Bovine Sperm Quality Research Articles

Impact of bull age, sperm processing, and microclimatic conditions on the viability and DNA integrity of cryopreserved bovine sperm.

Context Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0h and 3h incubation at 38°C post-thaw (3h) in 3422 batches. Semen was equilibrated for 24h before freezing if collected on Monday or Wednesday and 72h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results PMAI and %DFI (0h and 3h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3h) in summer. Conclusions Extending equilibration from 24 to 72h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing.

The Beneficial Effects of Pterostilbene on Post-Thawed Bovine Spermatozoa.

Reactive Oxygen Species (ROS), primarily produced by cellular metabolism, are highly reactive molecules that modify cellular compounds. During sperm preparation in Assisted Reproductive Techniques (ARTs), intrinsic and extrinsic sources of ROS can impact spermatozoa's oxidative status. The modification of the media with compounds that enhance sperm quality characteristics is of great significance. The current study investigated the effect of pterostilbene, a phenolic compound, on bovine sperm quality. Cryopreserved spermatozoa from six bulls were thawed, supplemented with pterostilbene (0, 10 μΜ, 25 μΜ) and incubated for 60 min and 240 min. Spermatozoa were analyzed in terms of motility, viability, acrosomal status and intracellular concentration of superoxide anion in each time point. The incubation of spermatozoa with 25 μΜ pterostilbene resulted in the preservation of quality parameters through superoxide anion mitigation, while its presence in capacitating conditions resulted in higher percentage of acrosome-reacted spermatozoa. The results of the present study indicate that the addition of pterostilbene prevents oxidative insult to spermatozoa and preserves the sperm quality parameters.

P-035 Exposure to high concentrations of non-esterified fatty acids impairs bovine sperm quality

Abstract Study question Do high concentrations of non-esterified fatty acids (NEFA) affect sperm quality? Summary answer In vitro exposure to high NEFA concentrations induced detrimental effects on acrosome integrity, mitochondrial membrane potential, plasma membrane integrity, and DNA damage in bovine spermatozoa. What is known already High NEFA levels present in obese individuals and in cows experiencing negative energy balance has been linked with impaired reproductive success. In vitro bovine models have showed that a high NEFA microenvironment (i.e. steric acid [SA], palmitic acid [PA], oleic acid [OA]) during oocyte maturation (Reproduction 2013, 145:33-44), fertilisation (ESHRE meeting 2021, P-176), and embryo culture (BMC Genomics 2016, 17:1004) is detrimental for blastocyst formation. However, the effect of high NEFA levels on sperm quality has been less explored. Study design, size, duration Bovine sperm from two bulls was prepared to a maximum concentration of 20x106/mL then incubated in in vitro fertilisation (IVF) medium for 4 h under different NEFA levels representing physiological (Control-1[C1], 28 μM SA, 23 μM PA, 21 μM OA) and pathophysiological (High-NEFA, 280μM SA, 230μM PA, 210μM OA), serum levels found in cows experiencing negative energy balance) concentrations. A second control (C2) group contained solvent. Participants/materials, setting, methods Fluorescence assays (Toxicity 2018, 393:52-50) were used to evaluate simultaneously acrosome integrity (FITC-PSA), mitochondrial membrane potential (JC-1) and plasma membrane integrity (DRAQ7, Hoechst 33342). Spermatozoa (n = 800 per group, four replicates) were assessed with epifluorescence microscopy and Ziess ZEN software. Sperm DNA damage was also assessed with the Halosperm G2® kit (C1; n = 812, C2; n = 879, High-NEFA; n = 930, six replicates). Data were arcsine transformed before analysis (ANOVA, followed by LSD post hoc test). Main results and the role of chance There was no significant difference between the control groups or between bulls in any of the variables analysed. The percentage (mean ± SEM) of spermatozoa with intact acrosome was higher in the control groups compared with high-NEFA (C1=64.50 ± 6.34%, C2=69.13 ± 4.08%, High-NEFA=49.75 ± 6.14%), but it reached statistical significance only when compared to C2 (C2 vs High-NEFA P = 0.037, C1 vs High-NEFA P = 0.096). Similarly, the percentage of spermatozoa with low mitochondrial membrane potential was increased following exposure to high concentrations of NEFA (C1=50.88 ± 5.69%, C2=45.00 ± 4.03%, High-NEFA=61.62 ± 3.38%), but a significant difference was only observed when compared to C2 (C2 vs High-NEFA P = 0.027, C1 vs High-NEFA P = 0.124). The high-NEFA group showed a higher plasma membrane damage (59.50 ± 1.06%) compared to controls (C1=47.50 ± 4.07% P = 0.01, C2=46.75 ± 1.79% P = 0.008). DNA damage was also increased in the High-NEFA group (20.00 ± 0.64%) compared to controls (C1=8.90 ± 0.71% P < 0.001, C2=6.82 ± 0.78% P < 0.001). Limitations, reasons for caution In vitro animal models do not reflect accurately in vivo conditions and cannot be directly extrapolated to humans, but they are useful for the development of conceptual models that could eventually be tested in the target species (e.g. obese individuals). Wider implications of the findings Our results indicate that high NEFA concentrations can impair sperm quality, and given that sperm with DNA damage can achieve fertilization (Human Reproduction 2010, 25:1594-1608), our data partially explain the decreased blastocyst formation previously observed in this model of high NEFA exposure (ESHRE meeting 2021, P-176). Trial registration number 000

Impact of Heat Stress on Bovine Sperm Quality and Competence.

Simple SummaryLivestock farming is facing growing challenges due to climate change. Both animal welfare and male fertility are parameters easily affected by thermal stress. Spermatogenesis is a highly thermosensitive process, and therefore heat stress leads to a marked decrease in the fertilizing capacity of spermatozoa. There is an urgent need to find sustainable ways of mitigating the effects of global warming on animal welfare and fertility, and consequently protect the profitability of farms.Global warming has negatively influenced animal production performance, in addition to animal well-being and welfare, consequently impairing the economic sustainability of the livestock industry. Heat stress impact on male fertility is complex and multifactorial, with the fertilizing ability of spermatozoa affected by several pathways. Among the most significative changes are the increase in and accumulation of reactive oxygen species (ROS) causing lipid peroxidation and motility impairment. The exposure of DNA during the cell division of spermatogenesis makes it vulnerable to both ROS and apoptotic enzymes, while the subsequent post-meiotic DNA condensation makes restoration impossible, harming later embryonic development. Mitochondria are also susceptible to the loss of membrane potential and electron leakage during oxidative phosphorylation, lowering their energy production capacity under heat stress. Although cells are equipped with defense mechanisms against heat stress, heat insults that are too intense lead to cell death. Heat shock proteins (HSP) belong to a thermostable and stress-induced protein family, which eliminate protein clusters and are essential to proteostasis under heat stress. This review focuses on effects of heat stress on sperm quality and on the mechanisms leading to defective sperm under heat stress.

A Non-Synonymous Point Mutation in a WD-40 Domain Repeat of EML5 Leads to Decreased Bovine Sperm Quality and Fertility.

This study is part of a concerted effort to identify and phenotype rare, deleterious mutations that adversely affect sperm quality, or convey high developmental and fertility potential to embryos and ensuing progeny. A rare, homozygous mutation in EML5 (EML5 R1654W ), which encodes a microtubule-associated protein with high expression in testis and brain was identified in an Angus bull used extensively in artificial insemination (AI) for its outstanding progeny production traits. The bull’s fertility was low in cross-breeding timed AI (TAI) (Pregnancy/TAI = 25.2%; n = 222) and, in general, AI breeding to Nellore cows (41%; n = 822). A search of the 1,000 Bull Genomes Run9 database revealed an additional 74 heterozygous animals and 8 homozygous animals harboring this exact mutation across several different breeds (0.7% frequency within the 6,191 sequenced animals). Phenotypically, spermatozoa from the homozygous Angus bull displayed prominent piriform and tapered heads, and outwardly protruding knobbed acrosomes. Additionally, an increased retention of EML5 was also observed in the sperm head of both homozygous and heterozygous Angus bulls compared to wild-type animals. This non-synonymous point mutation is located within a WD40 signaling domain repeat of EML5 and is predicted to be detrimental to overall protein function by genomic single nucleotide polymorphism (SNP) analysis and protein modeling. Future work will examine how this rare mutation affects field AI fertility and will characterize the role of EML5 in spermatogenesis.

Staphylococcus-Induced Bacteriospermia In Vitro: Consequences on the Bovine Spermatozoa Quality, Extracellular Calcium and Magnesium Content.

Bacterial contamination of bovine ejaculates intended for artificial insemination may be reflected in a significant economic loss due to unsuccessful fertilization as well as health issues of the recipients. The Staphylococcus genus represents a large part of bacteriocenosis of bovine ejaculates. Therefore, this study aims to get a closer look on the effects of Staphylococcus-induced bacteriospermia under in vitro conditions on bovine sperm quality. Prior to inducing bacteriospermia, spermatozoa were separated from each ejaculate using Percoll® Plus gradient medium in order to limit the effects only to the selected bacterial species. Seven Staphylococcus species previously isolated from bovine semen were used for our experiments at a turbidity of 0.5 McFarland (equivalent to 1.5 × 108 colony-forming units per mL). The contaminated semen samples were incubated at 37 °C and at times of 0, 2, and 4 h, motility, mitochondrial membrane potential, reactive oxygen species (ROS) generation, sperm DNA fragmentation, and magnesium (Mg) and calcium (Ca) extracellular concentration were analyzed and compared with the control group (uncontaminated). The results showed no significant changes at the initial measurement. However, significant adverse effects were observed after 2 h and 4 h of incubation. Most notably, the presence of S. aureus, S. warneri, S. kloosii, and S. cohnii caused a significantly increased ROS production, leading to sperm DNA fragmentation, changes in the mitochondrial membrane potential, and a decreased sperm motility. Furthermore, the presence of Staphylococcus species led to lower extracellular concentrations of Mg and Ca. In conclusion, the overgrowth of Staphylococcus bacteria in bovine semen may contribute to oxidative stress resulting in sperm DNA fragmentation, altered mitochondrial membrane potential, and diminished sperm motility.

Mitochondria Content and Activity Are Crucial Parameters for Bull Sperm Quality Evaluation.

Standard sperm evaluation parameters do not enable predicting their ability to survive cryopreservation. Mitochondria are highly prone to suffer injuries during freezing, and any abnormalities in their morphology or function are reflected by a decline of sperm quality. Our work focused on describing a link between the number and the activity of mitochondria, with an aim to validate its applicability as a biomarker of bovine sperm quality. Cryopreserved sperm collected from bulls with high (group 1) and low (group 2) semen quality was separated by swim up. The spermatozoa of group 1 overall retained more mitochondria (MitoTrackerGreen) and mtDNA copies, irrespective of the fraction. Regardless of the initial ejaculate quality, the motile sperm contained significantly more mitochondria and mtDNA copies. The same trend was observed for mitochondrial membrane potential (ΔΨm, JC-1), where motile sperm displayed high ΔΨm. These results stay in agreement with transcript-level evaluation (real-time polymerase chain reaction, PCR) of antioxidant enzymes (PRDX1, SOD1, GSS), which protect cells from the reactive oxygen species. An overall higher level of glutathione synthetase (GSS) mRNA was noted in group 1 bulls, suggesting higher ability to counteract free radicals. No differences were noted between basal oxygen consumption rate (OCR) (Seahorse XF Agilent) and ATP-linked respiration for group 1 and 2 bulls. In conclusion, mitochondrial content and activity may be used as reliable markers for bovine sperm quality evaluation.

Effects of Dietary Supplementation of Conjugated Linoleic Acids and Their Inclusion in Semen Extenders on Bovine Sperm Quality.

Simple SummarySuboptimal fertility in males accounts for about two-thirds of infertility cases, thus being of serious concern for the dairy industry, where optimal fertility is fundamental for farm profitability. Although genetic defects responsible for subfertility have been identified, the role of seminal compounds on fertility remain unclear. Feeding rumen-protected isomers of conjugated linoleic acid (CLA) to dairy cows reportedly enhances circulating insulin-like growth factor I (IGF-I) levels. In breeding bulls, the IGF-I concentration in seminal plasma has been positively correlated with fertility rates. Therefore, the objective of the study was to evaluate the effect of dietary CLA supplementation and of their inclusion to the semen extender on bovine semen quality and freezability.Isomers of conjugated linoleic acid (CLA) enhances circulating insulin-like growth factor I (IGF-I) levels. Furthermore, fertility rate of breeding bulls is positively correlated to seminal plasma IGF-I concentration. Our objective was to evaluate the effect of dietary CLA supplementation and inclusion to the semen extender on bovine semen quality and freezability. Fourteen bulls, randomly assigned to control (CTL) and CLA (50 g/day) groups, were supplemented for 10 weeks. Samples were collected at Weeks −2 (before supplementation), 0, 4, 6 (during supplementation), 10, and 11 (after supplementation). Blood and seminal plasma were analyzed for IGF-I; the ejaculates were frozen in the following subgroups: CTL (no addition to semen extender), CLA c9, t11 (50 µM), CLA c9, t11 (100 µM), CLA t10, c12 (50 µM), CLA t10, c12 (100 µM), and CLA mix (50 µM each of CLA c9, t11 and CLA t10, c12). Sperm motility, morphology, viability, mitochondrial membrane potential, and reactive oxidative species were assessed. CLA supplementation decreased ejaculates’ total volume, increased sperm concentration, beat cross frequency, and decreased oxidative stress; it also increased plasma and seminal plasma IGF-I levels compared to the CTL. The inclusion of CLA c9, t11 100 µM and CLA mixture in the extender increased live spermatozoa percentage post-thawing compared to other groups. Our results show a beneficial effect of CLA supplementation on semen quality; however, further studies evaluating fertilization rates are necessary to corroborate the results.

Transfection of exogenous DNA complexed to cationic dendrimer induces alterations of bovine sperm microRNAome

MicroRNAs have been hypothesized to be involved in the regulation of male fertility potential. The primary aim of our study was to demonstrate the effects of transfection with dendrimer nanostructure on the parameters of bovine sperm quality and to investigate whether the microRNA profile could be disturbed after cationic dendrimer-mediated exogenous DNA transfection of bovine spermatozoa. The binding of exogenous DNA was significantly increased when dendrimer-based transfection was implemented. However, cationic dendrimer transfection induced detrimental changes in the kinetics and sperm quality parameters, such as membrane integrity, acrosome reaction, and mitochondrial membrane potential, when compared to the control group. Sperm microRNA sequencing revealed 218 known and 106 novel microRNAs in the sperm samples, among which nine were dysregulated after transfection (one was upregulated and eight were downregulated), in comparison to the non-transfected sperm. All the dysregulated microRNAs were related to sperm quality and embryonic development. These results suggest that the transfection process using the dendrimer nanostructure has an impact on the quality and microRNA profile of bovine sperm.

Inclusion of Sericin in Tris Egg Yolk Extender Improves Bovine Sperm Quality during Cryopreservation (-196°C)

Inclusion of Sericin in Tris Egg Yolk Extender Improves Bovine Sperm Quality during Cryopreservation (-196°C)

Impact of the Spirulina maxima extract addition to semen extender on bovine sperm quality

The present study was undertaken to investigate an effect of various concentrations of Spirulina maxima extract (SME) in BIOXcell® extender on Simmental bulls spermatozoa quality. Semen was collected from 20 healthy bulls using an artificial vagina. Semen was evaluated in terms of concentration, motility, viability and morphology. Samples that showed more than 70% motility, 60% viability and 70% morphologically accepted spermatozoa were selected for the experiment. SME at the concentrations of 2, 4, 6 and 8 µg/mL was added to bovine commercial BIOXcell® extender. The semen aliquots were cooled and preserved at 4 °C. Their qualities were evaluated during pre-freezing, then the cooled semen samples were packaged into 0.25 ml straws. The straws were frozen in the vapour of liquid nitrogen, and stored at −196 °C in container. The straws were thawed one day later and the characteristics of spermatozoa were examined. Results showed significant effect of SME on characteristics such as viability, Reactive Oxygen Species synthesis, DNA fragmentation and motility of spermatozoa in post freezing conditions (p < 0.05) and revealed that supplementation of SME extender improved the post-thaw spermatozoa quality in bulls.HighlightsSpirulina maxima extract has antioxidant effect on bovine sperm cellsThe addition of Spirulina maxima extract improves the quality of bovine semenThe extender supplemented with 4 µg/mL Spirulina maxima extract improves the motility, viability and DNA integrity of the bovine spermatozoa

Effects of arginine and trehalose on post-thawed bovine sperm quality.

The present study was conducted to examine the protective role of arginine and trehalose on post-thaw bull sperm and oxidative stress parameters. Five ejaculates for each bull were used in the study. Each ejaculate, split into three equal aliquots and diluted at 37 °C with base extenders containing 2 mM arginine, 25 mM trehalose and no antioxidant (control) was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. Supplementation of the semen extender with arginine decreased the percentages of post-thawed subjective motility (29 ± 8.21%), CASA motility (12.2 ± 5.69%) and progressive motility (3.52 ± 2.13%), compared with the controls (43 ± 2.73%, 55.4 ± 6.78% and 33.48 ± 4.14%, respectively, P < 0.05). Supplementation of the semen extender with trehalose produced a higher mitochondrial activity and sperm viability (36.3 ± 3.99% and 44.1 ± 2.18%) compared with the control (13 ± 8.15 and 31.7 ± 3.94%, respectively, P < 0.05). It was established that trehalose (95.1%) and arginine (92.8%) protect DNA integrity compared to the control (90.4%) (P < 0.05). Trehalose supplementation in semen extenders provided great benefit in terms of viability, mitochondrial activity, and intact sperm DNA on frozen-thawed bull sperm.

Effects of single layer centrifugation (SLC) on bull spermatozoa prior to freezing on post-thaw semen characteristics.

Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen-thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species-specific colloid, Bovicoll. After centrifugation for 20min at 300g, the resulting sperm pellet was resuspended in OPTIXcell® (IMV Technologies, l'Aigle, France); the SLC-selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC-treated samples had a higher percentage of live, superoxide-positive spermatozoa than uncentrifuged samples (27.9±5.1% versus 21.7±6.7%; p=.03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9±8.2% versus 40.5±15.1%; p=.03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non-treated samples (42.0±8.5% versus 55.9±14.4%; p=.04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide-positive spermatozoa and live, hydrogen peroxide-negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC-selected spermatozoa compared to unselected samples.

Protein biomarkers for male artificial insemination subfertility in bovine spermatozoa.

BackgroundAlthough artificial insemination (AI) technique is an established biotechnology for bovine reproduction, the results of AI (conception rates) have a tendency to decline gradually. To our annoyance, moreover, AI‐subfertile bulls have been occasionally found in the AI centers. To resolve these serious problems, it is necessary to control the sperm quality more strictly by the examinations of sperm molecules.MethodsWe reviewed a number of recent articles regarding potentials of bovine sperm proteins as the biomarkers for bull AI‐subfertility and also showed our unpublished supplemental data on the bull AI‐subfertility associated proteins.Main findingsBull AI‐subfertility is caused by the deficiency or dysfunctions of various molecules including regulatory proteins of ATP synthesis, acrosomal proteins, nuclear proteins, capacitation‐related proteins and seminal plasma proteins.ConclusionIn order to control the bovine sperm quality more strictly by the molecular examinations, it is necessary to select suitable sperm protein biomarkers for the male reproductive problems which happen in the AI centers.

Effect of the herbicide atrazine and its metabolite DACT on bovine sperm quality

Atrazine (ATZ), one of the most extensively used herbicides, is considered a ubiquitous environmental contaminant. ATZ is a known endocrine disruptor, and deleterious effects on reproductive function have been shown, even at low, ecologically relevant doses (0.1–3μg/L). Once it enters the body, ATZ is metabolized to various metabolites, which are further detected in the urine, serum and tissues. In mammals, the major ATZ metabolite is diaminochlorotriazine (DACT). The current study focuses on direct effects of low doses of ATZ and DACT on bovine sperm isolated from ejaculates or epididymis compartments (head, body and tail). Sperm were incubated under capacitation conditions with or without 0.1–10μM ATZ or 1–100μM DACT. The integrity and functionality of sperm membranes (plasma, acrosomal and mitochondrial) were examined simultaneously by fluorescence staining at 0, 2 and 4h of incubation. Acrosome reaction (AR) was induced by Ca++ ionophore, after capacitation. The findings indicated that both ATZ and DACT adversely affect sperm, expressed by damaged sperm membranes. ATZ had a prominent effect on epididymal-tail sperm, expressed as disruption of all examined membranes, mostly at low (0.1 or 1μM) concentrations; pseudo-AR and that induced by Ca++ ionophore were both affected by exposure to 0.1μM ATZ (P<0.05 and P<0.00004, respectively). A similar pattern was documented for sperm isolated from ejaculates (P<0.002 and P<0.001, respectively). ΔYm was affected by ATZ in sperm isolated from the epididymis tail (1μM, P<0.0009), but not in that isolated from ejaculates. DACT reduced sperm viability at all examined concentrations and in all fractions. DACT at 1μM impaired ΔΨm in sperm isolated from the epididymis tail and ejaculate (P<0.005). DACT at 100μM did not induce pseudo-AR in sperm isolated from the ejaculate, but did in sperm isolated from the epididymis tail (P<0.05). Induction of AR by Ca++ ionophore was impaired in sperm isolated from ejaculate and exposed to 10 or 100μM DACT (P<0.05) and in sperm isolated from the epididymis tail and exposed to 1, 10 or 100μM DACT (P<0.0004). These findings reveal the harmful effect of exposure to ATZ and DACT, mainly at low ecologically relevant doses, on sperm viability, AR and mitochondrial function. We conclude that sperm at advanced stages of spermatogenesis, through its passage and storage in the epididymis compartments as well as in the ejaculate, is sensitive to herbicide. The results suggest that ATZ- or DACT-induced disruptions of sperm membranes might impair sperm fertilization competence.

Protective effect of crocetin on bovine spermatozoa against oxidative stress during invitro fertilization.

Oxidative stress is one of the major factors that contribute to poor semen quality and low rates of invitro fertilization. Crocetin, a main constituent of saffron (Crocus sativus L.) possesses potent antioxidant activity, by scavenging reactive oxygen species (ROS) and/or enhancing the activity of intracellular antioxidant enzymes. The aim of this study was to investigate, for the first time, the effect of crocetin on the quality characteristics of bull spermatozoa and fertilization rate. For this reason, frozen/thawed bovine spermatozoa were incubated with crocetin (1, 2.5, and 5μm), for 120 or 240min, in the presence of a negative control, and evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. In order to evaluate the impact of crocetin on cleavage and blastocyst rate, the compound was added in the IVF medium at the previously identified optimal concentration (2.5μm). The results indicate that incubation of spermatozoa with 2.5μm of crocetin resulted in a statistically significant lower production of superoxide anion and hydrogen peroxide, lower lipid peroxidation, and in better maintenance of motility parameters, viability, and acrosomal integrity, with a very small number of cells with DNA fragmentation, compared to the other groups (p<0.05). The presence of crocetin (2.5μm) in the fertilization medium also resulted in a significant increase in acrosome-reacted spermatozoa and blastocyst production, compared to the control group (p<0.01). These data indicate that crocetin (2.5μm) positively affects bovine sperm quality characteristics during a 240-min incubation and improves its fertilizing ability, directly and/or indirectly, by regulating ROS concentration and lipid peroxidation.

Preliminary study on effects of bovine frozen semen storage using a liquid nitrogen-independent method on the quality of post-thaw spermatozoa

Frozen semen of eight bulls was used to assess effects of storage temperature and length of storage time on frozen-thawed bovine sperm quality. In experiment 1, 25 straws of frozen semen of each bull were allocated to 3 groups. The control was still maintained in liquid nitrogen (LN2). The rest were abruptly moved from LN2 to −80°C and −30°C mechanical freezers, respectively. After thawing, it was found that the sperm motility, vitality and membrane integrity were comparable (P>0.05) between the control and the −80°C samples and were significantly inferior (P<0.001) in the −30°C samples, irrespective of storage time (1-day, 1-week and 1-month). In experiment 2, two out of the three parts (16–18 straws) of frozen semen of each bull were rapidly removed from LN2 and further kept in the freezer (−80°C). One day before being thawed, half of the samples in the freezer were promptly put back to LN2. The results showed that the frozen-thawed sperm quality was not significantly affected (P>0.05) both by storage temperature (−196°C, −80°C and −80 & −196°C) and storage time [day-2, day-8 (1-week) and day-31 (1-month)]. At the same storage times, the quality measures at different temperatures were not significantly different from one another (P>0.05). In conclusion, a −80°C mechanical freezer was as effective as LN2 in preserving in vitro quality of frozen-thawed bovine spermatozoa throughout 1-month of storage. When required for use, frozen semen stored in the freezer could be thawed immediately or transferred to the LN2 tank for thawing elsewhere.

Effects of arginine and trehalose on post-thawed bovine sperm quality

Effects of arginine and trehalose on post-thawed bovine sperm quality

Combination of cysteamine and lipoic acid can improve post-thawed bovine sperm quality

Combination of cysteamine and lipoic acid can improve post-thawed bovine sperm quality

Effects of feeding omega-3-fatty acids on fatty acid composition and quality of bovine sperm and on antioxidative capacity of bovine seminal plasma

The aim of the present study was to examine the effects of feeding alpha-linolenic (ALA) acid on fatty acid composition and quality of bovine sperm and on antioxidative capacity of seminal plasma. Nine bulls (ALA bulls) were fed with 800g rumen-resistant linseed oil with a content of 50% linolenic acid and eight bulls with 400g palmitic acid (PA bulls). Sperm quality was evaluated for plasma membrane and acrosome intact sperm (PMAI), the amount of membrane lipid peroxidation (LPO), and the percentage of sperm with a high DNA fragmentation index (DFI). Fatty acid content of sperm was determined using gas chromatography. Total antioxidant capacity, glutathione peroxidase, and superoxide dismutase activity were determined in seminal plasma. Feeding ALA increased (P<0.05) the docosahexaenoic acid (DHA) content in bulls whereas in PA bulls did not change. PMAI increased after cryopreservation in ALA bulls as well as in PA bulls during the experiment period (P<0.005). LPO of sperm directly after thawing did not change during the study period in ALA group, but decreased in PA group (P<0.006). After 3h of incubation LPO increased in the ALA group (P<0.02), while LPO did not differ between phases within groups. In conclusion, feeding of neither saturated nor polyunsaturated fatty acids affect the antioxidant levels in seminal plasma. Both saturated as well as polyunsaturated fatty acids had positive effects on quality of cryopreserved bovine sperm, although the content of docosahexaenoic acid in sperm membranes increased only in ALA bulls.