Long‐Term Storing of Frozen Semen at −196°C does not Affect the Post-Thaw Sperm Quality of Bull Semen

Today, it is theoretically assumed that frozen storage of semen doses in liquid nitrogen guarantees sperm functionality indefinitely. However, there are few studies that objectively evaluate the effects of long‐term storage on sperm quality parameters. In this study, we show a freezability analysis of bull semen stored for 1, 10, 25, 40 and 45 years at −196°C. Sperm viability and full sperm motility were analyzed by CASA system, and acrosome integrity was assessed with Coomassie blue staining. Our results showed that sperm viability and total sperm motility were not affected by long‐term cryopreservation at −196°C. Specifically, we did not find any significant differences (p > 0.05) associated between different long‐time storing analyzed; both parameters showed optimal values of sperm viability and total sperm motility (both over 60%). Additionally, the acrosomal integrity parameter was not affected, showing an optimal range (87±1.6 - 95±0.5%). We conclude that the sperm quality of bovine semen is not affected by long-term storage at −196°C. However, future field trials will be necessary in order to validate that both fertility and embryo viability are maintained for the times analyzed

Variations of early postmortem pH in carcasses of grass-fed steers and its relationship with glycolytic potential and meat quality traits

This study aimed to investigate the multivariate relationship between the glycolytic potential, and meat quality traits from grass-fed steers carcasses with variations in early postmortem pH. From a contemporary group of steers (n=70) from the same production unit and slaughtered under similar conditions, thirty carcasses (10/group) were selected based on pH values measured at 3h (pH3h): Low (< 6.2), Intermediate pH3h (6.2–6.5), and High (> 6.5). Carcasses segregated by pH3h groups were different (p < 0.05) in muscular glycogen (MCG), glucose (G+G6P) content, glycolytic potential (GP) and GPstrict (GP without lactate content). The interaction pH3h groups × sampling time was significant only for lactate content (LC) (p < 0.05). Quality traits, except redness, did not vary (p > 0.05) among pH3h groups. Color variables had a positive and moderate correlation with MGC, G+G6P, LC, and GP. Results does not allow to recommend early carcass segregation by very early pH (3h postmortem); however, important bivariate and multivariate relationships between G+G6P, lactate content and instrumental color parameters in LL muscle from grass-fed cattle were demonstrated

Variations of early postmortem pH in carcasses of grass-fed steers and its relationship with glycolytic potential and meat quality traits

This study aimed to investigate the multivariate relationship between the glycolytic potential, and meat quality traits from grass-fed steers carcasses with variations in early postmortem pH. From a contemporary group of steers (n=70) from the same production unit and slaughtered under similar conditions, thirty carcasses (10/group) were selected based on pH values measured at 3h (pH3h): Low ( 6.5). Carcasses segregated by pH3h groups were different (p 0.05) among pH3h groups. Color variables had a positive and moderate correlation with MGC, G+G6P, LC, and GP. Results does not allow to recommend early carcass segregation by very early pH (3h postmortem); however, important bivariate and multivariate relationships between G+G6P, lactate content and instrumental color parameters in LL muscle from grass-fed cattle were demonstrated

Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm : Dopamine Modulates Sperm Motility and Acrosomal Integrity

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP+ ), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility

Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm : Dopamine Modulates Sperm Motility and Acrosomal Integrity

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP+ ), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility

Temperature, but not excess of glycogen, regulates "in vitro" AMPK activity in muscle samples of steer carcasses.

Postmortem muscle temperature affects the rate of pH decline in a linear manner from 37.5°C to 0-2°C. The pH decline is correlated with the enzymatic degradation of glycogen to lactate and this process includes the metabolic coupling between glycogenolysis and glycolysis, and that are strongly upregulated by the AMPK. In this study, we used 12 samples previously characterized by have different muscle glycogen concentration, lactate and AMPK activity, selected from 38 steers that produced high final pH (>5.9) and normal final pH ( 0.05) and we did not detect structural differences in the polymers present in samples from both categories (p > 0.05), suggesting that postmortem AMPK activity may be highly sensitive to temperature and not to in vitro changes in glycogen concentration (p > 0.05). Our results allow concluding that normal concentrations of muscle glycogen immediately at the time of slaughter (0.5 h) and an adequate cooling managing of carcasses are relevant to let an efficient glycogenolytic/glycolytic flow required for lactate accumulation and pH decline, through the postmortem AMPK signalling pathway

High doses of dopamine reduce sperm motility and acrosomal integrity without affecting the sperm viability over time.

<p><b>A)</b> Effect of dopamine on sperm viability at 1, 3 and 6 hours of incubation. Equine sperm were incubated at 37°C with different concentrations of dopamine (0, 0.01, 0.1 and 1 mM) in Withenn’s buffer and the quantity of viability sperm was calculated using a CASA system, with a minimum of 500 sperm analyzed per experiment. Sperm viability at different times of analysis was normalized to time zero of the control. Results are the mean ± SEM of four independent experiments. <b>B)</b> Effect of dopamine on acrosomal integrity after 1, 3 and 6 hours of incubation. Equine sperm were incubated at 37°C with different dopamine concentrations (0, 0.01, 0.1 and 1 mM) in Withenn’s buffer (enrichment with BSA and bicarbonate) and the percentage of sperms with intact acrosomes was assessed with PSA-FITC staining and by cell count under an epifluorescence microscope. A minimum of 200 sperm were counted. The percentage of intact acrosomes was normalized to time zero of the control without dopamine. Results are the mean ± SEM of four independent experiments. *p<0.05 with respect to the control. <b>C)</b> Effect of dopamine on total sperm motility after incubation for 1, 3 and 6 hours. Equine sperm were incubated at 37°C with different concentrations of dopamine (0, 0.01, 0.1 and 1 mM) in Withenn’s buffer and the quantity of mobile sperm was calculated using a CASA system. A minimum of 500 sperm were analyzed per experiment. Total motility at different times of analysis was normalized to time zero of the control. Results are the mean ± SEM of four independent experiments. **p<0.01 with respect to the control.</p

Dopamine transporter inactivation attenuates the inhibitory effect of 1 mM dopamine on total sperm motility.

<p>Equine sperm were incubated at 37°C with 1 mM dopamine, with and without the specific DAT inhibitor, 10 µM GBR12909 for 1 hour, in Withenn’s buffer. Total motility was calculated using the CASA system, with a minimum of 500 sperm analyzed per experiment. Total motility was normalized to time zero of the control. Results are the mean ± SEM of four independent experiments. The different letters show significant changes between each treatment with p<0.01. We have considered in each experiment a control to normalize the results to the basal sperm condition before the incubation with dopamine and its respective vehicle.</p

SERT and NET are present in ejaculated sperm. The presence and location of other monoamine transporters was determined in equine sperm.

<p>A) The presence of SERT and NET transporters were verified in equine sperm (line 2 and 4) by Western blot assays. A protein extract from whole rat brain (line 1 and 3) was used as a control. Images are representative of 3 independent experiments. B) The localization of SERT and NET was performed by indirect immunofluorescence assays. Images are representative of 3 independent experiments. Bar scale is 10 µm.</p

The dopamine transporter present in equine sperm is functional and sensitive to selective inhibitors.

<p><b>A)</b> In freshly ejaculated equine sperm, ASP⁺ transport was used to assess the functionality of the DAT transporter. Fresh sperm were incubated with 8 µM ASP⁺ in capacitation medium and were analyzed with fluorimetry. Accumulated fluorescence was plotted as arbitrary units of fluorescence (AUF) as a function of transport time. Measurements were made every 35 seconds over a total assay time of 15 minutes. Results were plotted as the mean ± standard error (SEM) of eight independent assays. <b>B)</b> Kinetic characteristics of the DAT transporter present in equine sperm were established by incorporation of ASP⁺. Fresh sperm were incubated with different ASP⁺ concentrations (0 to 30 µM) in capacitation medium for 20 minutes. The difference between fluorescence at time zero and at twenty minutes was plotted as velocity with respect to the different ASP⁺ concentrations used in the assay. Results correspond to the mean ± standard error of an average of eight independent experiments. <b>C)</b> DAT transporter sensitivity was assessed in response to selective inhibition with 50 µM nomifensine and 10 µM bupropion. Fresh sperm were incubated with 8 µM ASP⁺ in capacitation medium for 20 minutes and the difference between fluorescence at time zero and at twenty minutes was recorded. Transport of ASP⁺ was considered to be 100% and inhibitor treatments were normalized with respect to this. Results correspond to the mean ± standard error (SEM) of an average of eight independent experiments, *p<0.05.</p