New seminal plasma removal method for freezing stallion semen

doi:10.1016/j.theriogenology.2013.01.014

Theriogenology

Volume 79, Issue 7, 15 April 2013, Pages 1120-1123.e1

https://doi.org/10.1016/j.theriogenology.2013.01.014 Get rights and content

Abstract

Seminal plasma removal, an indispensable step in equine semen cryopreservation, is usually done by centrifugation, but this might cause mechanical damage to sperm. A new method for seminal plasma removal from stallion semen, namely a filter composed of a synthetic hydrophilic membrane (Sperm Filter, BotuPharma, Botucatu, Sao Paulo, Brazil), was recently proposed. The objective of this study was to test the use of the Sperm Filter in the removal of seminal plasma before freezing stallion semen. Ejaculates from 31 stallions were divided into two groups and cryopreserved. In group 1 (G1), seminal plasma was removed with the Sperm Filter, and in group 2 (G2), seminal plasma was removed by centrifugation (600× g for 10 minutes). There were no differences (P < 0.05) between G1 and G2 in sperm kinetic parameters or plasma membrane integrity before or after cryopreservation. However, sperm recovery rate was higher (P < 0.05) for G1 versus G2 (mean ± SD, 89.4 ± 7.4% vs. 80.9 ± 5.5%). Therefore, the Sperm Filter was as efficient as centrifugation in removing seminal plasma from the stallion ejaculate. However, filtering was more practical and had significantly fewer sperm lost than the centrifugation technique.

Introduction

Semen freezing is a very important technique, because it can prevent the spread of diseases, eliminate geographical barriers and, above all, preserve genetic material indefinitely. However, damage to sperm during the semen freezing process can affect its subsequent fertility. This damage to sperm, when combined with the low resistance of the sperm of some stallions to cryopreservation [1], is one of the greatest barriers to widespread use of this biotechnology [2].

Post-thaw semen quality and fertility are influenced by several factors, namely the individual characteristics of each animal, technique used for seminal plasma removal, type of extender used, medium used for sperm dilution, freezing curve for the semen straws, type of straw storage used, semen thawing time and temperature, semen dose, and insemination technique used [3], [4]. Because so many factors influence the success of sperm cryopreservation, no ideal protocol for all cases exists [5], although numerous studies have been conducted with the aim of improving the quality of equine frozen semen [6].

Of the steps involved in freezing equine semen, seminal plasma removal from the ejaculate before freezing, has been a major focus of research [7], [8]. This step is essential for sperm cryopreservation [9], [10]. The technique typically used in the removal of seminal plasma from stallion ejaculate is centrifugation; however, this procedure might cause mechanical damage to sperm [11]. After analyzing centrifugal force and duration, Dell'aqua et al. [12] reported that the sperm loss was minimized and sperm quality and integrity were maximized when semen was centrifuged at 600× g for 10 minutes.

To concentrate semen without centrifugation and with less damage to sperm, Alvarenga et al. [13] proposed a new method of removing seminal plasma from stallion ejaculate by filtering the semen through a filter composed of a synthetic hydrophilic membrane (Sperm Filter, BotuPharma, Botucatu, Sao Paulo, Brazil). The objective of the present study was to test the efficacy of the Sperm Filter in removing seminal plasma from stallion semen before freezing.

Section snippets

Seminal plasma removal using the Sperm Filter

The Sperm Filter is a filter composed of a synthetic hydrophilic membrane with 2-μm pores; the filter is attached to a plastic ring that holds 50 mL of semen for filtration. The semen, diluted 1:1 with extender, was deposited on the membrane, and the filter was gently tapped over a 15-cm Petri dish. Because of the size of the pores and capillary action, 90 to 95% of the seminal plasma passed through the filter, and the sperm were retained. Then, an appropriate volume of extender was added to

Results

Before freezing, there were no differences (P > 0.05) among fresh semen in skim milk-based extender (G0), filtered semen (G1), and centrifuged semen (G2) for kinetic parameters and sperm plasma membrane integrity (Table 1). Additionally, for these parameters, there were no differences (P > 0.05) between G1 and G2 after thawing (Table 1). However, there were more sperm recovered in G1 versus G2 (89.4 ± 7.4% vs. 80.9 ± 5.5%).

Discussion

In the present study, the filtering technique (G1) effectively removed seminal plasma from stallion semen before freezing. This step in equine semen cryopreservation is essential, because seminal plasma can be detrimental to sperm [17]; therefore, the sperm is concentrated, thereby facilitating storage of large numbers of sperm in small volumes [18]. Although centrifugation is commonly used to remove equine seminal plasma, this might damage sperm and thus reduce motility and fertility [2], [11].

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Reduced bacterial load in stallion semen by modified single layer centrifugation or sperm washing
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The presence of bacteria poses a significant challenge to the quality of stallion semen used in artificial insemination. The bacterial content of insemination doses arises from various sources, such as the healthy stallion, environment, and collection equipment, and is implicated in fertility problems as well as reduced sperm quality during storage. The conventional approach of adding antibiotics to semen extenders raises concerns about antimicrobial resistance and potential negative effects on sperm characteristics, and may not be effective in inhibiting all bacteria. The objective of this study was to determine whether an innovative alternative to antibiotic usage – centrifugation through a single layer of a low density colloid (SLC) – could reduce the bacterial load in stallion semen, and to compare sperm characteristics in samples arising from this procedure, or simple extension of the ejaculate in semen extender, or from sperm washing, i.e. adding extender and then centrifuging the sample to allow the removal of most of the seminal plasma and extender. Eighteen semen samples were collected from six stallions. The semen samples were split and extended prior to washing or SLC, or received no further treatment other than extension. After preparation aliquots from each type of sample were sent for bacteriological examination; the remaining samples were stored for up to 72 h, with daily checks on sperm quality. The low density colloid SLC outperformed sperm washing or extension for bacterial reduction, effectively removing several bacterial species. The bacterial load in the samples was as follows: extended semen, 16 ± 6.7 × 10⁵; washed, 5.8 ± 2.0 × 10⁵; SLC, 2.3 ± 0.88 × 10⁵, p < 0.0001. In addition, SLC completely removed some bacterial species, such as Staphylococcus xylosus. Although there is no selection for robust spermatozoa with the low density colloid, sperm motility, membrane integrity, and DNA fragmentation were not different to washed sperm samples. These findings suggest that SLC with a low density colloid offers a promising method for reducing bacterial contamination in stallion semen without resorting to antibiotics.
Postthaw Addition of Autologous Seminal Plasma Improves Sperm Motion Characteristics in Fair and Poor Freezer Stallions
2019, Journal of Equine Veterinary Science
Citation Excerpt :
Therefore, optimum protocols for semen processing and insemination of mares need to be developed to provide appropriate protocols for individual stallions [4,7,8]. Before stallion semen can be frozen, most of the seminal plasma is removed by centrifugation or filtration of the semen and discarding the supernatant [9–12]. However, a proportion of 5%–20% of seminal plasma to semen has been recommended [9].
During semen processing for cryopreservation, most seminal plasma is usually removed, and components with protective effects on sperm may be missing after thawing and within the female reproductive tract. The present study evaluated the effect of postthaw addition of autologous seminal plasma on motion characteristics of stallion sperm with fair (n = 4) or poor (n = 3) freezability. Therefore, pure seminal plasma (group SP1), seminal plasma combined with fresh semen extender (group SP2), or seminal plasma mixed with freezing extender (group SP3) were used to fill 0.5 mL straws and frozen similar to stallion semen. Postthawing, semen samples (n = 42) were diluted either with semen extender (group FT) or with seminal plasma (n = 126) of groups SP1 to SP3 to 25 × 10⁶ sperm/mL. In fair freezer stallions, total and progressive motilities were higher in group FT than in group SP1 (P < .05), but there was no difference in poor freezing stallions among groups (P > .05). However, comparing individual stallions, positive effects of seminal plasma on total or progressive motility were detected in two stallions. Curvilinear velocity increased in groups SP2 and SP3 in fair freezer stallions and in all groups with seminal plasma compared with group FT in poor freezer stallions (P < .05). Although straightness was higher in groups SP2 and SP3 compared with group FT in fair freezer stallions (P < .05), there was no difference among groups in stallions with poor freezability (P > .05). Average lateral head displacement did not change among groups of fair freezer stallions (P > .05) but was higher in groups SP2 and SP3 than in group FT in poor freezer stallions (P < .05). Beat cross frequency was higher in all groups diluted with seminal plasma postthawing in fair freezer stallions (P < .05), but only in group SP1 than in group FT in poor freezer stallions (P < .05). The addition of autologous seminal plasma to frozen-thawed semen can improve motion characteristics of stallions with fair and poor freezability. This is a valuable additional protocol for laboratories dealing with cryopreservation of stallion semen and for veterinarians working with fair or poor freezer stallions.
Susceptibility of Stallion Spermatozoa to Different Oxidative Challenges: Role of Seminal Plasma
2017, Journal of Equine Veterinary Science
Citation Excerpt :
However, the removal of SP is a key step for sperm cryopreservation in horses. This step is essential mainly to concentrate the number of spermatozoa [14]. However, although this procedure is essential to a successful cryopreservation of equine sperm, removal of SP can increase sperm susceptibility to ROS due to the consequent removal of antioxidant protection [15].
Seminal cryopreservation provides several advantages in horse breeding. However, improvement on post-thaw sperm survival is still necessary. One of the main factors known to impair post-thaw quality of stallion's sperm is the oxidative damage caused by reactive oxygen species (ROS). In this context, the aim of this study was evaluated the effects of ROS on stallion sperm and the possible influence of seminal plasma (SP). Toward this aim, 13 ejaculates from adult stallions (n = 13) were divided into two aliquots which were centrifuged (600g/10 minutes), and the SP was removed and reserved. Pellets were suspended in physiological saline solution (A) or SP (B). Each of these solutions was divided into four aliquots and subjected to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical [OH⁻]) and the toxic by-product of lipid peroxidation (malondialdehyde [MDA]). Samples were then evaluated for the susceptibility to lipid peroxidation (thiobarbituric acid reactive substances) plasma membrane integrity (eosin–nigrosin), acrosome integrity (Fast Green/rose bengal), mitochondrial activity (3′3 diaminobenzidine), and DNA integrity. Samples incubated in the presence of SP were highly impaired by OH⁻ with regard to motility, plasma membrane integrity, mitochondrial activity, and DNA integrity. On the other hand, in the absence of SP, MDA was highly deleterious, especially with regard to motility, plasma membrane integrity, and mitochondrial activity. Thus, these results indicate that SP may have an important role on the protection of stallion sperm against the damages caused by MDA, an important product of lipid peroxidation.
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Research articleNew seminal plasma removal method for freezing stallion semen

Abstract

Introduction

Section snippets

Seminal plasma removal using the Sperm Filter

Results

Discussion

Anim Reprod Sci

Theriogenology

Journal of Equine Veterinary Science

Theriogenology

Theriogenology

Journal of Equine Veterinary Science

Fertil Steril

Vet Clin North Am Equine Pract

Cooled and frozen stallion semen: animal reproduction and biotechnology laboratory. Bulletin no. 9

The predictive value of semen analysis in the evaluation of stallion fertility

Reprod Domest Anim

Opitimal concentration of cryopreservative agents for semen from stallions that are classified “good” or “poor” freezing

Anim Reprod Sci

Centrifugation of stallion semen and its storage in large volume straws

J Reprod Fertil Suppl

Application of techniques for sperm selection in fresh and frozen-thawed stallion semen

Reprod Domest Anim

Effect of centrifugation and packing system on sperm parameters of equine frozen semen

Animal Reproduction Science

Research article
New seminal plasma removal method for freezing stallion semen