Evaluación de la composición proteica y lipídica de la membrana plasmática del espermatozoide de llama y su relación con el proceso de refrigeración
Currently, there is no cryopreservation protocol for South American camelid (SAC) semen that achieves acceptable pregnancy rates following artificial insemination (AI). This issue can be attributed to the limited understanding of the biochemical composition of llama spermatozoa, which remains undocu...
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Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2025
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_8055 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_8055.dir/8055.PDF |
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I28-R145-HWA_8055 |
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| record_format |
dspace |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-145 |
| collection |
Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
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spa |
| topic |
Lípidos Proteínas Membrana plasmática Espermatozoide Llama Refrigeración Lipids Proteins Plasma membrane Sperm Llama Refrigeration Espermatozoides Semen refrigerado Llama Ciencias Veterinarias |
| spellingShingle |
Lípidos Proteínas Membrana plasmática Espermatozoide Llama Refrigeración Lipids Proteins Plasma membrane Sperm Llama Refrigeration Espermatozoides Semen refrigerado Llama Ciencias Veterinarias Bertuzzi, Mariana Lucía Evaluación de la composición proteica y lipídica de la membrana plasmática del espermatozoide de llama y su relación con el proceso de refrigeración |
| topic_facet |
Lípidos Proteínas Membrana plasmática Espermatozoide Llama Refrigeración Lipids Proteins Plasma membrane Sperm Llama Refrigeration Espermatozoides Semen refrigerado Llama Ciencias Veterinarias |
| description |
Currently, there is no cryopreservation protocol for South American camelid (SAC) semen that achieves acceptable pregnancy rates following artificial insemination (AI). This issue can be attributed to the limited understanding of the biochemical composition of llama spermatozoa, which remains undocumented. In this context, given the known impact of the lipid and protein composition of the cell membrane on both fertilization processes and sperm cryotolerance, we considered its study and characterization to be the main focus of this thesis. Moreover, due to the unique rheological characteristics of SAC ejaculates, different treatments have been studied to reduce thread formation and thereby improve sample handling. One of the most widely used methods is semen incubation with proteolytic enzymes, such as collagenase. The use of this enzyme not only reduces thread formation but also induces a change in sperm motility pattern from oscillatory to progressive, while increasing the percentage of sperm with functional membranes. Based on these findings, this thesis aimed to assess the lipid and protein profiles of llama sperm incubated with collagenase as an attempt to determine the potential impact of the enzyme on sperm membrane lipids and proteins. Although pregnancy rates are low when inseminating with cooled llama semen, sperm survival rates after refrigeration are comparatively higher than after freezing, making cooling a more appealing preservation method. At the time this thesis project was proposed, there were no reports evaluating either in vitro or in vivo effects of using commercial extenders on cooled llama sperm. Therefore, researching this topic was of interest, as these extenders offer certain advantages over hand-made ones. Regarding the addition of egg yolk (EY) to sperm cryopreservation extenders, beneficial effects have been reported in SACs as well as in other species of productive interest. The mechanisms by which EY protects cells from cold shock remain incompletely understood. It has been proposed that lipid components from EY bind tightly to spermatozoa and are not removed even after several washes, suggesting that EY lipoproteins could associate irreversibly with the sperm plasma membrane. For this reason, it became relevant to determine whether the addition of EY to the commercial extender modified the protein and/or lipid composition and/or cholesterol distribution in the plasma membrane of cooled llama sperm. Finally, as with any sperm preservation procedure, the final step to confirm its actual efficiency is to perform an in vivo test through AI, which was conducted as the final objective of this thesis. The objectives of the thesis plan were: 1) To determine the lipid and protein composition of the plasma membrane of raw llama spermatozoa, as well as the distribution of cholesterol within it; 2) To evaluate whether enzymatic treatment (collagenase) of raw llama semen modifies the composition of the sperm plasma membrane; 3) To assess the effect of semen cooling, using a commercial extender with the addition of EY, on the protein and lipid composition and the cholesterol distribution in the plasma membrane of llama sperm; and 4) To evaluate, through AI, the in vivo fertilizing capacity of cooled llama sperm using a commercial extender that preserves sperm quality characteristics. Objectives 1 and 2: Lipids: It was determined that in order to accurately measure the lipids of llama spermatozoa, samples containing at least 300 million total spermatozoa were required. In addition to evaluating lipids in sperm from raw semen and semen incubated with collagenase (0.02%), differences in lipid composition were investigated between sperm collected in summer versus winter, and between young and adult animals. The following parameters were evaluated: cholesterol (Chol), phospholipids (PL), the Chol/PL ratio, fatty acid (FA) groups, individual FAs, and PL classes in spermatozoa from raw semen and after incubation with collagenase. No significant differences were found in Chol, Chol/PL, FAs, or PL classes between raw and collagenase-treated sperm, although a trend toward increased PL content was observed with enzyme use. No significant differences were observed for these same parameters when comparing by collection season. Young males showed significantly lower PL content in spermatozoa from raw semen, along with a tendency toward higher Chol levels, a higher Chol/PL ratio, and lower percentages of polyunsaturated fatty acids (PUFA) compared to adult males. These findings were also associated with higher percentages of total sperm motility and live sperm with intact acrosomes in younger individuals. Chol and the Chol/PL ratio positively correlated with total sperm motility and live sperm with intact acrosomes. Cholesterol distribution in the plasma membrane: Four staining patterns were identified using Filipin labelling: F1) fluorescence throughout the sperm head, F2) fluorescence in the acrosomal region, F3) fluorescence in the post-acrosomal region, and F4) absence of fluorescence in the entire head or fluorescence limited to the equatorial segment. No differences in these patterns were observed between sperm from raw semen and those incubated with collagenase. Proteins: Total protein content was similar between spermatozoa from raw semen and those incubated with collagenase. Spermatozoa from young males showed lower protein content compared to those from adult males. In addition to quantification, electrophoresis was performed on sperm from raw semen, semen incubated with 0.02% collagenase (the concentration normally used in our laboratory), and with 0.2% collagenase (positive control). No differences were observed in the number of protein bands between electrophoresis profiles of raw sperm and those treated with 0.02% collagenase. However, in samples incubated with the higher enzyme concentration, a 95 kDa band present in raw semen spermatozoa disappeared, and a new lower molecular weight band (about 45 kDa) appeared, which was absent in untreated samples. The 95 kDa band corresponded to two extracellular proteins of the same family: Heat shock protein HSP 90-alpha and HSP 90-beta. The second sequenced band at 45 kDa matched two extracellular proteins: Heat shock protein HSP 90-alpha isoform and Lactotransferrin. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were also used to evaluate the samples, revealing that incubation of semen with high concentrations of collagenase not only induces damage to the outer acrosome membrane but also causes severe damage to the sperm plasma membrane ultrastructure. Objective 3: Based on a preliminary study, the commercial extenders AndroMed® and Androstar® Plus were selected. These extenders were later tested with and without the addition of 20% EY for llama semen cooling. The study determined that AndroMed® (AM), with and without EY, would be used to evaluate the lipid and protein composition as well as the cholesterol distribution in membranes of cooled spermatozoa. Additionally, it was confirmed that males differ in their susceptibility to the cooling process, with their response varying according to the extender used (AM vs. AM-EY). It was also determined that the hypo-osmotic swelling (HOS) test, for assessing membrane functionality, is related to sperm response to cooling. Lipids: Lower cholesterol content and Chol/PL ratio were observed in sperm refrigerated with AM compared to those refrigerated with AM-EY, and both compared to spermatozoa from raw semen. Another interesting finding was that the membranes of cooled sperm incorporated lipids with monounsaturated (MFA) and dienoic fatty acids (DFA) present in the extenders, such as oleic acid (18:1) and linoleic acid (18:2). This led to a decrease in polyunsaturated fatty acids (PUFA) and an increase in MFA and DFA in cooled sperm compared to raw sperm. Free fatty acids (FFA) were also detected in sperm cooled with AM, which were not present in the extender alone (cell-free), suggesting that sperm produced these FFAs during the cooling process. In contrast, sperm cooled with AM-EY were found to incorporate triacylglycerols (TAG) from EY, without the presence of FFAs in these samples. This is consistent with the high TAG levels observed in AM-EY extender. These results favouring sperm cooled with AM-EY, aligned with the higher values of total sperm motility and live sperm with intact acrosomes observed in these samples compared to those cooled with AM. Cholesterol distribution: A change in cholesterol distribution was observed in cooled sperm, with a decrease in the F1 pattern and an increase in F2. Proteins: No differences in total protein content were observed among raw semen sperm, those diluted with AM and AM-EY, and those cooled with either extenders. The study focused on spermadsorbed proteins (SAP), analyzing band patterns and their respective molecular weights. It was determined that EY contributes proteins to the sperm, and that this occurs during sample dilution prior to cooling. In the resolution gel, high molecular weight bands derived from EY were visible, although they could not be identified using the applied methodology. Additionally, electrophoresis comparing diluted vs. cooled samples with AM-EY showed that after cooling, some bands disappeared and the density of others changed, suggesting that refrigeration may affect SAP. Furthermore, SEM and TEM revealed that cooling not only causes acrosome damage (as seen in routine evaluation), but also produces damage to the sperm plasma membrane. Objective 4: The plan proposed performing insemination using the best cooling results. Accordingly, 21 females were artificially inseminated with semen cooled using AM-EY, but no pregnancies were achieved. Additionally, insemination was attempted in 4 females using semen cooled with AM, also without achieving pregnancy. Based on the previously described results, 14 females were inseminated with semen diluted at 37 °C with AM-EY, again with no pregnancies achieved. Conclusions: For the first time, the lipid profile of llama spermatozoa from raw semen was revealed, as well as the distribution of cholesterol in the plasma membrane. Likewise, the protein profile of llama spermatozoa was described for the first time, determining the molecular weights of the constituent protein bands. A trend toward higher sperm PL values was observed following enzymatic treatment of raw semen with collagenase, without changes in other lipid determinations. Moreover, treatment with the enzyme at a concentration ten times higher than the normally used one could lyse or alter important sperm proteins, such as HSP90 or lactotransferrin. The presence of EY in extenders protects llama sperm from cooling-induced damage by supplying PL and TAG, but most importantly, cholesterol. This helps maintaining membrane functionality and integrity, yielding in vitro better sperm traits than samples refrigerated without EY. Additionally, EY would also contribute high molecular weight proteins. AI with cooled llama semen using commercial extenders, with or without EY, did not improve pregnancy rates compared to previously achieved outcomes. |
| author2 |
Carretero, María Ignacia |
| author_facet |
Carretero, María Ignacia Bertuzzi, Mariana Lucía |
| format |
Tesis doctoral Tesis doctoral acceptedVersion |
| author |
Bertuzzi, Mariana Lucía |
| author_sort |
Bertuzzi, Mariana Lucía |
| title |
Evaluación de la composición proteica y lipídica de la membrana plasmática del espermatozoide de llama y su relación con el proceso de refrigeración |
| title_short |
Evaluación de la composición proteica y lipídica de la membrana plasmática del espermatozoide de llama y su relación con el proceso de refrigeración |
| title_full |
Evaluación de la composición proteica y lipídica de la membrana plasmática del espermatozoide de llama y su relación con el proceso de refrigeración |
| title_fullStr |
Evaluación de la composición proteica y lipídica de la membrana plasmática del espermatozoide de llama y su relación con el proceso de refrigeración |
| title_full_unstemmed |
Evaluación de la composición proteica y lipídica de la membrana plasmática del espermatozoide de llama y su relación con el proceso de refrigeración |
| title_sort |
evaluación de la composición proteica y lipídica de la membrana plasmática del espermatozoide de llama y su relación con el proceso de refrigeración |
| publisher |
Universidad de Buenos Aires. Facultad de Ciencias Veterinarias |
| publishDate |
2025 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_8055 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_8055.dir/8055.PDF |
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AT bertuzzimarianalucia evaluaciondelacomposicionproteicaylipidicadelamembranaplasmaticadelespermatozoidedellamaysurelacionconelprocesoderefrigeracion |
| _version_ |
1867868342027550720 |
| spelling |
I28-R145-HWA_80552026-06-17 Currently, there is no cryopreservation protocol for South American camelid (SAC) semen that achieves acceptable pregnancy rates following artificial insemination (AI). This issue can be attributed to the limited understanding of the biochemical composition of llama spermatozoa, which remains undocumented. In this context, given the known impact of the lipid and protein composition of the cell membrane on both fertilization processes and sperm cryotolerance, we considered its study and characterization to be the main focus of this thesis. Moreover, due to the unique rheological characteristics of SAC ejaculates, different treatments have been studied to reduce thread formation and thereby improve sample handling. One of the most widely used methods is semen incubation with proteolytic enzymes, such as collagenase. The use of this enzyme not only reduces thread formation but also induces a change in sperm motility pattern from oscillatory to progressive, while increasing the percentage of sperm with functional membranes. Based on these findings, this thesis aimed to assess the lipid and protein profiles of llama sperm incubated with collagenase as an attempt to determine the potential impact of the enzyme on sperm membrane lipids and proteins. Although pregnancy rates are low when inseminating with cooled llama semen, sperm survival rates after refrigeration are comparatively higher than after freezing, making cooling a more appealing preservation method. At the time this thesis project was proposed, there were no reports evaluating either in vitro or in vivo effects of using commercial extenders on cooled llama sperm. Therefore, researching this topic was of interest, as these extenders offer certain advantages over hand-made ones. Regarding the addition of egg yolk (EY) to sperm cryopreservation extenders, beneficial effects have been reported in SACs as well as in other species of productive interest. The mechanisms by which EY protects cells from cold shock remain incompletely understood. It has been proposed that lipid components from EY bind tightly to spermatozoa and are not removed even after several washes, suggesting that EY lipoproteins could associate irreversibly with the sperm plasma membrane. For this reason, it became relevant to determine whether the addition of EY to the commercial extender modified the protein and/or lipid composition and/or cholesterol distribution in the plasma membrane of cooled llama sperm. Finally, as with any sperm preservation procedure, the final step to confirm its actual efficiency is to perform an in vivo test through AI, which was conducted as the final objective of this thesis. The objectives of the thesis plan were: 1) To determine the lipid and protein composition of the plasma membrane of raw llama spermatozoa, as well as the distribution of cholesterol within it; 2) To evaluate whether enzymatic treatment (collagenase) of raw llama semen modifies the composition of the sperm plasma membrane; 3) To assess the effect of semen cooling, using a commercial extender with the addition of EY, on the protein and lipid composition and the cholesterol distribution in the plasma membrane of llama sperm; and 4) To evaluate, through AI, the in vivo fertilizing capacity of cooled llama sperm using a commercial extender that preserves sperm quality characteristics. Objectives 1 and 2: Lipids: It was determined that in order to accurately measure the lipids of llama spermatozoa, samples containing at least 300 million total spermatozoa were required. In addition to evaluating lipids in sperm from raw semen and semen incubated with collagenase (0.02%), differences in lipid composition were investigated between sperm collected in summer versus winter, and between young and adult animals. The following parameters were evaluated: cholesterol (Chol), phospholipids (PL), the Chol/PL ratio, fatty acid (FA) groups, individual FAs, and PL classes in spermatozoa from raw semen and after incubation with collagenase. No significant differences were found in Chol, Chol/PL, FAs, or PL classes between raw and collagenase-treated sperm, although a trend toward increased PL content was observed with enzyme use. No significant differences were observed for these same parameters when comparing by collection season. Young males showed significantly lower PL content in spermatozoa from raw semen, along with a tendency toward higher Chol levels, a higher Chol/PL ratio, and lower percentages of polyunsaturated fatty acids (PUFA) compared to adult males. These findings were also associated with higher percentages of total sperm motility and live sperm with intact acrosomes in younger individuals. Chol and the Chol/PL ratio positively correlated with total sperm motility and live sperm with intact acrosomes. Cholesterol distribution in the plasma membrane: Four staining patterns were identified using Filipin labelling: F1) fluorescence throughout the sperm head, F2) fluorescence in the acrosomal region, F3) fluorescence in the post-acrosomal region, and F4) absence of fluorescence in the entire head or fluorescence limited to the equatorial segment. No differences in these patterns were observed between sperm from raw semen and those incubated with collagenase. Proteins: Total protein content was similar between spermatozoa from raw semen and those incubated with collagenase. Spermatozoa from young males showed lower protein content compared to those from adult males. In addition to quantification, electrophoresis was performed on sperm from raw semen, semen incubated with 0.02% collagenase (the concentration normally used in our laboratory), and with 0.2% collagenase (positive control). No differences were observed in the number of protein bands between electrophoresis profiles of raw sperm and those treated with 0.02% collagenase. However, in samples incubated with the higher enzyme concentration, a 95 kDa band present in raw semen spermatozoa disappeared, and a new lower molecular weight band (about 45 kDa) appeared, which was absent in untreated samples. The 95 kDa band corresponded to two extracellular proteins of the same family: Heat shock protein HSP 90-alpha and HSP 90-beta. The second sequenced band at 45 kDa matched two extracellular proteins: Heat shock protein HSP 90-alpha isoform and Lactotransferrin. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were also used to evaluate the samples, revealing that incubation of semen with high concentrations of collagenase not only induces damage to the outer acrosome membrane but also causes severe damage to the sperm plasma membrane ultrastructure. Objective 3: Based on a preliminary study, the commercial extenders AndroMed® and Androstar® Plus were selected. These extenders were later tested with and without the addition of 20% EY for llama semen cooling. The study determined that AndroMed® (AM), with and without EY, would be used to evaluate the lipid and protein composition as well as the cholesterol distribution in membranes of cooled spermatozoa. Additionally, it was confirmed that males differ in their susceptibility to the cooling process, with their response varying according to the extender used (AM vs. AM-EY). It was also determined that the hypo-osmotic swelling (HOS) test, for assessing membrane functionality, is related to sperm response to cooling. Lipids: Lower cholesterol content and Chol/PL ratio were observed in sperm refrigerated with AM compared to those refrigerated with AM-EY, and both compared to spermatozoa from raw semen. Another interesting finding was that the membranes of cooled sperm incorporated lipids with monounsaturated (MFA) and dienoic fatty acids (DFA) present in the extenders, such as oleic acid (18:1) and linoleic acid (18:2). This led to a decrease in polyunsaturated fatty acids (PUFA) and an increase in MFA and DFA in cooled sperm compared to raw sperm. Free fatty acids (FFA) were also detected in sperm cooled with AM, which were not present in the extender alone (cell-free), suggesting that sperm produced these FFAs during the cooling process. In contrast, sperm cooled with AM-EY were found to incorporate triacylglycerols (TAG) from EY, without the presence of FFAs in these samples. This is consistent with the high TAG levels observed in AM-EY extender. These results favouring sperm cooled with AM-EY, aligned with the higher values of total sperm motility and live sperm with intact acrosomes observed in these samples compared to those cooled with AM. Cholesterol distribution: A change in cholesterol distribution was observed in cooled sperm, with a decrease in the F1 pattern and an increase in F2. Proteins: No differences in total protein content were observed among raw semen sperm, those diluted with AM and AM-EY, and those cooled with either extenders. The study focused on spermadsorbed proteins (SAP), analyzing band patterns and their respective molecular weights. It was determined that EY contributes proteins to the sperm, and that this occurs during sample dilution prior to cooling. In the resolution gel, high molecular weight bands derived from EY were visible, although they could not be identified using the applied methodology. Additionally, electrophoresis comparing diluted vs. cooled samples with AM-EY showed that after cooling, some bands disappeared and the density of others changed, suggesting that refrigeration may affect SAP. Furthermore, SEM and TEM revealed that cooling not only causes acrosome damage (as seen in routine evaluation), but also produces damage to the sperm plasma membrane. Objective 4: The plan proposed performing insemination using the best cooling results. Accordingly, 21 females were artificially inseminated with semen cooled using AM-EY, but no pregnancies were achieved. Additionally, insemination was attempted in 4 females using semen cooled with AM, also without achieving pregnancy. Based on the previously described results, 14 females were inseminated with semen diluted at 37 °C with AM-EY, again with no pregnancies achieved. Conclusions: For the first time, the lipid profile of llama spermatozoa from raw semen was revealed, as well as the distribution of cholesterol in the plasma membrane. Likewise, the protein profile of llama spermatozoa was described for the first time, determining the molecular weights of the constituent protein bands. A trend toward higher sperm PL values was observed following enzymatic treatment of raw semen with collagenase, without changes in other lipid determinations. Moreover, treatment with the enzyme at a concentration ten times higher than the normally used one could lyse or alter important sperm proteins, such as HSP90 or lactotransferrin. The presence of EY in extenders protects llama sperm from cooling-induced damage by supplying PL and TAG, but most importantly, cholesterol. This helps maintaining membrane functionality and integrity, yielding in vitro better sperm traits than samples refrigerated without EY. Additionally, EY would also contribute high molecular weight proteins. AI with cooled llama semen using commercial extenders, with or without EY, did not improve pregnancy rates compared to previously achieved outcomes. Fil: Bertuzzi, Mariana Lucía.Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Argentina Carretero, María Ignacia Bertuzzi, Mariana Lucía 2025-10-24 Actualmente no existe un protocolo de criopreservación de semen de camélidos sudamericanos (CSA) que permita obtener tasas de preñez aceptables luego de la inseminación artificial (IA). Este problema puede atribuirse a la comprensión limitada de la composición bioquímica de los espermatozoides de llama, que sigue sin estar documentada. En este sentido, dado el impacto conocido de la composición lipídica y proteica de la membrana celular tanto en los procesos de fertilización como en la criotolerancia de los espermatozoides, consideramos su estudio y caracterización como la temática principal de la presente tesis. Por otra parte, debido a las características reológicas únicas que presentan los eyaculados de CSA se han estudiado diferentes tratamientos para reducir la filancia y con ello mejorar la manipulación de las muestras. Uno de los métodos más utilizados es la incubación del semen con enzimas proteolíticas, como la colagenasa. El uso de esta enzima no solo reduce la filancia, sino que también produce un cambio en el patrón de movilidad de los espermatozoides de oscilatorio a progresivo y a su vez aumenta el porcentaje de espermatozoides con funcionalidad de membrana. A partir de estos resultados, en la presente tesis se propuso evaluar los lípidos y proteínas en espermatozoides de llama incubados con colagenasa como un intento de determinar la posible implicancia de la enzima sobre los lípidos y proteínas de membrana. Aunque los porcentajes de preñez son bajos cuando se insemina con semen refrigerado de llama, las tasas de supervivencia de los espermatozoides refrigerados son comparativamente más altas que las del congelamiento, resultando la refrigeración en una metodología de preservación más atractiva. Al momento de presentar el plan de tesis, no existían reportes que evaluaran los efectos tanto in vitro como in vivo, del uso de diluyentes comerciales sobre espermatozoides refrigerados de llama. Por lo tanto, investigar acerca de esta temática resultó atractivo ya que estos diluyentes presentan ciertas ventajas respecto a los artesanales. En cuanto al agregado de yema de huevo (YH) a los diluyentes de criopreservación de espermatozoides, se han reportado beneficios tanto en los CSA como en otras especies de interés productivo. Los mecanismos mediante los cuales la YH protege a las células del shock por frío no están completamente aclarados. Se ha propuesto que componentes lipídicos de la YH se unen firmemente a los espermatozoides y que los mismos no se remueven luego de varios lavados, sugiriendo que las lipoproteínas de la YH podrían asociarse a la membrana de los espermatozoides en forma irreversible. Por este motivo, resultó interesante determinar si el agregado de YH al diluyente comercial modificó la composición proteica y/o lipídica y/o la distribución del colesterol en la membrana plasmática del espermatozoide refrigerado de llama. Finalmente, en todo procedimiento de conservación espermática el paso final para corroborar su eficiencia real es realizar una prueba in vivo mediante la IA, lo que se realizó en el último objetivo de la tesis. Los objetivos del plan de tesis fueron: 1) Determinar la composición lipídica y proteica de la membrana del espermatozoide de semen fresco de llama, así como la distribución del colesterol en la misma, 2) Evaluar si el tratamiento enzimático (colagenasa) del semen fresco de llama modifica la composición de la membrana plasmática del espermatozoide, 3) Evaluar el efecto de refrigerar semen, utilizando un diluyente comercial con el agregado de YH, sobre la composición proteica y lipídica y la distribución del colesterol de la membrana plasmática del espermatozoide de llama y 4) Evaluar mediante IA la capacidad fecundante in vivo de espermatozoides refrigerados de llama utilizando un diluyente comercial que conserve las características espermáticas. Objetivos 1 y 2: Lípidos: Se determinó que para medir los lípidos del espermatozoide de llama en forma correcta era necesario tener muestras con al menos 300 millones de espermatozoides totales. Además de evaluar los lípidos en el espermatozoide de semen fresco e incubado con colagenasa (0,02%), se investigaron las diferencias en la composición lipídica de los espermatozoides recolectados en verano frente a los de invierno, y entre animales jóvenes y adultos. Se evaluaron el colesterol (Chol), los fosfolípidos (PL), la relación Chol/PL, los grupos de ácidos grasos (AG), los AG individuales y las clases de PL en espermatozoides procedentes de semen fresco y luego de la incubación con colagenasa. No se encontraron diferencias significativas en los valores de Chol, Chol/PL, AG y clases de PL entre los espermatozoides de semen fresco y los incubados con colagenasa, aunque se observó una tendencia a mayor cantidad de PL con el uso de la enzima. Al comparar estos mismos parámetros por estación de colecta, no se observaron diferencias significativas. En cuanto a la edad, los machos jóvenes mostraron un contenido de PL significativamente menor en los espermatozoides del semen fresco con una tendencia a mayor Chol, una mayor relación Chol/PL y menores porcentajes de AG poliinsaturados en comparación con los machos adultos. Esto a su vez coincidió con mayores porcentajes de movilidad total y de espermatozoides vivos con acrosomas intactos en los individuos jóvenes. El Chol y la Chol/PL correlacionaron positivamente con la movilidad total y los espermatozoides vivos con acrosomas intactos. Distribución del colesterol en la membrana plasmática: Se determinaron 4 patrones de coloración con la tinción Filipin: F1) fluorescencia en toda la cabeza espermática, F2) fluorescencia en la región acrosomal, F3) fluorescencia a nivel de la región post-acrosomal y F4) ausencia de fluorescencia en toda la cabeza o fluorescencia a nivel del segmento ecuatorial; sin observar diferencias en los diferentes patrones entre los espermatozoides de semen fresco y aquellos incubados con colagenasa. Proteínas: Los valores de proteínas totales fueron similares entre los espermatozoides de semen fresco y los incubados con colagenasa. Los espermatozoides de machos jóvenes presentaron menores valores de proteínas que los de los adultos. Además de la cuantificación, se realizaron electroforesis de espermatozoides de semen fresco, incubados con 0,02% de colagenasa (concentración habitualmente utilizada en nuestro laboratorio), e incubados con 0,2% de colagenasa (control positivo). No se observaron diferencias respecto a la cantidad de bandas observadas entre las electroforesis de los espermatozoides de semen fresco y los incubados con colagenasa a la concentración habitual (0,02%). Al evaluar las muestras incubadas con enzima a mayor concentración, desapareció una banda de aproximadamente unos 95 kDa, presente en los espermatozoides de semen fresco, apareciendo además una banda de menor peso (aproximadamente 45 kDa) que no se observó en las muestras sin tratamiento. La banda de 95 kDa coincidió con dos proteínas extracelulares de la misma familia: la Heat shock protein HSP 90-alpha y la Heat shock protein HSP 90-beta. La segunda banda secuenciada de 45 kDa coincidió con dos proteínas extracelulares: la Heat shock protein HSP 90-alpha isoform y la Lactotransferrina. Al mismo tiempo se utilizó microscopia electrónica de barrido (MEB) y de transmisión (MET) para evaluar las muestras, determinando que la incubación del semen con colagenasa a alta concentración ocasiona no solo daños en la membrana acrosomal externa, sino también importantes daños en la ultraestructura de la membrana plasmática de los espermatozoides. Objetivo 3. A partir de un estudio preliminar se seleccionaron los diluyentes comerciales AndroMed® y Androstar® Plus. Posteriormente, se probaron estos diluyentes con y sin agregado de 20% de YH en la refrigeración de semen de llama. Dicho estudio determinó que se utilizaría el AndroMed® (AM), con y sin YH, para evaluar la composición lipídica y proteica, así como la distribución del colesterol de la membrana de los espermatozoides refrigerados. Adicionalmente, se comprobó que hay una susceptibilidad diferente de los machos al proceso de refrigeración, incluso variando la respuesta de acuerdo al diluyente utilizado (AM vs. AM-YH). También se determinó que la técnica hipoosmótica (HOS) para evaluar la funcionalidad de membrana está relacionada con la respuesta de los espermatozoides a la refrigeración. Lípidos: Se observó una menor cantidad de Chol y de la relación Chol/PL en los espermatozoides refrigerados con AM respecto a los refrigerados con AM-YH, y de ambos respecto a los espermatozoides de semen fresco. Otro resultado interesante fue que efectivamente las membranas de los espermatozoides refrigerados captan lípidos con ácidos grasos monoinsaturados (MFA) y dienoicos (DFA) presentes en los diluyentes como el oleico (18:1) y el linoleico (18:2). Esto genera una disminución de los porcentajes de ácidos grasos polinsaturados (PUFA) con un aumento de los MFA y DFA en los espermatozoides refrigerados respecto a los espermatozoides de semen fresco. También se detectaron ácidos grasos libres (FFA) en los espermatozoides refrigerados con AM que no se detectaron en el diluyente sólo (sin células), indicando que el espermatozoide generó estos FFA durante el proceso de refrigeración. Mientras que, se observó que los espermatozoides refrigerados con AM-YH captan triacilglicéridos (TAG) de la YH, sin observar FFA en estas muestras. Esto en concordancia con los altos niveles de TAG que se observa en el diluyente AM-YH. Estos resultados a favor de los espermatozoides refrigerados con AM-YH se condicen con los mayores valores de movilidad total y de espermatozoides vivos con acrosomas intactos observados en las muestras refrigeradas con AM-YH respecto a las de AM. Distribución del colesterol: Se evidenció que hay un cambio en la distribución del colesterol en los espermatozoides refrigerados, disminuyendo el patrón F1 y aumentando el F2. Proteínas: No se observaron diferencias en lo que respecta a la cantidad de proteínas totales entre los espermatozoides de semen fresco, los diluidos con AM y AM-YH y los refrigerados con ambos diluyentes. El foco del estudio se centró en el análisis de las proteínas adsorbidas por los espermatozoides (PAS), analizándose las bandas y los respectivos pesos moleculares. Se pudo determinar que evidentemente la YH aporta proteínas a los espermatozoides, y que esto sucede durante la dilución de las muestras previo al proceso de refrigeración. Asimismo, en el gel de resolución se pudieron observar bandas de alto peso molecular que aporta la YH y que no pudieron determinarse con la metodología utilizada. A su vez, las electroforesis que compararon las muestras diluidas vs las refrigeradas con AM-YH mostraron que luego de la refrigeración se pierden bandas, y se modifica la densidad de otras, indicando que la refrigeración podría afectar las PAS. Adicionalmente, se evaluó la ultraestructura de los espermatozoides mediante MEB y MET, observando que la refrigeración no sólo genera daño acrosomal (similar a lo observado en la evaluación de rutina) sino que también produce daños en la membrana plasmática de los espermatozoides. Objetivo 4. En el plan se propuso inseminar con los mejores resultados de refrigeración. En este sentido se inseminaron artificialmente 21 hembras con semen refrigerado con AM-YH, sin lograr preñez. A su vez, se inseminaron 4 hembras con semen refrigerado con AM, tampoco obteniéndose preñez. Debido a los resultados previamente descriptos, se inseminaron 14 hembras con semen diluido a 37 ºC con AM-YH, nuevamente sin lograr preñez. Conclusiones: Se reveló por primera vez el perfil lipídico del espermatozoide de semen fresco de llama, así como la distribución del colesterol en la membrana plasmática. También, se describió por primera vez el perfil proteico del espermatozoide de llama, determinando los pesos moleculares de las bandas que lo componen. Se observó una tendencia a mayores valores de fosfolípidos en los espermatozoides de semen fresco tratados con colagenasa, sin modificar el resto de las determinaciones lipídicas. A su vez, el tratamiento con la enzima a concentración diez veces más elevada a la utilizada habitualmente podría lisar o alterar proteínas espermáticas de importancia, como la HSP90 o la lactotransferrina. La presencia de YH en los diluyentes protege al espermatozoide de llama de los daños ocasionados por la refrigeración, aportando fosfolípidos y TAG, pero sobre todo colesterol, manteniendo así la funcionalidad e integridad de la membrana y obteniendo in vitro mejores valores de las características espermáticas que las muestras refrigeradas sin YH. A su vez, también aportaría proteínas de alto peso molecular. La IA con semen refrigerado de llama en diluyentes comerciales con y sin YH no mejoró los porcentajes de preñez hasta ahora alcanzados. application/pdf Lípidos Proteínas Membrana plasmática Espermatozoide Llama Refrigeración Lipids Proteins Plasma membrane Sperm Llama Refrigeration spa Universidad de Buenos Aires. Facultad de Ciencias Veterinarias info:eu-repo/semantics/openAccess 2025-10-24 htpps://creativecommons.org/licenses/by-nend/2.5/ar/ Espermatozoides Semen refrigerado Llama Ciencias Veterinarias Doctora de Universidad de Buenos Aires en Ciencias Veterinarias Evaluación de la composición proteica y lipídica de la membrana plasmática del espermatozoide de llama y su relación con el proceso de refrigeración info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_8055 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_8055.dir/8055.PDF |