Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs.

Mode of action of cryoprotectants for sperm preservation.

The sperm mitochondrion: Organelle of many functions.

Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to direct utilization within the mitochondria. Measurements were performed after incubation in modified BWW media with varying concentrations of lactate, pyruvate, and glucose. The effects of media and duration of incubation on sperm motility, ROS production, and oxygen consumption were determined using a linear mixed-effects model. Duplicate ejaculates from four stallions were used in three separate experiments to determine the effects of substrate availability and concentration on sperm motility and mitochondrial function and the relationship of oxygen consumption with cellular ROS production. The present results indicate that lactate and pyruvate are the most important sources of energy for stallion sperm motility and velocity, and elicit a dose-dependent response. Additionally, lactate and pyruvate are ideal for maximal mitochondrial function, as sperm in these media operate at a very high level of their bioenergetic capability due to the high rate of energy metabolism. Moreover, we found that addition of glucose to the media is not necessary for short-term storage of equine sperm, and may even result in reduction of mitochondrial function. Finally, we have confirmed that ROS production can be the result of mitochondrial dysfunction as well as intense mitochondrial activity.

https://bioone.org/journals/biology-of-reproduction/volume-95/issue-2/biolreprod.116.140707/Lactate-and-Pyruvate-Are-Major-Sources-of-Energy-for-Stallion/10.1095/biolreprod.116.140707.pdf

Lactate and Pyruvate Are Major Sources of Energy for Stallion Sperm with Dose Effects on Mitochondrial Function, Motility, and ROS Production

The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa More recently, the progressive introduction of flow cytometry is increasing the number of tests available However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation In this paper, mitochondrial structure and function will be reviewed in the stallion, when possible specific stallion studies will be discussed, and general findings on mammalian mitochondrial function will be argued when relevant Especial emphasis will be put on their role as source of reactive oxygen species and in their role regulating sperm lifespan, a possible target to investigate with the aim to improve the quality of frozen-thawed stallion sperm Later on, the impact of current sperm technologies, principally cryopreservation, on mitochondrial function will be discussed pointing out novel areas of research interest with high potential to improve current sperm technologies

The Impact of Reproductive Technologies on Stallion Mitochondrial Function.

Summary

Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) and poor (poor freezability ejaculates: PFE) freezability stallion ejaculates in other sperm parameters, including peroxide and superoxide levels, potential of mitochondrial membrane and nuclear integrity. With this purpose, a total of 24 stallion ejaculates were cryopreserved and classified into two groups (GFE vs. PFE), depending on their sperm membrane integrity and motility after freeze-thawing. From the total of 24 ejaculates, 13 were classified as GFE and the other 11 were classified as PFE. Apart from differences in sperm membrane permeability and lipid disorder after freeze-thawing, GFE presented significantly (p < 0.05) higher percentages of viable spermatozoa with high content of peroxides and of superoxides than PFE. In contrast, and despite cryopreservation of stallion spermatozoa increasing DNA fragmentation and disrupting disulphide bonds in sperm head proteins, no significant differences between GFE and PFE were seen. We can thus conclude that good and poor freezability stallion ejaculates differ in their reactive oxygen species levels after cryopreservation, but not in the damage extent on sperm nucleus.

Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus

In the donkey, artificial insemination (AI) with frozen-thawed semen is associated with low fertility rates, which could be partially augmented through adding seminal plasma (SP) and increasing sperm concentration. On the other hand, post-AI endometrial inflammation in the jenny is significantly higher than in the mare. While previous studies analyzed this response through recovering Polymorphonuclear Neutrophils (PMN) from uterine washings, successive lavages can detrimentally impact the endometrium, leading to fertility issues. For this reason, the first set of experiments in this work intended to set an in vitro model through harvesting PMN from the peripheral blood of jennies. Thereafter, how PMN, which require a triggering agent like formyl-methionyl-leucyl-phenylalanine (FMLP) to be activated, are affected by donkey semen was interrogated. Finally, we tested how four concentrations of spermatozoa (100 × 106, 200 × 106, 500 × 106 and 1000 × 106 spermatozoa/mL) affected their interaction with PMN. We observed that semen, which consists of sperm and SP, is able to activate PMN. Whereas there was a reduced percentage of spermatozoa phagocytosed by PMN, most remained attached on the PMN surface or into a surrounding halo. Spermatozoa not attached to PMN were viable, and most of those bound to PMN were also viable and showed high tail beating. Finally, only sperm concentrations higher than 500 × 106 spermatozoa/mL showed free sperm cells after 3 h of incubation, and percentages of spermatozoa not attached to PMN were higher at 3 h than at 1 h, exhibiting high motility. We can thus conclude that semen activates PMN in the donkey, and that the percentage of spermatozoa phagocytosed by PMN is low. Furthermore, because percentages of spermatozoa not attached to PMN were higher after 3 h than after 1 h of incubation, we suggest that PMN-sperm interaction plays an instrumental role in the reproductive strategy of the donkey.

https://www.mdpi.com/1422-0067/21/10/3478/pdf

Seminal Plasma, Sperm Concentration, and Sperm-PMN Interaction in the Donkey: An In Vitro Model to Study Endometrial Inflammation at Post-Insemination

Color Doppler provides a reliable and rapid means of monitoring luteolysis in female donkeys.

While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. Remarkably, sperm interacts with PMN, but the presence of seminal plasma reduces that binding. As seminal plasma is a complex fluid made up of different molecules, including proteins, this study aimed to evaluate how different seminal plasma fractions, separated by molecular weight ( 100 kDa), affect sperm–PMN binding. Sperm motility, viability, and sperm–PMN binding were evaluated after 0 h, 1 h, 2 h, 3 h, and 4 h of co-incubation at 38 °C. Two seminal plasma fractions, including 30–50 kDa or 50–100 kDa proteins, showed the highest sperm motility and viability. As viability of sperm not bound to PMN after 3 h of incubation was the highest in the presence of 30–50 and 50–100 kDa proteins, we suggest that both fractions are involved in the control of the jenny’s post-breeding inflammatory response. In conclusion, this study has shown for the first time that specific fractions rather than the entire seminal plasma modulate sperm–PMN binding within the donkey uterus. As several proteins suggested to be involved in the control of post-AI endometritis have a molecular weight between 30 and 100 kDa, further studies aimed at determining the identity of these molecules and evaluating their potential effect in vivo are much warranted.

Specific Seminal Plasma Fractions Are Responsible for the Modulation of Sperm–PMN Binding in the Donkey

Sperm selection techniques have became an important tool to improve sub fertile ejaculates. The main aim of this study was to assess the dynamics of motile sperm subpopulations of ejaculates from subfertile stallions subjected to density gradient centrifugation (DGC) involving a double layer colloid [DGC2], before and after storage for 24 and 48 hours. Ejaculates from eight stallions with fertility problems were subjected to the different treatments: 1) control [C]; 2) centrifugation [CT]; 3) density gradient centrifugation involving a double layer colloid [DGC2] and 4) post storage density gradient centrifugation involving a double layer colloid DGC2 post]. In the C, CT and DGC2 treatments, sperm variables were analysed at 0 h and after 24 and 48 h of storage at 4-7 ˚C. In the DGC2 post treatment, the semen was stored at 4-7˚Cand sperm samples selected by DGC2 at 24 and 48 h. Viability, sperm abnormalities and motility were then examined using a computerized system. Four motile sperm subpopulations with different motility characteristics were observed in the all treatments. The DGC2 sperm had faster, straighter moving and more active cells at 0 h. At 24 and 48 h, however, the motility variable values fell and the slower spermatozoa subpopulations increased in size. The DGC2 post treatment returned better overall sperm motility variable values at 24 and 48 h; a larger percentage of spermatozoa fell into the faster subpopulations. In conclusion, DGC2 could be used to improve semen from subfertile stallions as well as semen showing poor fertility after storage.

http://ijas.iaurasht.ac.ir/article_513313_5d5ace312a1a9873943bb9b02cfa9623.pdf

Stallion sperm selection by density gradient centrifugation involving a double layer colloid: effects on sperm subpopulation dynamics in fresh and stored semen.

H. Marin

Papers

Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus

Seminal Plasma, Sperm Concentration, and Sperm-PMN Interaction in the Donkey: An In Vitro Model to Study Endometrial Inflammation at Post-Insemination

Color Doppler provides a reliable and rapid means of monitoring luteolysis in female donkeys.

Specific Seminal Plasma Fractions Are Responsible for the Modulation of Sperm–PMN Binding in the Donkey

Stallion sperm selection by density gradient centrifugation involving a double layer colloid: effects on sperm subpopulation dynamics in fresh and stored semen.