Bovine Sperm Quality Research Articles

Antioxidant effect of crocin on bovine sperm quality and in vitro fertilization

Reactive oxygen species (ROS) production above critical levels affects the genetic and functional integrity of spermatozoa by causing oxidative stress. Spermatozoa are susceptible to oxidative stress in terms of motility and fertilization capacity. Crocin (crocetin di-gentiobiose ester), a main constituent of Crocus Sativus L. (saffron), is known for its antioxidant activity by scavenging ROS, especially superoxide anion. The aim of the present study is to evaluate the effect of crocin on the quality characteristics of spermatozoa and fertilization rate. Frozen–thawed and washed spermatozoa from four different bulls were incubated with three different concentrations of crocin (0.5, 1, and 2 mM), for 120 and 240 minutes, in the presence of a negative control, and were evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. The most potent concentration of crocin (1 mM) was also added in the fertilization medium to test its impact on fertilization outcome. The results indicate that the incubation of spermatozoa with 1 mM of crocin resulted in a statistically significant lower production of ROS, lower lipid peroxidation and in better maintenance of motility, viability, and acrosomal integrity, with a very small number of fragmented cells, compared to the control and the other treated groups (P < 0.05). Crocin concentration of 1 mM resulted in a significant increase of blastocyst rate, compared to the control group (P < 0.01). These data indicate that crocin (1 mM) improves bovine sperm quality and its fertilization capability, directly and/or indirectly, by modulating ROS concentration.

Efeito da inibição da óxido nítrico sintase induzível na capacitação in vitro de espermatozoides bovinos

Avaliaram-se o papel do óxido nítrico (NO) por meio da inibição da enzima óxido nítrico sintase induzível (iNOS), após a adição da aminoguanidina (AG), na motilidade, no vigor e na integridade da membrana plasmática nos tempos de 15, 60, 120, 180, 240 e 300min e a atividade mitocondrial e a capacitação de espermatozoides bovinos após 300min de cultivo. Adicionaram-se diferentes concentrações (0,001, 0,01 e 0,1M) de AG durante a capacitação induzida pela heparina e 500μM de nitroprussiato de sódio (SNP, doador de NO) à concentração deletéria. A adição de 0,1M de AG diminuiu a motilidade e o vigor espermático e a integridade da membrana (P&lt;0,05). A adição de SNP ao meio de cultivo com 0,1M de AG somente reverteu a integridade da membrana após 300min. A inibição da síntese de NO pela adição de AG não alterou a atividade mitocondrial. A percentagem de oócitos penetrados com espermatozoides tratados com 0,01 e 0,1M de AG diminuiu 20,3 e 100%, respectivamente, em relação aos não tratados (controle) (P&lt;0,05), contudo houve aumento de 15% na percentagem de oócitos desnudados penetrados com espermatozoides capacitados em presença de 0,1M de AG. Conclui-se que a inibição da síntese de NO pela AG diminuiu a qualidade espermática durante a capacitação de espermatozoides bovinos in vitro, exceto a atividade mitocondrial. Somente a integridade da membrana foi revertida após adição de NO, sugerindo diferentes vias de ação do NO na qualidade espermática ao longo da capacitação in vitro de espermatozoides bovinos.

Association between seasonal changes in fatty-acid composition, expression of VLDL receptor and bovine sperm quality

Reduced reproductive performance of dairy cows during the summer is often associated with elevated temperature. Semen collected and cryopreserved during the summer may be of low quality and might contribute to the compromised fertility of dairy cows during this season. The present study examined the association between seasonality, semen quality and its potential to survive cryopreservation. A comparison between semen collected during the summer (July to August) and that collected during the winter (November to December) revealed the summer semen to be inferior, as reflected by low motility and high mortality of sperm. Furthermore, samples that were defined as good quality had changes in lipid concentration and fatty-acid composition in both the seminal plasma and cell compartment. In particular, semen collected during the summer had reduced levels of polyunsaturated arachidonic acid (20:4; P < 0.05) and decreased levels of linoleic acid (18:2; P < 0.05) in the cell compartment; corresponding reductions in cholesterol ( P < 0.06) and fatty-acid concentrations ( P < 0.001) were detected in seminal plasma of semen collected during the summer. In addition, we provided the first evidence for the existence of a very-low-density lipoprotein receptor (VLDLr) in bovine sperm, suggesting a mechanism for sperm utilization of extracellular lipids. Interestingly, the expression of VLDLr was three-fold greater in samples collected during the winter than in those collected in the summer ( P < 0.01) and was negatively associated with saturated fatty-acid concentration ( P < 0.018) but not with that of cholesterol. An opposite pattern was noted for samples obtained during the summer; mRNA expression of VLDLr was negatively associated with cholesterol concentration ( P < 0.01) but not with that of saturated fatty acids. Such modifications associated with extracellular lipid utilization and fatty-acid composition might explain, in part, the reduced quality of summer semen.

113 EFFECT OF SPERM COATING ON THE QUALITY OF BOVINE FROZEN - THAWED SPERMATOZOA

The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing. Table 1. VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm This research was supported by IWT (no. IWT/020727).

184 THE EFFECT OF A NOVEL SEMEN DISINFECTION TREATMENT ON THE VIABILITY AND FERTILIZING CAPACITY IN VIVO OF BOVINE SPERMATOZOA

The goal of this investigation was to determine the effect of a novel procedure, consisting of an initial incubation in antibiotic cocktail followed by centrifugation through a trypsinized Percoll gradient, on the viability and fertilizing capacity in vivo of bovine sperm as compared to standard processing methods. Exp 1: Semen collected by electroejaculation from 12 bulls were aliquoted into four treatment groups: (1) control (standard method) using Biladyl A (Minitübe, Tiefenbach, Germany) at a 1:4 (semen:diluent) dilution; (2) antibiotic cocktail (gentamicin, spectinomycin, lincomycin, tylosin, and kanamycin at 250, 300, 150, 200, and 1000 μg/mL, respectively) at a 1:9 (semen:cocktail) dilution and incubation (38°C) for 15 min; (3) trypsinized Percoll (Sigma, St. Louis MO, USA) gradient treatment by layering 1 mL semen on the top of a 90, 45, and 30% Percoll (bottom to top) gradient, with the 90 and 30% segments of the column containing 10 μg/mL soy-based trypsin inactivator (Sigma) and the 45% segment containing 0.25% trypsin (Sigma), and then centrifuging at 700g for 30 min; and (4) a combination of treatments 2 and 3 by concentrating the sperm by gentle centrifugation (300g for 10 min) after incubation in antibiotic cocktail, and then layering 1 mL of the sperm suspension on the Percoll gradients. In treatments 2–4, the treated sperm pellets were resuspended in Biladyl A (1:4), and all treatments were refrigerated (4°C) and examined at 0, 24, 48, and 72 hours for progressive motility, viability, and acrosomal integrity. The results were analyzed statistically using the Student's t-test (P &lt; 0.05). Exp 2: Semen samples collected from two bulls were treated with either the control method or the combination antibiotic cocktail and trypsinized Percoll methods (1 and 4 above), and then used to AI a total of 5 (control) and 6 (combined treatment) superovulated cows three times at 12-h intervals. Day 7 embryos were recovered and assessed for stage and morphological quality. The results for Exp 1 indicated that sperm treated with either the trypsinized Percoll alone or in combination with the antibiotic cocktail had significantly higher (P &lt; 0.05) progressive motility than the control at 0 (87.5 and 89.2 vs. 75.4%), 24 (87.1 and 87.9 vs. 73.2%), 48 (87.1 and 86.7 vs. 70.7%), and 72 h (82.1 and 80.4 vs. 64.6%, all respectively) post-treatment. Likewise, treated sperm had significantly greater viable and acrosome-intact sperm than the control at 0 to 72 h post-treatment. Although not statistically significant, there were more transferable-quality embryos recovered from cows inseminated using sperm treated with the combined method as compared to the control (58.9 vs. 43%, respectively). In conclusion, the trypsinized Percoll gradient method for processing bovine semen (alone or in combination with antibiotic cocktail) improved the quality of bovine sperm refrigerated for up to 72 h and had no detrimental affect on the number of transferable-quality embryos collected after AI.

Effect of whole blood and serum on bovine sperm quality and in vitro fertilization capacity

Under physiologic conditions, low concentrations of blood may be present in the uterine fluid of the estrous cow at the moment of insemination. To decrease the insemination dose and to obtain good insemination results with less fertile semen, more invasive insemination methods such as utero tubal junction (UTJ) insemination can be used. More invasive insemination methods increase the risk of damaging the hyperemic endometrium, with blood in the uterine fluid as result. In this study, the effect of 0, 0.15 and 1.5% whole blood and serum on bovine sperm quality and in vitro fertilizing capacity was evaluated. Sperm quality as assessed by total motility, progressive motility, membrane integrity and acrosomal status was not affected by the presence of blood or serum ( P>0.05). However, the in vitro fertilizing capacity decreased with increasing concentrations of blood and serum ( P<0.01). The rate of polyspermy increased with increasing concentrations of serum ( P<0.01), but not with increasing concentrations of blood ( P=0.30). In conclusion, no immediate effect of blood and serum was visible on several sperm quality parameters, except for an increased prevalence of head to head agglutination (HHA). However, blood and serum did have a negative effect on in vitro fertilizing capacity.

The implications of a paternally derived centrosome during human fertilization: consequences for reproduction and the treatment of male factor infertility.

Successful fertilization in humans follows a complex series of events, including the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei, the restoration of the sperm centrosome, and the nucleation of microtubule-mediated motility necessary to bring the male and female pronuclei into close apposition. These events occur after both fertilization in vitro and after intracytoplasmic sperm injection (ICSI), a new technique which is currently being applied in many clinics to overcome severe male infertility. Defects in any of the events leading to fertilization can be lethal to the zygote and may prove to be causes of infertility. Imaging of inseminated human and rhesus oocytes using immunohistochemical techniques reveals several phases at which fertilization arrests. Oocytes from some infertile patients failed to complete fertilization due to failure of the sperm aster microtubules in uniting the sperm and egg nuclei. The rate of sperm aster formation, size, and organization during fertilization has been used as a measurement of bovine sperm quality. The development of an assay using Xenopus laevis oocyte extract can also be used to test sperm from various species for their ability to form esters and perform other centrosomal functions in vitro, as well as another indicator of sperm quality. Semen from men with questionable fertility was found to contain sperm which are generally incapable of producing sperm asters. In addition, the activity of centrosomal proteins such as gamma-tubulin and centrin have been detected in mammalian eggs and sperm. The levels of gamma-tubulin increase markedly after exposure to X. laevis egg extract. Defects in either male or female nucleus decondensation also resulted in the arrest of fertilization and was found to occur in both inseminated human oocytes and in rhesus oocytes fertilized by ICSI. These discoveries on the molecular basis of infertility in humans have important implications for infertility diagnosis and managing reproduction.