Relação entre a capacitação espermática e a fertilidade de sêmen criopreservado de bovinos / Relationship between sperm capacitation and fertility in bovine cryopreserved semen

A qualidade do sêmen criopreservado, utilizado na inseminação artificial em tempo fixo (IATF) em bovinos, é um dos principais fatores que impactam sobre a fertilidade, e está relacionada à capacidade de produção espermática dos touros, à criotolerância dos espermatozoides e aos critérios técnicos do processo de criopreservação adotados. Neste sentido, devemos destacar a importância do controle de qualidade das partidas de sêmen antes de serem liberadas para uso na IATF. Nos últimos anos várias técnicas vêm sendo desenvolvidas para avaliar com mais acurácia as partidas de sêmen e evitar o uso daquelas que possam resultar em prejuízos na fertilidade, mas mesmo assim, tem se notado alta variabilidade na taxa de prenhez entre touros e entre partidas de sêmen. Esta divergência se deve ao fato da habilidade fértil do espermatozoide ser multifatorial, ou seja, dependente de diversas características estruturais, morfofuncionais e moleculares. Ademais, os eventos que permitem os espermatozoides passarem por capacitação espermática, um pré-requisito para a fertilidade, têm intrigado os pesquisadores em vista da complexidade dos processos envolvidos e dos efeitos deletérios da criopreservação espermática. Esta revisão tem por objetivo compilar estudos que mostrem a relação entre a capacitação espermática e a fertilidade do sêmen bovino criopreservado, levando em consideração as técnicas de avaliação e os resultados até o momento.(AU)

The quality of cryopreserved semen, used in fixed-time artificial insemination (FTAI) in cattle, is one of the main factors that impact fertility and is related to the sperm production capacity of bulls, sperm cryotolerance, and the technical criteria for cryopreservation. In this sense, we must highlight the importance of quality control of semen batches before commercialization for the FTAI. In recent years, several techniques have progressed to more accurately evaluate semen batches and avoid using those that may result in impaired fertility. However, we still notice a high variability in the pregnancy rate between bulls and between semen batches. This divergence is due to the multifactorial sperm-fertilizing ability, i.e., it depends on several structural, morphofunctional, and molecular characteristics. In addition, the events that allow sperm to undergo sperm capacitation, a prerequisite for fertility, have intrigued researchers due to the complexity of the processes involved and the adverse effects of sperm cryopreservation. This review aims to compile studies that show the relationship between sperm capacitation and fertility in cryopreserved bovine semen, considering the evaluation techniques and results to date.(AU)

Sperm cell capacitation status of ram semen after cooling

Background: The use of conventional artificial insemination (AI) in sheep production is usually associated with lower fertility rates when frozen semen is used. Cooled ram semen has been an alternative over frozen semen due to the higher viability, seminal quality and fertility rates following AI. The semen preservation process promotes sperm cell modifications similar to capacitation (capacitation-like) that causes cell damage affecting viability and seminal quality, but such effects are unclear for cooled semen. The aim of this study was to determine the status of sperm cell capacitation (CA) and acrosome reaction (AR) during ram semen processing and cooling under different extenders, dilution factors, and aerobiosis conditions as a function of storage time at 5o C. Materials, Methods & Results: Two consecutive ejaculates per day per male were collected from 2 adult rams by artificial vagina at 48-72 h intervals, in three replications. After macro- and microscopic evaluations, semen was segregated into groups under 3 extenders (Tris-egg yolk or TY, citrate-egg yolk or CY, skimmed milk or SM), 2 dilution factors (1 x 109 or Bi, 100 x 106 or Mi cells/mL), and 2 aerobiosis conditions (aerobic or A, semi-anaerobic or SA). Diluted semen was cooled to 5ºC and stored for up to 72 h, with evaluations every 24 h. Aliquots of fresh ejaculates and of each cooled diluted subgroup, according to extender, dilution, and aerobiosis, were collected at times T0 and T72 for determination of acrosome status and membrane integrity by the chlortetracycline (CTC) and trypan blue-Giemsa stainings, respectively. No differences were detected in sperm cell motility (M) and motility vigor (V) between fresh and diluted semen. After cooling, a significant decrease in M was observed after 48 h in CY and SM compared with fresh semen and 0 h of cooling, while V started to decrease after 24 h in CY compared with TY. Likewise, M/V from different dilutions and aerobic conditions decreased more significantly after 48 and 24 h of cooling, respectively. The sperm capacitation status did not show differences in the proportion of non-capacitated (NCA), CA and AR sperm cells between TY, CY, and SM extenders (NCA: 75.0%, 71.3%, 74.0%; CA: 15.7%, 17.2%, 15.9%; AR: 9.3%, 11.5%, 10.2%) or between Bi and Mi dilutions (NCA: 74.0%, 72.9%; CA: 15.9%, 16.6%; AR: 10.1%, 10.5%), respectively. However, differences (P < 0.05) were observed between A and SA aerobic conditions, with CA (17.0% vs. 15.5%) and AR (11.9% vs. 8.7%) rates being higher in A than SA, respectively, with no differences in NCA (71.1% vs. 75.8%), irrespective of the storage time. Sperm cell viability decreased after 48 h, especially in CY (P < 0.05). Discussion: Ram sperm cells can suffer irreversible damage due to thermal shock during cooling. Egg yolk-based extenders provide phospholipids and cholesterol to protect the sperm cell membrane during the thermal shock caused by the change in temperature. In this study, sperm cells had irreversible decreases in M/V, with increase in acrosome and plasma membrane damage after cooling to 5ºC. The largest and smallest decreases in M and V over time were observed in the CY and TY extenders, respectively. In addition to the extender type, the semen preservation method and storage time promoted changes in the capacitation status, AR and in sperm cell viability, which per se were associated with a decrease in semen fertility. In fact, the proportions of CA and/or AR sperm cells gradually increased over time after dilution and storage at 5ºC, with a negative correlation between sperm cell viability and M/V over time. In summary, extender and cooling time affected mostly M/V, while aerobiosis condition and dilution factor were more associated with acrosome status and sperm survival, with the extender having less impact on the acrosome status as a function of time.

Effect of glycerol addition time on the cryopreserved Korean native brindle cattle (Chikso) sperm quality

Although cryopreservation is an efficient method for maintaining the biological and genetic resources of sperm, the sperm damage during the cryopreservation process cannot be ignored. It should be possible to obtain the most effective cryopreservation performance by accurately grasping the effects of various factors on the cryopreservation of sperm. The previous study demonstrated that a suitable standard protocol for cryopreservation of Korean native brindled cattle (Chikso) does not exist, based on the methods for semen cryopreservation of Chikso differ in each research center. The most obvious difference between most of protocols is the addition of glycerol before and after cooling during the Chikso cryopreserved semen process. Therefore we focused on the effects of glycerol addition time on the quality of cryopreserved Chikso sperm. In the present study, 27 individual Chikso samples were collected by transrectal massage and divided into two parts: the "cryopreservation method A" group (adding glycerol before cooling) and the "cryopreservation method B" group (adding glycerol after cooling). Meanwhile, the values of various sperm parameters were derived from each group, including sperm motility, kinematics, capacitation status, cell viability, and intracellular ATP levels, which we used to compare and evaluate sperm function. The results of this study indicated that during the semen cryopreservation process of the Chikso, the addition of glycerol after cooling yielded superior results in a variety of sperm parameters, such as sperm motility, progressive motility, rapid motility, VCL, VSL, VAP, ALH, capacitation status, viability, and intracellular ATP level after freezing and thawing. Our study is suggested that the glycerol addition time during the cryopreservation process for Chikso should be considered. In addition, our results may be provided reference to develop suitable the cryopreservation procedure of the Chikso sperm.(AU)

A L-arginina na produção in vitro de embriões bovinos: perspectivas para o futuro / L-arginine in the in vitro production of bovine embryos: perspectives for the future

A L-arginina (L-arg) é o principal precursor da síntese do NO, contudo, é precursora também da síntese de creatina, agmatina, ureia, síntese proteica, L-ornitina, poliaminas, L-prolina e L-glutamato. Nesta breve revisão, vamos falar de alguns resultados que estão sendo obtidos sobre o papel da L-arg na capacitação de espermatozoides bovinos e seu impacto na produção in vitro de embriões. Estudos in vitro mostraram que a adição de L-arg ao meio de capacitação espermática está associada a um aumento na produção de NO, que se correlaciona com aumento da motilidade e vigor, integridade da membrana plasmática e acrossomal, atividade mitocondrial, capacitação espermática, peroxidação lipídica, bem como com a produção de blastocistos. Além disso, a adição da L-arg ao meio de capacitação in vitro, altera o perfil de proteínas importantes ligadas ao processo de capacitação, fertilização e desenvolvimento embrionário inicial. Estes efeitos da L-arg são GMPc dependentes e independentes. Na maturação in vitro, entretanto, embora já tenham sido encontrados bons resultados com o uso do L-arg, mais estudos são necessários para determinar a concentração ideal a ser adicionada ao meio de maturação in vitro e seu impacto na produção de blastocistos. Visto que a pré-capacitação de espermatozoides induzida pela heparina em presença de L-arg foi o método mais eficiente na produção in vitro de embriões, sugerimos sua utilização. Mais pesquisas sobre o metabolismo da L-arg no espermatozoide e CCOs de bovinos durante eventos ligados à fertilização são necessários para se identificar novas vias que atuem nestas etapas in vitro visando o aumento da percentagem e qualidade de embriões bovinos produzidos in vitro.

L-arginine (L-arg) is the main source of NO synthesis; however, it is also a precursor of the synthesis of creatine, agmatine, urea, protein synthesis, L-ornithine, polyamines, L-proline, and Lglutamate. In this brief review, we will discuss some results obtained previously about the role of L-arg in the capacitation of bovine sperm and its impact on in vitro embryo production. In vitro studies have shown that the addition of L-arg to the sperm capacitation medium is associated with an increase in NO production, which in controlled levels is related to an increased motility and vigor, plasma and acrosomal membrane integrity, mitochondrial activity, sperm capacitation, peroxidation lipids, as well as with the blastocyst production. Furthermore, the addition of L-arg to the in vitro capacitation medium alters the profile of important proteins linked to the capacitation process, fertilization, and early embryonic development. These effects of L-arg are cGMP dependent and independent. In in vitro maturation, however, although good results have already been found with the use of L-arg, further studies are needed to determine the ideal concentration to be added to the in vitro maturation medium and its impact on the production of blastocysts. Since heparin-induced pre-capacitation of spermatozoa in the presence of L-arg was the most efficient method for in vitro embryo production, we suggest its use. More research on L-arg metabolism in bovine sperm and OCCs during events related to fertilization is needed to identify new pathways that act in these in vitro steps aiming to increase the percentage and quality of bovine embryos produced in vitro.

Criopreservação e crioprotetores na proteção do espermatozoide suíno / Cryopreservation and cryoprotectors in protection of the swine sperm

O uso de sêmen congelado apresenta menores índices de concepção e menor número de leitões nascidos por parto. Isso ocorre porque a criopreservação acarreta variações de temperatura, alteração da permeabilidade espermática e processos oxidativos, sendo que o sêmen suíno apresenta, naturalmente, baixa capacidade antioxidante e sofre estresse oxidativo. No entanto, vários crioprotetores são utilizados para diversas espécies e já é descrito o uso de colesterol, carreado com ciclodextrina, e de aminoácidos, visando a melhoria do sêmen após o descongelamento. A adição de colesterol ao sêmen, previamente à criopreservação, mantém a integridade da membrana acrossomal e retarda a capacitação espermática, feito que pode ser facilitado com o carreamento por ciclodextrina. Em paralelo, pesquisas apontam a importância do uso de aminoácidos no sêmen, que formam uma camada de proteção térmica que preserva a integridade da célula espermática, além de apresentarem função osmorregulativa. Nesse sentido, a viabilização da criopreservação do sêmen suíno favorece o melhoramento genético, minimiza a transmissão de patógenos e possibilita a formação de um banco de germoplasma. Para tanto, faz-se necessário o aprimoramento do uso de crioprotetores em uma proporção ideal, que mantenha a integridade da membrana espermática e não prejudique a capacitação espermática e a reação acrossomal, necessárias para a fecundação.

The use of frozen semen presents lower conception rates and fewer piglets born per birth. This is due to the fact that cryopreservation causes variations in temperature, changes in sperm permeability and oxidative processes, and the swine semen naturally presents low antioxidant capacity and undergoes oxidative stress. However, several cryoprotectants are used for several species, and the use of cyclodextrin and amino acid-loaded cholesterol is already described, aiming to improve the semen after thawing. The addition of cholesterol to the semen prior to cryopreservation maintains the integrity of the acrosomal membrane and slows sperm capacitation, which can be facilitated by cyclodextrin loading. In parallel, research indicates the importance of the use of amino acids in the semen, which form a layer of thermal protection that preserves the integrity of the sperm cell, in addition to presenting osmorregulatory function. In this sense, the viability of the cryopreservation of the swine semen favors the genetic improvement, minimizes the transmission of pathogens and allows the formation of a germplasm bank. Therefore, it is necessary to improve the use of cryoprotectants in an ideal proportion, which maintains the integrity of the sperm membrane and does not detract from the sperm capacitation and acrosomal reaction required for fertilization.

Criopreservação e crioprotetores na proteção do espermatozoide suíno / Cryopreservation and cryoprotectors in protection of the swine sperm

O uso de sêmen congelado apresenta menores índices de concepção e menor número de leitões nascidos por parto. Isso ocorre porque a criopreservação acarreta variações de temperatura, alteração da permeabilidade espermática e processos oxidativos, sendo que o sêmen suíno apresenta, naturalmente, baixa capacidade antioxidante e sofre estresse oxidativo. No entanto, vários crioprotetores são utilizados para diversas espécies e já é descrito o uso de colesterol, carreado com ciclodextrina, e de aminoácidos, visando a melhoria do sêmen após o descongelamento. A adição de colesterol ao sêmen, previamente à criopreservação, mantém a integridade da membrana acrossomal e retarda a capacitação espermática, feito que pode ser facilitado com o carreamento por ciclodextrina. Em paralelo, pesquisas apontam a importância do uso de aminoácidos no sêmen, que formam uma camada de proteção térmica que preserva a integridade da célula espermática, além de apresentarem função osmorregulativa. Nesse sentido, a viabilização da criopreservação do sêmen suíno favorece o melhoramento genético, minimiza a transmissão de patógenos e possibilita a formação de um banco de germoplasma. Para tanto, faz-se necessário o aprimoramento do uso de crioprotetores em uma proporção ideal, que mantenha a integridade da membrana espermática e não prejudique a capacitação espermática e a reação acrossomal, necessárias para a fecundação.(AU)

The use of frozen semen presents lower conception rates and fewer piglets born per birth. This is due to the fact that cryopreservation causes variations in temperature, changes in sperm permeability and oxidative processes, and the swine semen naturally presents low antioxidant capacity and undergoes oxidative stress. However, several cryoprotectants are used for several species, and the use of cyclodextrin and amino acid-loaded cholesterol is already described, aiming to improve the semen after thawing. The addition of cholesterol to the semen prior to cryopreservation maintains the integrity of the acrosomal membrane and slows sperm capacitation, which can be facilitated by cyclodextrin loading. In parallel, research indicates the importance of the use of amino acids in the semen, which form a layer of thermal protection that preserves the integrity of the sperm cell, in addition to presenting osmorregulatory function. In this sense, the viability of the cryopreservation of the swine semen favors the genetic improvement, minimizes the transmission of pathogens and allows the formation of a germplasm bank. Therefore, it is necessary to improve the use of cryoprotectants in an ideal proportion, which maintains the integrity of the sperm membrane and does not detract from the sperm capacitation and acrosomal reaction required for fertilization.(AU)

Effects of insulin on sperm cell quality in ram semen cooled at 5°C / Efeitos da insulina sobre a qualidade das células espermáticas no sêmen ovino refrigerado a 5oC

Insulin is present in the seminal plasma and is involved in sperm activities like motility and capacitation. However, the effects of insulin on the viability of cooled ram sperm are not fully understood. Therefore, the objective of the current study was to evaluate the effect of insulin addition on ram sperm maintained at 5ºC. Sperm samples were collected from six healthy, mature Santa Inês rams. The ejaculates were divided into two aliquots with (insulin group) or without (control group) insulin (3 IU mL-1) in the semen extender, and then cooled at 5°C for 48 hours. Subsequently, the sperm cells were evaluated for motility and kinetics using computer-assisted semen analysis. The samples were evaluated for acrosomal integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and membrane functionality by the hypoosmotic swelling test. The semen analysis was performed after 24 or 48 hours of cooling. There was an increased percentage of progressive sperm motility (%), straightness (%), linearity (%) and beat caudal frequency (Hz) in the insulin group after 24 and 48 hours of cooling (p < 0.05). However, insulin did not affect total sperm motility, sperm velocities (VSL, VAP and VCL) (µm seg-1), acrosomal integrity and membrane functionality during cooling (p > 0.05). In conclusion, the addition of 3 IU mL-1 insulin to ram semen extender improved the quality of sperm motility after cooling.(AU)

A insulina está presente no plasma seminal e participa de atividades espermáticas, como a motilidade e capacitação. Entretanto, os efeitos da insulina sobre a viabilidade do espermatozoide ovino resfriado ainda não estão elucidadas. Desta forma, o objetivo do presente estudo foi avaliar os efeitos da adição de insulina sobre o espermatozoide ovino durante o tempo de armazenamento à 5º C. Amostras espermáticas de seis carneiros da raça Santa Inês foram utilizadas. Os ejaculados foram divididos em duas aliquotas, com (grupo insulina) ou sem (grupo controle) adição de insulina (3 UI mL-1) no diluidor seminal e, posteriormente, resfriados até 5oC e mantidos armazenados por 48 horas. Em seguida, os espermatozoides foram avaliados quanto a motilidade e cinética utilizando um Sistema Computadorizado de Análise de Sêmen (CASA). Adicionalmente, as amostras espermáticas foram analizadas quanto a integridade acrosomal por meio de sondas fluorescentes (FITC-PNA) e, funcionalidade de membrana pelo teste hiposmótico. As análises seminais foram realizadas após 24 ou 48 horas de resfriamento. Foram verificados aumentos de espermatozoides com motilidade progressiva (%), retilinearidade (%), linearidade (%) e frequência de batimento caudal (BCF) (Hz) no grupo insulina após 24 ou 48 horas de resfriamento (p < 0.05). Entretanto, não houve efeito da adição de insulina sobre a porcentagem de espermatozoides moveis (%) e das velocidades espermáticas (VSL, VAP e VCL) (µm seg-1), integridade acrossomal e funcionalidade de membrana durante o resfriamento (p > 0.05). Conclui-se que adição de insulina (3 UI mL-1) no diluidor seminal melhora a qualidade da motilidade espermática durante o resfriamento.(AU)

Possibilidades do uso do RNA espermático como ferramenta na suinocultura / Possibilities of using sperm's RNA as a tool in pig farming

A seleção de macho mais férteis é essencial para a suinocultura moderna, a análise do transcriptoma espermático tem se mostrado como uma ferramenta interessante para podermos escolher animais com maior potencial de fertilidade ou congelabilidade de sêmen. O objetivo da presente revisão foi caracterizar os RNA encontrados em espermatozoides suínos e apresentar possíveis finalidades de seu uso na suinocultura. Por sofrer mudanças estruturais significativas, como meiose, compactação do DNA e perda de parte do citoplasma, as linhagens celulares que dão origem aos espermatozoides também apresentam uma população singular na população RNA. Os espermatozoides carregam um transcriptoma heterogêneo, com mRNA e muitos pequenos RNA truncados e de difícil avaliação. Eventos como congelamento, capacitação e estresse térmico causam alteração nos perfis de RNA espermático. Existem RNA que podemos relacionar como marcadores moleculares para diversas funções, além disso, há muitas evidências de que o RNA espermático contribua na modulação gênica durante a fertilização e desenvolvimento embrionário inicial, tornando estes em um importante alvo para estudos futuros.

The selection of fertile males is essential for modern pig farming, the sperm transcriptome analysis has been shown to be an interesting tool to be able to choose animals with greater fertility potential or semen freezeability. The aim of this review was to characterize the RNA found in boar spermatozoa and to present possible purposes for its use in pig farming. By undergoing significant structural changes, such as meiosis, DNA compaction and loss of part of the cytoplasm, spermatic cells also have a unique population in the RNA population. Sperm carry a heterogeneous transcriptome, with mRNA and many small and truncated RNAs. Events such as freezing, capacitation and heat stress cause changes in sperm RNA profiles. There are RNAs that we can relate as molecular markers for several functions, in addition, there is evidence that sperm RNA contributes to gene modulation during fertilization and early embryonic development, making these an important target for future studies.

Lectin spatial immunolocalization during in vitro capacitation in Tursiops truncatus spermatozoa

Spermatozoa interactions with the female reproductive tract and oocyte are regulated by surface molecules such as glycocalyx. The capacitation process comprises molecular and structural modifications which increase zona pellucida binding affinity. Lectins allowed us to describe glycocalyx changes during maturation, capacitation and acrosome reaction. This study had as its aim to identify lectin binding patterns using four lectins with different carbohydrate affinity in bottlenose dolphin (Tursiops truncatus) spermatozoa both before and after in vitro capacitation. Two semen samples from the same dolphin obtained on consecutive days were used, with four different lectin binding patterns becoming visible in both samples before and after capacitation. A highly stained equatorial segment with prolongations at the edges appeared as the most frequent pattern with Wheat germ agglutinin (WGA) in uncapacitated spermatozoa. However, it was homogeneously distributed over the acrosomal region after capacitation. Instead, the use of Peanut agglutinin (PNA) resulted in most spermatozoa showing high labelling in the acrosomal periphery region before capacitation and a homogeneous staining in the acrosomal region within the population of capacitated spermatozoa. Nevertheless, the most representative patterns with Concavalin A (ConA) and Aleuria aurantia agglutinin (AAA) lectins did not change before and after capacitation, labelling the acrosomal region periphery. These findings could contribute to the understanding of the reproductive biology of cetaceans and the improvement of sperm selection techniques.(AU)

Lectin spatial immunolocalization during in vitro capacitation in Tursiops truncatus spermatozoa

Spermatozoa interactions with the female reproductive tract and oocyte are regulated by surface molecules such as glycocalyx. The capacitation process comprises molecular and structural modifications which increase zona pellucida binding affinity. Lectins allowed us to describe glycocalyx changes during maturation, capacitation and acrosome reaction. This study had as its aim to identify lectin binding patterns using four lectins with different carbohydrate affinity in bottlenose dolphin (Tursiops truncatus) spermatozoa both before and after in vitro capacitation. Two semen samples from the same dolphin obtained on consecutive days were used, with four different lectin binding patterns becoming visible in both samples before and after capacitation. A highly stained equatorial segment with prolongations at the edges appeared as the most frequent pattern with Wheat germ agglutinin (WGA) in uncapacitated spermatozoa. However, it was homogeneously distributed over the acrosomal region after capacitation. Instead, the use of Peanut agglutinin (PNA) resulted in most spermatozoa showing high labelling in the acrosomal periphery region before capacitation and a homogeneous staining in the acrosomal region within the population of capacitated spermatozoa. Nevertheless, the most representative patterns with Concavalin A (ConA) and Aleuria aurantia agglutinin (AAA) lectins did not change before and after capacitation, labelling the acrosomal region periphery. These findings could contribute to the understanding of the reproductive biology of cetaceans and the improvement of sperm selection techniques.

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37°C in BTS without (CTR) or with (5, 10, 20 µg/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39°C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 µg/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen.

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37°C in BTS without (CTR) or with (5, 10, 20 µg/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39°C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 µg/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen.(AU)

Effects of the cryopreservation process on dog sperm integrity

Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P <0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.

Effects of the cryopreservation process on dog sperm integrity

Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P <0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.(AU)

Effect of the addition of sodium caseinate on the viability of cryopreserved buffalo semen / Efeito da adição de caseinato de sódio sobre a viabilidade do sêmen bubalino criopreservado

The use of cooled semen in artificial insemination operations results in higher pregnancy rates than the use of frozen semen. This result seems to be related to the more severe damage triggered by the freezing process than that observed during refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a substance capable of preventing early sperm capacitation, a significant cause of the decreased pregnancy rate resulting from the use of frozen semen. The first objective of this study was to evaluate whether a commercial egg yolk diluent developed for frozen bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent in combination with sodium caseinate during the procedures of buffalo sperm cryopreservation using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membrane integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study, it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the harmful effects related to seminal cryopreservation.

O uso de sêmen resfriado em operações de inseminação artificial resulta em taxas de prenhez mais altas do que o uso de sêmen congelado. Esse resultado parece estar relacionado aos danos mais severos desencadeados pelo processo de congelação, quando comparado com o de refrigeração. Devido à sua capacidade de se ligar às proteínas ligadoras de espermatozoides e íons cálcio, o caseinato de sódio foi estudado quanto a sua capacidade de prevenir a capacitação espermática precoce, uma causa significativa de diminuição da taxa de prenhez com o uso de sêmen congelado. O primeiro objetivo deste estudo foi avaliar se um diluidor comercial a base de gema de ovo, destinado à congelação de sêmen bovino, poderia ser usado para a criopreservação de sêmen bubalino; o segundo objetivo foi investigar o efeito da adição do caseinato de sódio ao diluidor estudado durante os procedimentos de criopreservação de espermatozoides bubalinos. Foram empregadas a citometria de fluxo e a análise computadorizada do movimento espermático como métodos de avaliação seminal. Na primeira fase do estudo, comparando-se os resultados da cinética espermática e da integridade das membranas plasmática e acrossomal, observou-se que o processo de congelação promoveu mais danos celulares que o processo de resfriamento. Na segunda fase do estudo, não foram observados efeitos benéficos da adição de caseinato de sódio ao diluente empregado. A partir dos resultados do presente estudo, foi possível concluir que o diluente à base de gema de ovo foi adequado para a criopreservação do sêmen de bubalino e que a adição do caseinato de sódio não diminuiu os efeitos deletérios desencadeados pelo processo de criopreservação seminal.

Effect of the addition of sodium caseinate on the viability of cryopreserved buffalo semen / Efeito da adição de caseinato de sódio sobre a viabilidade do sêmen bubalino criopreservado

The use of cooled semen in artificial insemination operations results in higher pregnancy rates than the use of frozen semen. This result seems to be related to the more severe damage triggered by the freezing process than that observed during refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a substance capable of preventing early sperm capacitation, a significant cause of the decreased pregnancy rate resulting from the use of frozen semen. The first objective of this study was to evaluate whether a commercial egg yolk diluent developed for frozen bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent in combination with sodium caseinate during the procedures of buffalo sperm cryopreservation using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membrane integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study, it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the harmful effects related to seminal cryopreservation.(AU)

O uso de sêmen resfriado em operações de inseminação artificial resulta em taxas de prenhez mais altas do que o uso de sêmen congelado. Esse resultado parece estar relacionado aos danos mais severos desencadeados pelo processo de congelação, quando comparado com o de refrigeração. Devido à sua capacidade de se ligar às proteínas ligadoras de espermatozoides e íons cálcio, o caseinato de sódio foi estudado quanto a sua capacidade de prevenir a capacitação espermática precoce, uma causa significativa de diminuição da taxa de prenhez com o uso de sêmen congelado. O primeiro objetivo deste estudo foi avaliar se um diluidor comercial a base de gema de ovo, destinado à congelação de sêmen bovino, poderia ser usado para a criopreservação de sêmen bubalino; o segundo objetivo foi investigar o efeito da adição do caseinato de sódio ao diluidor estudado durante os procedimentos de criopreservação de espermatozoides bubalinos. Foram empregadas a citometria de fluxo e a análise computadorizada do movimento espermático como métodos de avaliação seminal. Na primeira fase do estudo, comparando-se os resultados da cinética espermática e da integridade das membranas plasmática e acrossomal, observou-se que o processo de congelação promoveu mais danos celulares que o processo de resfriamento. Na segunda fase do estudo, não foram observados efeitos benéficos da adição de caseinato de sódio ao diluente empregado. A partir dos resultados do presente estudo, foi possível concluir que o diluente à base de gema de ovo foi adequado para a criopreservação do sêmen de bubalino e que a adição do caseinato de sódio não diminuiu os efeitos deletérios desencadeados pelo processo de criopreservação seminal.(AU)

Mini-Percoll technique induces similar capacitation features in domestic ruminant frozen-thawed spermatozoa regardless of the presence of heparin

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modificationsthat render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium,heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PMand would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes inbull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quicklythan buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similarwhether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by miniPercoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status andsperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed spermafter mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percolland supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes),capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P < 0.05) in most of the spermmotility parameters after mini-Percoll. Conversely, ovine samples presented...

Mini-Percoll technique induces similar capacitation features in domestic ruminant frozen-thawed spermatozoa regardless of the presence of heparin

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modificationsthat render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium,heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PMand would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes inbull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quicklythan buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similarwhether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by miniPercoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status andsperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed spermafter mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percolland supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes),capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P < 0.05) in most of the spermmotility parameters after mini-Percoll. Conversely, ovine samples presented...(AU)

Different approaches for assessing sperm function

Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction. In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the “one shot” parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways. Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their subpopulation distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways.

Different approaches for assessing sperm function

Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction. In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the “one shot” parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways. Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their subpopulation distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways.(AU)