RESUMEN
During the freeze-drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well-established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff-Quik (DQ) with two established procedures: acridine orange test (AOT) and sperm chromatin dispersion (SCD) on freeze-dried (FD) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze-dried in basic medium supplemented with two different chelating agents: EGTA or EDTA. After rehydration, the spermatozoa were subjected to DNA damage detection using a SCDt, AOT and DQ stain simultaneously. The results showed that the DNA damage levels in the EGTA group were significantly lower than those in the EDTA group. AOT detected a significantly higher proportion of spermatozoa with fragmented DNA than DQ and SCD. The results of the SCD test and DQ stain exhibited a significant positive correlation for DNA fragmentation (r = 0.528), whereas a negative correlation was observed between SCD, DQ and AOT (r = -0.134 and r = -0.332 respectively). The present study shows that both the SCD test and DQ assay are effective methods for detecting FD stallion sperm DNA fragmentation, whereas using of AOT is questionable.
Asunto(s)
Quelantes , Daño del ADN , Liofilización/veterinaria , Caballos , Preservación de Semen/veterinaria , Espermatozoides/metabolismo , Naranja de Acridina , Animales , Colorantes , Ácido Edético , Ácido Egtácico , Colorantes Fluorescentes , Liofilización/métodos , Técnicas In Vitro , Masculino , Preservación de Semen/efectos adversos , Preservación de Semen/métodosRESUMEN
Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion's breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose-egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters.
Asunto(s)
Criopreservación , Crioprotectores/farmacología , Dimetilformamida/farmacología , Preservación de Semen/métodos , Espermatozoides/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Yema de Huevo/química , Epidídimo/citología , Epidídimo/fisiología , Glicerol/farmacología , Caballos , Masculino , Concentración Osmolar , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Espermatozoides/fisiologíaRESUMEN
In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n=11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n=16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p<0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid.
Asunto(s)
Medios de Cultivo , Técnicas de Cultivo de Embriones/veterinaria , Oocitos/fisiología , Ovinos/embriología , Animales , Blastocisto/fisiología , Células Cultivadas , Criopreservación/veterinaria , Transferencia de Embrión , Desarrollo Embrionario , Femenino , Fertilización In Vitro/veterinaria , Hormona Folículo Estimulante , Líquido Folicular , EmbarazoRESUMEN
The effect of supplementation of sex-sorted and non-sorted spermatozoa with seminal plasma protein (SPP) on fertility after cervical insemination was examined in the present study. Spermatozoa were sorted into high purity X and Y chromosome-bearing spermatozoa or not sorted and then either supplemented with SPP (>10 kDa) before freezing and/or after thawing (non-sorted only) or processed without supplementation. Inseminations were performed over 2 days with ewes receiving 100 or 25 million motile non-sorted spermatozoa in the cervix or uterus, respectively, or two cervical inseminations of 3.5 million motile sorted spermatozoa. Pregnancy rates in cervically inseminated ewes were unaffected by supplementation of sorted or non-sorted spermatozoa with SPP before freezing compared with no supplementation. The effect of post-thaw supplementation of non-sorted spermatozoa with SPP on pregnancy rates after cervical insemination varied with the day of insemination (P < 0.05); fertility was similar to laparoscopic insemination on Day 1 (56.0 +/- 10.2% v. 58.6 +/- 10.1%), but not on Day 2 (23.1 +/- 7.4% v. 66.7 +/- 9.2%). In conclusion, under the conditions of the present study, SPP did not consistently improve pregnancy rates after cervical insemination with frozen-thawed ram spermatozoa. This is the first report of pregnancies (5/56 ewes inseminated) after cervical insemination with frozen-thawed sex-sorted ram spermatozoa. Although the success rate is low, the findings are encouraging because ewes inseminated with the sex-sorted spermatozoa received only 7% of the recommended dose (100 million motile) for cervical insemination of frozen-thawed spermatozoa.
Asunto(s)
Fertilidad/fisiología , Inseminación Artificial/veterinaria , Proteínas de Plasma Seminal/fisiología , Preselección del Sexo/veterinaria , Ovinos/fisiología , Espermatozoides/fisiología , Animales , Criopreservación/veterinaria , Femenino , Inseminación Artificial/métodos , Inseminación Artificial/normas , Modelos Logísticos , Masculino , Preservación de Semen/métodos , Preservación de Semen/veterinariaRESUMEN
To determine whether flow sorting increased the susceptibility of spermatozoa to reactive oxygen species (ROS), ram semen was either diluted with Tris medium (100 x 10(6) spermatozoa mL(-1); D) or highly diluted (10(6) spermatozoa mL(-1)) before being centrifuged (DC) at 750g for 7.5 min at 21 degrees C or flow-sorted (S) before cryopreservation. Thawed spermatozoa were resuspended in graded concentrations of hydrogen peroxide to induce oxidative stress. In Experiment 1, following exposure to 30 or 45 muM hydrogen peroxide (H(2)O(2)), the total motility (%) of DC (41.0 +/- 7.3 or 25.7 +/- 6.7, respectively) and S spermatozoa (33.8 +/- 6.3 or 20.1 +/- 6.3, respectively) was lower (P < 0.001) than that of D spermatozoa (58.7 +/- 5.6 or 44.5 +/- 6.7, respectively). In Experiment 2, supplementation of samples containing H(2)O(2) with catalase (150 IU mL(-1)) or seminal plasma proteins (4 mg protein per 10(8) spermatozoa) negated oxidative stress, resulting in comparable values to samples receiving no H(2)O(2)in terms of the proportion of spermatozoa with stable plasmalemma (as determined using merocyanine-540 and Yo-Pro-1) in the D and S groups, the proportion of viable, acrosome-intact spermatozoa (as determined by fluorescein isothiocyanate and propidium iodide staining) in the D group and the motility of control (undiluted) and S spermatozoa. Neither H(2)O(2) nor sperm type (i.e. D, DC or S) had any effect on intracellular concentrations of ROS. These results show that flow sorting increases the susceptibility of spermatozoa to ROS, but the inclusion of anti-oxidants or seminal plasma as part of the sorting protocol improves resistance to oxidative stress.
Asunto(s)
Catalasa/farmacología , Citometría de Flujo/veterinaria , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo/fisiología , Ovinos/fisiología , Espermatozoides/fisiología , Animales , Catalasa/metabolismo , Membrana Celular/fisiología , Criopreservación/veterinaria , Peróxido de Hidrógeno/metabolismo , Masculino , Proteínas de Plasma Seminal/farmacología , Espermatozoides/metabolismoRESUMEN
Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n=four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37 degrees C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze-thawing.
Asunto(s)
Separación Celular/veterinaria , Criopreservación/veterinaria , Crioprotectores/farmacología , Citometría de Flujo/veterinaria , Preservación de Semen/veterinaria , Proteínas de Plasma Seminal/farmacología , Espermatozoides/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Masculino , Técnicas Reproductivas/veterinaria , Preservación de Semen/efectos adversos , Ovinos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/patologíaRESUMEN
Two experiments have been performed to clone the bucardo, an extinct wild goat. The karyoplasts were thawed fibroblasts derived from skin biopsies, obtained and cryopreserved in 1999 from the last living specimen, a female, which died in 2000. Cytoplasts were mature oocytes collected from the oviducts of superovulated domestic goats. Oocytes were enucleated and coupled to bucardo's fibroblasts by electrofusion. Reconstructed embryos were cultured for 36h or 7d and transferred to either Spanish ibex or hybrid (Spanish ibex malex domestic goat) synchronized recipients. Embryos were placed, according to their developmental stage, into the oviduct or into the uterine horn ipsilateral to an ovulated ovary. Pregnancy was monitored through their plasmatic PAG levels. In Experiment 1, 285 embryos were reconstructed and 30 of them were transferred at the 3- to 6-cells stage to 5 recipients. The remaining embryos were further cultured to day 7, and 24 of them transferred at compact morula/blastocyst stage to 8 recipients. In Experiment 2, 154 reconstructed embryos were transferred to 44 recipients at the 3- to 6-cells stage. Pregnancies were attained in 0/8 and 7/49 of the uterine and oviduct-transferred recipients, respectively. One recipient maintained pregnancy to term, displaying very high PAG levels. One morphologically normal bucardo female was obtained by caesarean section. The newborn died some minutes after birth due to physical defects in lungs. Nuclear DNA confirmed that the clone was genetically identical to the bucardo's donor cells. To our knowledge, this is the first animal born from an extinct subspecies.
Asunto(s)
Clonación de Organismos/métodos , Extinción Biológica , Cabras/genética , Nacimiento Vivo/veterinaria , Animales , Secuencia de Bases , Cesárea/veterinaria , Conservación de los Recursos Naturales , Criopreservación/veterinaria , ADN Mitocondrial/análisis , ADN Mitocondrial/química , Técnicas de Cultivo de Embriones/veterinaria , Transferencia de Embrión/veterinaria , Femenino , Fibroblastos/ultraestructura , Glicoproteínas/sangre , Pulmón/anomalías , Datos de Secuencia Molecular , Técnicas de Transferencia Nuclear/veterinaria , Oocitos/ultraestructura , Embarazo , Proteínas Gestacionales/sangreRESUMEN
Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta superfamily, is specifically expressed in oocytes and is essential for sheep prolificacy. Reported mutations in this gene cause increased ovulation rate and infertility in a dosage-sensitive manner. In this work, a new naturally occurring mutation in the BMP15 gene from the ovine Rasa Aragonesa breed is described. This mutation is a deletion of 17 bp that leads to an altered amino acid sequence and introduces a premature stop codon in the protein. Highly significant associations (P < 0.0001) were found between the estimated breeding value for prolificacy and the genotype of BMP15 in Rasa Aragonesa animals with high and low breeding values for this trait. As for other mutations in BMP15, this new mutation is associated with increased prolificacy and sterility in heterozygous and homozygous ewes respectively.
Asunto(s)
Fertilidad/genética , Infertilidad Femenina/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Eliminación de Secuencia , Ovinos/genética , Animales , Codón de Terminación , Femenino , Factor 9 de Diferenciación de Crecimiento , EmbarazoRESUMEN
Seminal oxidative stress status is emerging as a significant prognostic tool in assisted reproductive technology. A dynamic interplay between pro- and anti-antioxidant substances in the ejaculate is essential. In this study, we determined seasonal changes in the activity of the antioxidant enzyme defense system comprising superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and catalase (CAT) in seminal plasma (SP) of mature Rasa Aragonesa rams. This breed corresponds to a local Spanish genotype with a short seasonal anoestrus between May and July. In addition, the activity of these enzymes was measured in protein fractions isolated from ram SP by exclusion chromatography. Total protein content in ram SP was higher during the breeding season (October-February) with a significantly higher value in first ejaculates. Antioxidant enzyme activities were higher during the non-breeding season (March-September). Comparing first and second ejaculates, SOD and CAT activities were higher in the first of all months. However, GR and GPx activities changed throughout the year. Thus, GR activity was higher in July and August in first ejaculates, this difference being significant in July (4.53 versus 2.37 nmol substrate/minmg protein, P<0.05). Conversely, GPx activity was significantly higher in September and November in second ejaculates (21.1 versus 6.81 and 10.91 versus 5.33, respectively, P<0.05). After SP fractionation by exclusion chromatography, GR activity was located in fractions 1 and 2 being irrelevant in the following peaks, and CAT activity was not detected all along the chromatographic profile. GPx and SOD activities were spread out along all fractions with a main peak in fractions 6 and 7. Given that these two fractions showed the greatest capacity to recover and prevent cold-shock membrane injury [Barrios B, Pérez-Pé R, Gallego M, Tato A, Osada J, Muino-Blanco T, Cebrián-Pérez JA. Seminal plasma proteins revert the cold-shock damage on ram sperm membrane. Biol Reprod 2000;63:1531-7, Barrios B, Fernández-Juan M, Muino-Blanco T, Cebrián-Pérez J. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock. J Androl 2005;26:539-49], we could suggest that the protective effect might be, at least partially, due to the antioxidant enzyme activity.
Asunto(s)
Oxidorreductasas/fisiología , Estaciones del Año , Semen/enzimología , Ovinos/fisiología , Animales , Cromatografía en Gel/veterinaria , Masculino , Oxidorreductasas/análisis , Proteínas/análisis , Factores de TiempoRESUMEN
This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.
Asunto(s)
Preservación de Semen/veterinaria , Ovinos , Interacciones Espermatozoide-Óvulo , Espermatozoides/fisiología , Animales , Supervivencia Celular , Fertilización In Vitro/veterinaria , Gelatina , Masculino , Leche , Preservación de Semen/métodos , Recuento de Espermatozoides , Motilidad Espermática , Temperatura , Factores de TiempoRESUMEN
The acrosome reaction is an important marker for sperm function. Because different laboratory techniques may be used to detect this exocytotic process, the objective of this study was to investigate the use of fluoresceinated lectins to assess the acrosomal status of nonpermeabilized ram spermatozoa. In addition, we used centrifugal countercurrent distribution (CCCD) in an aqueous 2-phase system to assess the sperm surface modifications associated with the acrosome reaction by observing changes in their partition behavior. We analyzed the binding of 5-fluorescein isothiocyanate (FITC)-conjugated lectins to ram sperm to select a lectin that bound preferentially to the acrosomal region, which would allow differentiation of acrosome-intact from acrosome-damaged ram spermatozoa. Ricinus communis agglutinin (RCA) bound intensely to the anterior and weakly to the equatorial acrosomal regions. Acrosomal labeling changed when spermatozoa were induced to acrosome-react with calcium ionophore A23187. RCA acrosomal labeling significantly increased (P < .0001) after incubation (84% versus 28% in control samples). To determine if RCA lectin labeling could be used to assess the acrosomal status of fresh ram spermatozoa in suspension, we compared the percentage of acrosome-reacted sperm detected by the carboxyfluorescein diacetate/propidium iodide (CFDA/PI) double-fluorescent staining with the percentage detected by FITC-RCA labeling. The incidence of acrosome-reacted spermatozoa detected by CFDA/PI was not significantly different (P = .704; 13 comparisons in 6 different experiments) from the incidence of spermatozoa detected by FITC-RCA staining. The evaluation of the spontaneous acrosome reaction by RCA labeling (5.83%) was not significantly different (P = .644) from that assessed by CFDA/PI (6.88%). The percentage of induced acrosome reactions detected by CFDA/PI staining (56%) significantly correlated (P < .0001; r = 0.876) with that detected by RCA labeling (56.67%). We simultaneously carried out a comparative CCCD in an aqueous 2-phase system to analyze sperm surface changes associated with the acrosome reaction. Results revealed that sperm surface hydrophobicity decreased in samples that had been incubated with ionophore compared with the untreated-control samples. Likewise, RCA binding after CCCD showed that all acrosome-reacted cells were stained, whereas only 42% of cells were lectin-labeled in the untreated semen sample. This change in lectin reactivity of acrosome-reacted spermatozoa signals the presence of some deep membrane or intracellular residues that would affect partitioning. Therefore, the FITC-RCA-labeling procedure can be used to accurately assess the acrosomal status of ram spermatozoa in suspension.
Asunto(s)
Reacción Acrosómica/fisiología , Lectinas/metabolismo , Lectinas de Plantas , Ovinos/fisiología , Espermatozoides/fisiología , Animales , Calcimicina/farmacología , Fluoresceína-5-Isotiocianato , Fluoresceínas , Colorantes Fluorescentes , Indicadores y Reactivos , Ionóforos/farmacología , Masculino , Propidio , Espermatozoides/efectos de los fármacosRESUMEN
The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing-thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen-thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen-thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk-yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk-yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.
Asunto(s)
Separación Celular , Espermatozoides/fisiología , Reacción Acrosómica , Animales , Distribución en Contracorriente , Masculino , Microscopía Electrónica de Rastreo , Ovinos , Capacitación Espermática , Espermatozoides/citología , Espermatozoides/ultraestructuraRESUMEN
The aim of this study was to evaluate the effect of four extenders (Sucrose (S), Galactose (G), milk-yolk (MY), and Fiser (F)) on the motility, membrane integrity, and functional integrity of ram spermatozoa during liquid storage at 15 degrees C. The use of either S or MY for the selection of high quality spermatozoa by a swim-up procedure was comparatively analyzed. Additionally, the activity of three antioxidant enzymes, superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) was evaluated in both swim-up selected samples maintained at 15 degrees C for 6h. Sperm motility was better preserved in MY and was significantly higher after 6h of incubation than in either S or F (P<0.0001) and G (P<0.0005). Likewise, the incidence of spermatozoa with integral and functional membranes was higher in samples diluted in MY, with no significant decrease after 6h of incubation. The comparative analysis of the swim-up procedure performed with either MY or S revealed that not only was total sperm recovery significantly (P<0.001) higher (67.3%+/-3.21 versus 47.6%+/-3.78), but also that the best survival rate of spermatozoa was found in the MY stored sample. Sperm motility, viability and response to a hypoosmotic swelling (HOS) test were also significantly higher in the MY extended sample, maintaining still significantly higher values after 6h of incubation. In addition, this sample showed higher activity values for the antioxidant defense enzyme system.
Asunto(s)
Antioxidantes/metabolismo , Preservación de Semen/veterinaria , Ovinos , Motilidad Espermática , Espermatozoides/enzimología , Espermatozoides/fisiología , Animales , Separación Celular , Supervivencia Celular , Dextranos , Galactosa , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Masculino , Leche , Preservación de Semen/métodos , Soluciones , Sacarosa , Superóxido Dismutasa/metabolismoRESUMEN
Lectins, high molecular weight glycoproteins with different sugar-binding specificity, are able to agglutinate different cell types. The recovery of high-quality spermatozoa can be facilitated by the agglutination induced by the lectin binding. The objective of this study was to combine sperm-lectin agglutination with a dextran/swim-up procedure for developing a new selection technique for ram spermatozoa. To study sperm quality, cell viability (plasma membrane integrity), the HOS-test response and progressive individual motility were assessed. Simultaneously, centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system was carried out to analyze sperm surface heterogeneity. Semen from 3 mature Saltz rams was pooled, and 0.5-mL aliquots were incubated with 4 fluorescein-labelled lectins (ECL, JAC, PSA, RCA). Then, a dextran solution was gently added and overlaid with medium. The top layer of the medium containing the spermatozoa was collected and replaced by careful addition of fresh medium. The incubation sequence was repeated 3 times at 10-min intervals. The consecutive 4 top layers obtained were pooled to give the swim-up combined sample. The highest rate of improvement in sperm quality was obtained after incubation with RCA, with a 50% increase in progressive individual motility, 21.6% in HOS value and 39.5% in viability. Total cell recovery was 64% (1.56x10(9) cells), with a viable cell recovery rate of 86%. The obtained sample showed 82% motility, 80% HOS score and 77% viability, up from the pre-swim-up values of 51, 60 and 57 %, respectively. Comparative CCCD analysis revealed a very high heterogeneous population in the RCA/swim-up sample obtained, while a much more homogeneous population was obtained in the sample after the dextran/swim-up procedure previously developed byus With this simple method, a large proportion of highly-motile spermatozoa with preserved plasma membrane and high heterogeneity can be obtained. These results strongly suggest that this selection procedure could result in a high fertility rate.
Asunto(s)
Lectinas/fisiología , Ovinos/fisiología , Aglutinación Espermática , Motilidad Espermática/fisiología , Animales , Centrifugación/veterinaria , MasculinoRESUMEN
Sperm morphologic assessment is considered an irreplaceable part of standard laboratory routine analyses in the diagnosis of male fertility. Thus, in an attempt to quantify the effects of season on sperm morphology and its functional significance in relation to sperm quality parameters, sperm head morphometric traits were analyzed by using an objective computerized analysis combined with principal components analysis (PCA) cluster analysis to establish the relationship between the distribution of the subpopulations found and sperm quality in each season. There were slight variations on sperm motility and sperm membrane integrity indexes (P > 0.05). However, the mean values for sperm concentration substantially changed among seasons in all individuals studied (P < 0.01). There were significant differences in sperm morphometric parameters (P < 0.01) as well as in the distribution of morphometric subpopulations between seasons (P < 0.001). In conclusion, this study confirmed that there was an important seasonal effect on sperm morphometric traits. In addition, the distribution of these subpopulations seems to be related to the season studied and the ejaculate quality which would be a very important indicator of sperm function. The substantial information derived from these morphometric subpopulations has provided new knowledge which can be used in future studies using sperm morphometry as a seasonal indicator in ram ejaculates.
Asunto(s)
Estaciones del Año , Ovinos/fisiología , Espermatozoides/fisiología , Espermatozoides/ultraestructura , Animales , Cruzamiento , Membrana Celular/ultraestructura , Fertilidad , Inseminación Artificial/veterinaria , Masculino , Semen/citología , Recuento de Espermatozoides , Cabeza del Espermatozoide/ultraestructura , Motilidad Espermática , Espermatozoides/clasificaciónRESUMEN
The importance of understanding the sperm changes after the cryopreservation process has been emphasized in human and veterinary andrology. In previous studies, we have shown that the morphometric characteristics assessed by computer-assisted analysis following the freeze-thawing process revealed differences in terms of dimension and shape between individuals that may be related to bio-physiologic factors such as sexual maturity. The purpose of this study was to determine if there are differences associated with cryoresistance and sperm head morphometric dimensions in individuals with different sexual maturity ratings (SMRs; 12, 30 and 96 months of age). Ejaculates from nine normospermic fertile rams with different SMRs were analyzed in an attempt to quantify the morphometric dimensions and the shape of sperm heads from each group after the cryopreservation process. The mean values of sperm concentration among individuals with different SMRs were significantly different (P < 0.01). Cryopreservation substantially reduced sperm motility and plasma membrane integrity irrespective of SMR assessed, with young animals being the most affected (P < 0.01). Sperm quality at thawing for all sperm parameters evaluated was significantly higher for old individuals than for middle-aged or young individuals (P < 0.01). There were no significant differences in the sperm head dimension or shape among middle-aged and old individuals (P > 0.05). However, significant differences were detected in area, perimeter and width (lower values) and length, ellipticity and elongation (higher values) in old or middle-aged individuals compared with young individuals (P < 0.01). In conclusion, this study confirms that ram age is related to sperm morphometric dimensions, and sperm size and shape may affect spermatozoa survival, being good indicators of freezability. Therefore, the present study provides information on the morphometric maturation of ram sperm and supports the idea that the dimensions of spermatozoa may be taken as an approximate indication of its relative maturity.
Asunto(s)
Criopreservación/veterinaria , Preservación de Semen/veterinaria , Maduración Sexual/fisiología , Ovinos/crecimiento & desarrollo , Cabeza del Espermatozoide/ultraestructura , Envejecimiento/fisiología , Animales , Membrana Celular/ultraestructura , Masculino , Recuento de Espermatozoides , Motilidad Espermática , Espermatozoides/fisiología , Espermatozoides/ultraestructuraRESUMEN
It is widely accepted that sperm morphology is a good indicator of fertility and it has been proposed that sperm quality may be related to subtle changes in sperm head morphology. However, a precise estimation of the morphology of ram sperm would be very useful to improve reproductive success in ovine. Computer-assisted morphometric analysis and clustering analysis have been important tools to study sperm subpopulations in domestic animals. However, to the best of our knowledge, no data exist studing morphometric differences regarding to sperm subpopulations within the ovine ejaculate. The aim of this study was to test the presence and distribution of sperm morphometric subpopulations in cryopreserved ejaculates from yearling and mature rams using an objective method by computer analysis system and to establish the relationship between the distribution of the subpopulations found and sperm quality in each individual ram. Principal component analysis revealed that three principal components for yearlings and four components for mature rams that represented more than 84% of the cumulative variance in both cases. After cluster analysis, three sperm morphometric subpopulations for yearlings (CLY) and four for mature (CLM) rams were identified with defined sperm dimensions and shapes. CLY1 included big, round and short sperm (37%), CLY2 included average size and slightly elliptical and elongated sperm (48%), CLY3 included small, long, elliptical and elongated sperm cells (15%). CLM1 consisted of average size and moderate elliptical and elongated (26%), CLM2 consisted of small, long, elliptical and elongated (31%), CLM3 consisted of small and round (32%) and CLM4 included big, short and round (8%) spermatozoa respectively. There were significant differences in the distribution of the three subpopulations (P < 0.001) as well as in the sperm concentration, total motility (%), sperm viability (%) and the overall (P < 0.05) in the ejaculates among the four yearling rams tested. Same results were found for the four subpopulations and the different sperm quality parameters in the ejaculates among the four mature rams tested. In conclusion, cryopreserved ram semen showed a specific structure with regard to sperm morphometric subpopulations. In addition, the distribution of these subpopulations seems to be related to stud maturity age and the ejaculate quality which would be a very important indicator of sperm function. Thus, analysis of sperm morphometric subpopulation structure together with functional tests could provide valuable information to assess the cryoresistence of ram spermatozoa.
Asunto(s)
Criopreservación/veterinaria , Análisis de Semen/veterinaria , Preservación de Semen/veterinaria , Ovinos/crecimiento & desarrollo , Espermatozoides/citología , Factores de Edad , Animales , Procesamiento de Imagen Asistido por Computador , Masculino , Maduración SexualRESUMEN
Membrane proteins orchestrate key events required for participation of sperm in fertilisation. These proteins may be removed or altered due to the mechanical and dilution stressors associated with sex-sorting of sperm. Ram sperm were incubated with Hoechst 33342 and flow-sorted. Sex-selected (viable, orientated) and waste (separated into non-viable or non-orientated) sperm populations were collected, or sperm were not sorted. Sperm membrane proteins were extracted and characterised by one- and two-dimensional PAGE. Densiometric analysis of protein bands separated by one-dimensional PAGE showed proteins of 30 and 28 kDa as doublet bands on non-sorted sperm, and single bands on sex-sorted sperm, and the proportion of a 14 kDa protein was 3-fold higher in non-sorted compared to sorted sperm. Proteins in the 14 kDa band were identified by mass spectroscopy as a bovine Fibronectin type-2 protein (Fn-2), cytochrome oxidase 5a (Cox5a) and a sperm membrane associated protein (SLLP1). The abundance of these proteins in the two-dimensional gels was lowest in the sorted sperm population identified as viable during sorting (orientated and non-orientated sperm) and highest in the non-viable sperm population (P < 0.001). We concluded that the membrane protein profile was different for sex-sorted compared with non-sorted sperm, due to the selection of plasma membrane-intact cells in the flow-sorted population. This provided further evidence that sex-sorting selected a homogenous population of sperm with superior function to non-sorted sperm. Furthermore, this was apparently the first time sperm membrane acrosome associated protein was reported in ram sperm, and it was demonstrated that seminal plasma proteins remained on the sperm membrane after sex-sorting.
Asunto(s)
Electroforesis en Gel Bidimensional/veterinaria , Proteínas de la Membrana/análisis , Ovinos , Espermatozoides/química , Espermatozoides/citología , Animales , Separación Celular/veterinaria , Citometría de Flujo/veterinaria , Masculino , Proteínas de la Membrana/aislamiento & purificación , Sexo , Preselección del Sexo/veterinaria , Espermatozoides/fisiologíaRESUMEN
Although the use of fresh semen in the Irish dairy AI industry only accounts for 5% of total AI usage, this may peak to over 25% during the spring breeding season due to the increased demand for Irish proven sires of high genetic merit. The aim of this study was to examine the effect of storage of fresh semen for up to 7 d at ambient temperature on fertilization and embryo development in vitro, and on the ability of sperm to penetrate artificial mucus in vitro. In vitro matured bovine oocytes were inseminated with fresh semen stored in a caprogen-based diluent, with or without prior Percoll separation. Irrespective of sire, storage of fresh semen at ambient temperature for up to 7 d post collection had no effect on cleavage rate or blastocyst development after IVF. In addition, blastocyst quality, as assessed by the proportion of blastocysts hatching from the zona, was not affected by semen storage. Higher numbers of fresh sperm migrated through artificial mucus on Day 0 (day of semen collection) compared with frozen-thawed sperm. On Day 1 and 2 postcollection there was no difference in the number of sperm migrating through the mucus, but storage of sperm at ambient temperature for longer than 2 d resulted in a significant decline in their ability to penetrate mucus compared with frozen sperm from the same ejaculate. In conclusion, bovine sperm retain the ability to fertilize oocytes in vitro for up to 7 d following storage at ambient temperature. However, the ability of sperm to migrate through artificial mucus in vitro is severely depressed after 2 d storage which may have significant implications for the ability of these sperm to reach the site of fertilization in vivo after AI.
Asunto(s)
Bovinos , Fertilización/fisiología , Preservación de Semen/veterinaria , Espermatozoides/fisiología , Animales , Desarrollo Embrionario , Fertilización In Vitro , Masculino , Preservación de Semen/métodos , Interacciones Espermatozoide-ÓvuloRESUMEN
The response of ram spermatozoa to seminal plasma is highly variable, in part due to the presence of both stimulatory and inhibitory factors. The aim of this study was to assess variation in the protection of ram spermatozoa during freezing by seminal plasma. The seminal plasma variables studied were season of collection, fractionation and method of supplementation. Spermatozoa were supplemented before freezing with 4 mg of seminal plasma proteins (SPPs) per 10(8) cells, and their motility and viability were assessed during post-thaw incubation (37 degrees C). In Experiment 1, semen was (a) frozen with no supplementation (Control) (b) extended with a Tris-based diluent (C + TRIS), or (c) supplemented with seminal plasma collected throughout the year (in the Southern Hemisphere) and pooled for January-March, April-June, July-August and October-December, and either fractionated to produce a concentrated >10 kDa seminal plasma protein retentate (>10 kDa SPP), or kept as crude seminal plasma (CP). There was no effect of season or seminal plasma type (CP or >10 kDa SPP) on motility of spermatozoa. CP and >10 kDa SPP improved the viability of spermatozoa when collected from January-September compared to Control. Supplementation with >10 kDa SPP increased viability of spermatozoa, compared to CP, when collected from January to July. In Experiment 2, >10 kDa SPP were either added directly to the spermatozoa or included in the cryodiluent or >10 kDa SPP were not supplemented (Control). Both supplementation methods improved the motility and the proportion of viable, acrosome-intact spermatozoa but direct supplementation resulted in more viable, acrosome-intact spermatozoa compared with supplementation of the cryodiluent. These results show that supplementation of ram spermatozoa with CP, or its protein component (>10 kDa SPP), before freezing protects them from freeze-thaw damage. The protective effect is greatest when seminal plasma is collected during the breeding season, fractionated with >10 kDa filters and added directly to the spermatozoa.