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The in vitro production of equine embryos via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) has increased rapidly. There is a marked effect of the individual mare on the outcome of OPU-ICSI, but little is known about the influence of the mare's health condition. This study aimed to investigate the potential associations between the concentrations of interleukin-6 (IL-6), reactive oxygen metabolites (d-ROMs), and biological antioxidant potential (BAP) in serum of oocytes' donor mares and the subsequent embryonic development. Just before OPU, a blood sample was collected from 28 Warmblood donor mares, that were subjected to a routine OPU-ICSI program. The serum concentrations of IL-6, d-ROMs, and BAP were assayed photometrically. The maturation, cleavage and blastocyst rate as well as the kinetics of blastocyst development were recorded. The average blastocyst rate was 24.68 ± 5.16% and the average concentrations of IL-6, d-ROMs, and BAP were 519.59 ± 157.08 pg/mL, 171.30 ± 4.55 carratelli units (UCARR), and 2711.30 ± 4.55 μmol/L, respectively. Serum concentrations of IL-6, d-ROMs, and BAP were not significantly different between mares yielding at least one blastocyst (552.68 ± 235.18 pg/mL, 168.36 ± 5.56 UCARR, and 2524.80 ± 159.55 μmol/L) and mares yielding no blastocysts (468.47 ± 179.99 pg/mL, 175.85 ± 7.89 UCARR, and 2999.50 ± 300.13 μmol/L, respectively). Serum concentrations of d-ROMs were significantly lower in mares with fast growing (at day 7-8 post ICSI; 148.10 ± 8.13 UCARR) compared to those with slow growing blastocysts (≥ day 9 post ICSI; 179.41 ± 4.89 UCARR; P = 0.003). Taken together, the serum concentration of IL-6, d-ROMs, and BAP do not determine the mare's ability to produce blastocysts in vitro. Although it may be questioned whether a single sample is representative of the mare's health status, changes in serum metabolites related to oxidative stress at the time of oocyte retrieval were linked to a delayed blastocyst development in a clinical OPU-ICSI outcome., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality parameters were analyzed immediately after light-stimulation (0 min) and after 120 min of incubation at 38 °C. Sperm motility was evaluated using a Computerized Semen Analysis System (CASA), and flow cytometry and different fluorochromes were used to evaluate the integrity and lipid disorder of plasma membrane, mitochondrial membrane potential and intracellular levels of peroxides and superoxides. Irradiation significantly increased the percentages of spermatozoa with high mitochondrial membrane potential (1-1-1 pattern) and the intracellular levels of peroxides (2-2-2 pattern) at 0 min. In addition, sperm kinematic parameters (2-2-2 and 3-3-3 patterns) and percentages of viable spermatozoa with low membrane lipid disorder (3-3-3 pattern) were significantly higher in irradiated samples than in the control at 120 min. Our results indicate that red-light stimulation could help increase the resilience of frozen-thawed stallion sperm to withstand post-thaw incubation at 38 °C for 120 min and that these effects rely on the irradiation pattern. Further research should evaluate whether light-stimulation could also have a positive on fertility rates after artificial insemination., Competing Interests: Declaration of competing interest J.E.R.G. and M.Y. are inventors of a patent entitled ‘Method and apparatus for improving the quality of mammalian sperm’ (European Patent Office, No. 16199093.2; EP-3-323-289-A1), which is owned by Instruments Utils de Laboratori Geniul, SL (Barcelona, Spain)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This study sought to determine whether sperm irradiation using a light emission diode (LED) at 620-630 nm affects the motility, membrane integrity (viability), mitochondrial activity and intracellular levels of reactive oxygen species (ROS) in fresh diluted and liquid-stored donkey semen. With this purpose, sixteen ejaculates (eight fresh diluted and eight cooled-stored) were collected from eight adult jackasses. Fresh semen samples were diluted in Kenney extender and stimulated with red-light after collection, whereas cooled semen was stored at 4 °C for 24 h after dilution and then irradiated. In all cases, semen samples were packed into 0.5-mL transparent straws, which were then randomly divided into control and 19 treatments: six consisted of single red-light exposure, and the other 13 involved irradiation at light-dark-light intervals. Upon irradiation, sperm motility, membrane integrity mitochondrial membrane potential (MMP) and intracellular levels of superoxide anion (·O 2 - ) and hydrogen peroxide (H 2 O 2 ) were evaluated. While specific light-patterns increased both sperm motility and mitochondrial activity, they did not affect sperm membrane integrity and had no clear impact on intracellular ROS levels. The effects of irradiation patterns differed between fresh and cooled semen since, whereas 1 and 4 min patterns induced the greatest increments in the total and progressive motility of fresh semen, 4 min, 4-1-4 and 4-4-4 were the most suitable for cooled-stored samples. In both fresh diluted and cooled-stored semen, the motility increase observed after light-stimulation for 4 min was concomitant with changes in the percentages of spermatozoa with high mitochondrial membrane potential. In summary, this study shows, for the first time, that specific irradiation patterns increase sperm motility and mitochondrial activity in the donkey. Furthermore, the precise effect of red-light appears to depend on the specific functional status of cells, with separate effects on fresh and cooled samples., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported herein., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This study investigated whether the activities of four antioxidant enzymes present in jackass seminal plasma (SP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) and glutathione reductase (GSR), are related to the sperm ability to withstand cryopreservation. Eighteen ejaculates from 16 healthy jackasses were collected and split into two aliquots. The first one was centrifuged (3,000×g, 4 °C for 10 min) and used to determine the activities of these four enzymes in SP, whereas the other was diluted in a skim-milk extender and then cryopreserved. Assessment of sperm motility and membrane integrity was performed before and after cryopreservation. Based on the percentages of total motile and viable spermatozoa at post-thaw, samples were classified as good (GFE) or poor (PFE) freezability ejaculates through cluster analyses. Total and specific activities of SOD in seminal plasma were higher (P < 0.05) in GFE than in PFE, whereas no significant differences between GFE and PFE were observed regarding total and specific activities of CAT, GPX and GSR. However, post-thaw sperm parameters were positively correlated with total and specific activities of CAT and negatively correlated with those of GSR. In conclusion, determination of total and specific activities of SOD in the seminal plasma of a given jackass ejaculate may predict the sperm ability to withstand cryopreservation. In addition, our results warrant further research on addressing whether SOD activity in seminal plasma does not only allow predicting the sperm cryotolerance of a given ejaculate but also that of all ejaculates from a given jackass., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study compared the activities of four antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX; and glutathione reductase, GSR) in the seminal plasma of stallions and jackasses. Eighteen stallion ejaculates and 24 jack ejaculates were collected through an artificial vagina. Seminal plasma was obtained by several centrifugations at 3000×g and 4 °C for 10 min, and activities of SOD, CAT, GPX and GSR were subsequently determined. We also evaluated whether the collecting season had any influence on the activities of these four enzymes in both stallions and jackasses. Antioxidant capacity of seminal plasma was significantly higher in jackasses than in stallions (mean ± SEM, SOD: 1707.7 ± 195.9 U/mL vs. 231.9 ± 29.6 U/mL; CAT: 9094.7 ± 1292.9 U/L vs.1682.7 ± 525.9 U/L; GPX 845.4 ± 106.0 U/L vs. 469.7 ± 60.3 U/L; GSR: 50.3 ± 5.1 U/L vs. 20.7 ± 4.6 U/L). Furthermore, whereas season had no effect on the activity of these four enzymes in stallions, the activities of CAT and GPX in jack seminal plasma were significantly higher in the summer than in the other seasons. In addition, the activities of SOD and CAT were found to be significantly correlated with the percentages of progressively motile spermatozoa, and with the percentages of linearity and straightness, respectively, in jackasses. In contrast, the activities of these four enzymes were not correlated with sperm quality parameters in stallions. Finally, while SOD, CAT, and GPX activities but not those of GSR were correlated in jackasses, the activities of all four enzymes were correlated each other in stallions. We can thus conclude that the activities of SOD, CAT, GPX and GSR differ between the seminal plasma of stallions and donkeys, and vary between seasons in jackasses., (Copyright © 2019 Elsevier Inc. All rights reserved.)