Anel E., Boixo J.C., Mata-Campuzano M., Martinez-Pastor F., Anel L. & de Paz P. 2012. Sperm concentration at freezing affects post-thaw quality and fertility of ram semen. Theriogenology 77(6):1111-1118. <http://dx.doi.org/10.1016/j.theriogenology.2011.10.013> <PMid:22192392>
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Publication Dates

  • Publication in this collection
    Sept 2018

History

  • Received
    20 Sept 2017
  • Accepted
    06 Nov 2017
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