Tube Penetration Research Articles

Two functionally interchangeable Vps9 isoforms mediate pollen tube penetration of style.

Style penetration by pollen tubes is essential for reproductive success, a process requiring canonical Rab5s in Arabidopsis. However, functional loss of Arabidopsis Vps9a, the gene encoding for guanine nucleotide exchange factor (GEF) of Rab5s, did not affect male transmission, implying the presence of a compensation program or redundancy. By combining genetic, cytological, and molecular approaches, we report that Arabidopsis Vps9b is a pollen-preferential gene, redundantly mediating pollen tube penetration of style with Vps9a. Vps9b is functionally interchangeable with Vps9a, whose functional distinction results from distinct expression profiles. Functional loss of Vps9a and Vps9b results in the mis-targeting of Rab5-dependent tonoplast proteins, defective vacuolar biogenesis, disturbed distribution of post-Golgi vesicles, increased cellular turgor, cytosolic acidification, and disrupted organization of actin microfilaments (MF) in pollen tubes, which collectively lead to the failure of pollen tubes to grow through style.

Arabidopsis Sar1b is critical for pollen tube growth.

Pollen tube growth is an essential step leading to reproductive success in flowering plants, in which vesicular trafficking plays a key role. Vesicular trafficking from endoplasmic reticulum to the Golgi apparatus is mediated by the coat protein complex II (COPII). A key component of COPII is small GTPase Sar1. Five Sar1 isoforms are encoded in the Arabidopsis genome and they show distinct while redundant roles in various cellular and developmental processes, especially in reproduction. Arabidopsis Sar1b is essential for sporophytic control of pollen development while Sar1b and Sar1c are critical for gametophytic control of pollen development. Because functional loss of Sar1b and Sar1c resulted in pollen abortion, whether they influence pollen tube growth was unclear. Here we demonstrate that Sar1b mediates pollen tube growth, in addition to its role in pollen development. Although functional loss of Sar1b does not affect pollen germination, it causes a significant reduction in male transmission and of pollen tube penetration of style. We further show that membrane dynamics at the apex of pollen tubes are compromised by Sar1b loss-of-function. Results presented provide further support of functional complexity of the Sar1 isoforms.

Unlocking a ‘lock–key’ mechanism governing pollen–pistil interactions

Pollen–pistil interactions ensure genetic diversity and shape the reproductive success of plants. Lan et al. recently revealed that the interaction among various receptor-like kinases, cell-wall proteins, and stigmatic RALF peptides (sRALFs) or pollen RALF peptides (pRALFs) on the stigma surface govern the penetration of pollen tubes in members of the Brassicaceae.

Karsten tube analysis for joint mortar identification in masonry

The present work examines the Karsten tube penetration test to identify mortar types in existing masonry, for mortars containing air-lime or hydraulic binders. Mortar type identification is e.g. of relevance for the reusability of the fired clay bricks in the masonry, where a hydraulic binder content makes the mortar removal process significantly more difficult. The Karsten tube test makes it possible to make a simple and non-destructive mortar analysis on-site assessing the water absorption of the mortar, via the surface of the mortar joint alone. The present approach considers both the effect of the surrounding bricks and the surface finish of the pointing. Furthermore, the setup masks the brick surface exposed under the Karsten tube to focus the exposure on the mortar. Three different mortar types in existing masonry were tested: air-lime mortar, air-lime/hydraulic lime mortar and air-lime/cement mortar. The results showed linear water absorptions, which were clearly distinguishable for the hydraulic and non-hydraulic binders during the 15-minute tests. The measurement results in the present work document how the Karsten tube penetration test can be used for simple and non-destructive identification of joint mortar types determining whether the masonry was constructed with air-lime mortar or mortar containing hydraulic binders.

Antagonistic RALF peptides control an intergeneric hybridization barrier on Brassicaceae stigmas

Pollen-pistil interactions establish interspecific/intergeneric pre-zygotic hybridization barriers in plants. The rejection of undesired pollen at the stigma is crucial to avoid outcrossing but can be overcome with the support of mentor pollen. The mechanisms underlying this hybridization barrier are largely unknown. Here, in Arabidopsis, we demonstrate that receptor-like kinases FERONIA/CURVY1/ANJEA/HERCULES RECEPTOR KINASE 1 and cell wall proteins LRX3/4/5 interact on papilla cell surfaces with autocrine stigmatic RALF1/22/23/33 peptide ligands (sRALFs) to establish a lock that blocks the penetration of undesired pollen tubes. Compatible pollen-derived RALF10/11/12/13/25/26/30 peptides (pRALFs) act as a key, outcompeting sRALFs and enabling pollen tube penetration. By treating Arabidopsis stigmas with synthetic pRALFs, we unlock the barrier, facilitating pollen tube penetration from distantly related Brassicaceae species and resulting in interspecific/intergeneric hybrid embryo formation. Therefore, we uncover a "lock-and-key" system governing the hybridization breadth of interspecific/intergeneric crosses in Brassicaceae. Manipulating this system holds promise for facilitating broad hybridization in crops.

Study of the hygroscopic properties of environmentally friendly lightened composites through waste recovery

Gypsum-plaster is becoming an ideal binder in the valorisation and recovery of waste for designing more sustainable composites. Nevetheless, the main limitation of plaster lies in its susceptibility to the action of water. In this research, a complete study of the hygroscopic properties of a new material has been carried out, in which plaster material has been partially substituted in mass by dissolved expanded polystyrene waste in percentages of 8.8% to 26.5%. Additionally, fibres from end-of-life tyres have been incorporated at 1.2% by mass to improve the strength and durability of the compounds, achieving a total reduction of up to 28% of the original raw materials. The experimental campaign delves into the water performance of this new plaster composites through mercury porosimetry tests, water absorption by capillarity, total water absorption, water vapour permeability, Karsten Tube penetration, water-stove cycles, as well as the analysis of images obtained by scanning electron microscopy. The main results obtained show a reduction of 97.8% in surface water absorption, as well as a decrease of up to 17.44% in water vapour permeability compared to traditional plaster. Moreover, the developed materials exhibit good mechanical behaviour after undergoing several accelerated ageing cycles. This work highlights the potential of the new plaster composites designed to produce a more sustainable alternative to commercial prefabricated boards and panels, especially suitable for use in interior rooms where high resistance to the action of water is required.

Pollination, pollen tube growth, and fertilization independently contribute to fruit set and development in tomato.

In flowering plants, pollination, pollen tube growth, and fertilization are regarded as the first hierarchical processes of producing offspring. However, their independent contributions to fruit set and development remain unclear. In this study, we examined the effect of three different types of pollen, intact pollen (IP), soft X-ray-treated pollen (XP) and dead pollen (DP), on pollen tube growth, fruit development and gene expression in "Micro-Tom" tomato. Normal germination and pollen tube growth were observed in flowers pollinated with IP; pollen tubes started to penetrate the ovary at 9h after pollination, and full penetration was achieved after 24h (IP24h), resulting in ~94% fruit set. At earlier time points (3 and 6h after pollination; IP3h and IP6h, respectively), pollen tubes were still in the style, and no fruit set was observed. Flowers pollinated with XP followed by style removal after 24h (XP24h) also demonstrated regular pollen tubes and produced parthenocarpic fruits with ~78% fruit set. As expected, DP could not germinate and failed to activate fruit formation. Histological analysis of the ovary at 2 days after anthesis (DAA) revealed that IP and XP comparably increased cell layers and cell size; however, mature fruits derived from XP were significantly smaller than those derived from IP. Furthermore, there was a high correlation between seed number and fruit size in fruit derived from IP, illustrating the crucial role of fertilization in the latter stages of fruit development. RNA-Seq analysis was carried out in ovaries derived from IP6h, IP24h, XP24h and DP24h in comparison with emasculated and unpollinated ovaries (E) at 2 DAA. The results revealed that 65 genes were differentially expressed (DE) in IP6h ovaries; these genes were closely associated with cell cycle dormancy release pathways. Conversely, 5062 and 4383 DE genes were obtained in IP24h and XP24h ovaries, respectively; top enriched terms were mostly associated with cell division and expansion in addition to the 'plant hormone signal transduction' pathway. These findings indicate that full penetration of pollen tubes can initiate fruit set and development independently of fertilization, most likely by activating the expression of genes regulating cell division and expansion.

Disruption of pollen tube homogalacturonan synthesis relieves pollen tube penetration defects in the Arabidopsis O-FUCOSYLTRANSFERASE1 mutant.

During angiosperm sexual reproduction, pollen tubes must penetrate through multiple cell types in the pistil to mediate successful fertilization. Although this process is highly choreographed and requires complex chemical and mechanical signaling to guide the pollen tube to its destination, aspects of our understanding of pollen tube penetration through the pistil are incomplete. Our previous work demonstrated that disruption of the Arabidopsis thaliana O-FUCOSYLTRANSFERASE1 (OFT1) gene resulted in decreased pollen tube penetration through the stigma-style interface. Here, we demonstrate that second site mutations of Arabidopsis GALACTURONOSYLTRANSFERASE 14 (GAUT14) effectively suppress the phenotype of oft1 mutants, partially restoring silique length, seed set, pollen transmission, and pollen tube penetration deficiencies in navigating the female reproductive tract. These results suggest that disruption of pectic homogalacturonan (HG) synthesis can alleviate the penetrative defects associated with the oft1 mutant and may implicate pectic HG deposition in the process of pollen tube penetration through the stigma-style interface in Arabidopsis. These results also support a model in which OFT1 function directly or indirectly modifies structural features associated with the cell wall, with the loss of oft1 leading to an imbalance in the wall composition that can be compensated for by a reduction in pectic HG deposition.

Homeostasis of flavonoids and triterpenoids most likely modulates starch metabolism for pollen tube penetration in rice.

In angiosperms, the timely delivery of sperm cell nuclei by pollen tube (PT) to the ovule is vital for double fertilization. Penetration of PT into maternal stigma tissue is a critical step for sperm cell nuclei delivery, yet little is known about the process. Here, a male-specific and sporophytic mutant xt6, where PTs are able to germinate but unable to penetrate the stigma tissue, is reported in Oryza sativa. Through genetic study, the causative gene was identified as Chalcone synthase (OsCHS1), encoding the first enzyme in flavonoid biosynthesis. Indeed, flavonols were undetected in mutant pollen grains and PTs, indicating that the mutation abolished flavonoid biosynthesis. Nevertheless, the phenotype cannot be rescued by exogenous application of quercetin and kaempferol as reported in maize and petunia, suggesting a different mechanism exists in rice. Further analysis showed that loss of OsCHS1 function disrupted the homeostasis of flavonoid and triterpenoid metabolism and led to the accumulation of triterpenoid, which inhibits significantly α-amylase activity, amyloplast hydrolysis and monosaccharide content in xt6, these ultimately impaired tricarboxylic acid (TCA) cycle, reduced ATP content and lowered the turgor pressure as well. Our findings reveal a new mechanism that OsCHS1 modulates starch hydrolysis and glycometabolism through modulating the metabolic homeostasis of flavonoids and triterpenoids which affects α-amylase activity to maintain PT penetration in rice, which contributes to a better understanding of the function of CHS1 in crop fertility and breeding.

Do egg cell‐secreted aspartic proteases promote gamete attachment?

During the double fertilization process of flowering plants (angiosperms), pollen tubes transport a pair of immobile sperm cells towards and inside the embryo sac for fusion with egg and central cell, respectively. Male-female gamete interactions during fertilization are critical for reproductive success and seed development, and thus have interested biologists since decades. Compared to animals, little is known about gamete interaction mechanisms in angiosperms. This article is protected by copyright. All rights reserved.

Assessing Als3 Peptide-Binding Cavity and Amyloid-Forming Region Contributions to Candida albicans Invasion of Human Oropharyngeal Epithelial Cells.

Although it is widely recognized that disruption of ALS3 reduces the invasion of Candida albicans germ tubes into mammalian oral epithelial cells, the mechanism of this interaction was unexplored. C. albicans strains with structurally informed mutations to remove adhesive activity of the peptide-binding cavity (PBC) or aggregative activity mediated by the amyloid-forming region (AFR) were assessed for their ability to invade cultured human oropharyngeal epithelial cells. Initial assays utilized untreated fungal and epithelial cells. Subsequent work used epithelial cells treated with cytochalasin D and C. albicans cells treated with thimerosal to investigate invasion mediated by active penetration of germ tubes and epithelial cell induced endocytosis, respectively. Results demonstrated the importance of the PBC for the invasion process: loss of PBC function resulted in the same reduced-invasion phenotype as a C. albicans strain that did not produce Als3 on its surface. Invasion via active penetration was particularly compromised without PBC function. Loss of AFR function produced a wild-type phenotype in the untreated and thimerosal-treated invasion assays but increased invasion in cytochalasin D-treated epithelial cells. In previous work, reduced AFR-mediated Als3 aggregation increased C. albicans adhesion to cultured epithelial cell monolayers, presumably via increased PBC accessibility for ligand binding. Collectively, results presented here demonstrate that Als3 PBC-mediated adhesion is integral to its invasive function. These new data add to the mechanistic understanding of the role of Als3 in C. albicans invasion into mammalian oral epithelial cells.

Comparisons among populations and individuals to evaluate pollen-pistil interaction as a mechanism of reproductive interference in Taraxacum.

Reproductive interference (RI), an interspecific mating interaction that reduces the fitness of at least one of the species involved, can lead to exclusive distributions in closely related species. A hypothesis previously proposed is that RI in plants may occur by ovule usurpation, in which pistils lack interspecific incompatibility and mistakenly accept heterospecific pollen, thereby losing an opportunity for conspecific pollen fertilization. However, few comparative studies have evaluated the consistency of the inferred mechanism within and among individuals and populations. We conducted hand-pollination experiments in six populations of three native Taraxacum species that suffered from different levels of RI from an alien congener, T. officinale, and compared pollen-pistil interactions among populations. We also investigated the interactions for eight individual T. japonicum plants whose response to heterospecific pollen deposition had been previously measured. Our results revealed that pollen tubes often penetrated native ovaries following heterospecific pollination in populations suffering from strong RI, whereas they seldom did in populations suffering from marginal RI. However, the relative frequency of the pollen tube penetration was not significantly related to the strength of alien RI. Not all pistils on an individual plant showed the same pollen receptivity following heterospecific pollination; rather, some accepted and some refused the pollen tubes. The relationship between pollen tube penetration following heterospecific pollination and the strength of the alien RI was also not significant among individuals. Our present results generally support the ovule usurpation hypothesis, but suggest that other factors, such as competition for pollinator services, variation in the effects of heterospecific pollen donors, and condition of the native inflorescences, might also affect the observed RI strength.

Pollen-Pistil Interactions as Reproductive Barriers.

Pollen-pistil interactions serve as important prezygotic reproductive barriers that play a critical role in mate selection in plants. Here, we highlight recent progress toward understanding the molecular basis of pollen-pistil interactions as reproductive isolating barriers. These barriers can be active systems of pollen rejection, or they can result from a mismatch of required male and female factors. In some cases, the barriers are mechanistically linked to self-incompatibility systems, while others represent completely independent processes. Pollen-pistil reproductive barriers can act as soon as pollen is deposited on a stigma, where penetration of heterospecific pollen tubes is blocked by the stigma papillae. As pollen tubes extend, the female transmitting tissue can selectively limit growth by producing cell wall-modifying enzymes and cytotoxins that interact with the growing pollen tube. At ovules, differential pollen tube attraction and inhibition of sperm cell release can act as barriers to heterospecific pollen tubes.

Enabling the Application of Large Footprint Open‐Bottom Permeameters Through New Shape Factors

AbstractOpen‐bottom permeameters (OBP) are commonly used for estimating the hydraulic properties of sediment beds under water bodies and fluxes across the sediment‐water interface. The specific shape factor for an OBP flow situation is necessary for data interpretation. It is determined by the ratio of the permeameter tube radius to the tube's penetration depth within the sediment. Previous permeameter designs favored small radii which allowed for deeper penetration and stability of the tube. The shape factor is provided here for large footprint OBPs that allows for shallow penetration and greater radius. Through a numerical and analytical modeling study of the OBP hydrodynamics, previous knowledge on the OBP shape factor is generalized to the entire range of radius‐to‐penetration ratios that might be practically applied. The results reveal the advantages of the large footprint design, including better sensitivity due to the ability to impose larger discharge over a shorter test duration.

Solidago canadensis, a Flower or Weed?

Figure 1. Flowers or weeds? S. canadensis is one of the worst exotic weeds in China (A). Yet, farmers grow it as a cut flower (B). Because diploid cytotypes grow faster and flower earlier than other polyploids, all cultivated S. canadensis cytotypes for cut flowers are diploids. We found that diploids are sterile under hot-summer temperature south of 24°C-isotherm range and cultivation of S. canadensis will not lead to naturalization. However, the risk for naturalization increases in regions north of 20ºC-isotherm range. Photo credit: Sheng Qiang. Figure 2. Population of Solidago canadensis in native and introduced range in North America and China, respectively. S. canadensis plants are significantly taller and denser in the introduced range in China (Jiangsu) (A) than in their native range of North America (Pennsylvania, USA) (B). S. canadensis plants often outcompete native plants in invasive habitats and essentially become monoculture-type communities. Photo credit: Sheng Qiang. Figure 3. Dramatic difference in height of Solidago canadensis between native and invasive populations. S. canadensis plants can often be over 4m in height in China (Nanjing) (A), but are usually below 2m in North America (Ottawa, Canada) (B). Photo credit: Sheng Qiang. Figure 4. Field sampling site diagram. At each site, sampling plots were separated by at least 10 m to minimize the probability of sampling multiple ramets from individual genets. Photo credit: Guoqi Chen. Figure 5. Pollen germination and pollen tube growth on a stigma. (A) Pollen germination; (B) pollen tube growth; (C) penetration of pollen tubes into an ovary. Photo credit: Beibei Yao. These photographs illustrate the article “Solidago canadensis, a flower or weed?” by Sheng Qiang, Jiliang Cheng, Guoqi Chen, Beibei Yao published in Ecological Monographs. https://doi.org/10.1002/ecm.1445.

Interaction between Metarhizium anisopliae and Its Host, the Subterranean Termite Coptotermes curvignathus during the Infection Process.

Simple SummaryThe use of Metarhizium anisopliae as a biological control of insect pests has been experimented in the laboratory as well as in field trials. This includes against the termite Coptotermes curvignathus, however the results have varying degrees of success. One reason could be due to the lack of detailed knowledge on the molecular pathogenesis of M. anisopliae. In the current study, the conidial suspension of M. anisopliae isolate PR1 was first inoculated on the C. curvignathus, after which the pathogenesis was examined using two different approaches: electron microscopy and protein expression. At the initiation stage, the progression observed and documented including adhesion, germination, and penetration of the fungus on the cuticle within 24 h after inoculation. Later, this was followed by colonization and spreading of the fungus at the cellular level. Proteomics of C. curvignathus witnessed the expression of proteins related to cell regulation and defense, while in M. anisopliae, protein related to transport and fungal virulence were expressed throughout the infection. These findings offer relevant knowledge for use in the development of M. anisopliae as a prospective biological control agent for termites in the future.Metarhizium anisopliae (Metchnikoff) Sorokin, a pathogenic fungus to insects, infects the subterranean termite, Coptotermes curvignathus Holmgren, a devastating pest of plantation trees in the tropics. Electron microscopy and proteomics were used to investigate the infection and developmental process of M. anisopliae in C. curvignathus. Fungal infection was initiated by germ tube penetration through the host’s cuticle as observed at 6 h post-inoculation (PI), after which it elongated into the host’s integumental tissue. The colonization process continued as seen from dissemination of blastospores in the hemocoel at 96 h PI. At this time point, the emergent mycelia had mummified the host and forty-eight hours later, new conidia were dispersed on the termites’ body surface. Meanwhile, hyphal bodies were observed in abundance in the intercellular space in the host’s body. The proteomes of the pathogen and host were isolated separately using inoculated termite samples withdrawn at each PI-time point and analyzed in two-dimensional electrophoresis (2-DE) gels. Proteins expressed in termites showed evidence of being related to cell regulation and the immune response, while those expressed in M. anisopliae, to transportation and fungal virulence. This study provides new information on the interaction between termites and its entomopathogen, with potential utilization for developing future biopesticide to control the termite population.

Membrane receptor-mediated mechano-transduction maintains cell integrity during pollen tube growth within the pistil.

Invasive or penetrative growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading or penetrating cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here, we show that Arabidopsis pollen tubes sense and/or respond to mechanical changes via a cell-surface receptor kinase Buddha's Paper Seal 1 (BUPS1) while emerging from compressing female tissues. BUPS1-defective pollen tubes fail to maintain cell integrity after emergence from these tissues. The mechano-transduction function of BUPS1 is established by using a microfluidic channel device mimicking the mechanical features of the invivo growth path. BUPS1-based mechano-transduction activates Rho-like GTPase from Plant 1 (ROP1) GTPase to promote exocytosis that facilitates secretion of BUPS1's ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-based mechano-transduction system for cells to cope with the physical challenges during invasive or penetrative growth.

A Membrane Receptor-Mediated Mechanosensing System Maintains Cell Integrity During Invasive Growth

Invasive growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here we show that Arabidopsis pollen tubes sense mechanical changes via a cell-surface receptor kinase CUPID1 while penetrating compressing female tissues. Pollen tubes carrying cupid1 mutations fail to maintain cell integrity and burst after exiting these tissues. The mechanosensing function of CUPID1 is established in a microfluidic channel device that mimics the mechanical features of the in vivo growth path. CUPID1-based mechanosensing activates ROP1 GTPase signaling to promote exocytosis that facilitates the secretion of CUPID1's ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-mediated mechanosensing system for cells to cope with the physical challenges during invasive growth.

Pollen tube growth and fruit set in quince (Cydonia oblonga Mill.)

Aim of study: To determine the self-compatibility level of eight quince cultivars.Area of study: The region of Belgrade (Central Serbia).Material and methods: Pollen tube growth in vivo and fruit set in two pollination variants (self- and open-pollination) were studied in eight quince cultivars. The quantitative parameters of pollen tube growth (average number of pollen tubes in the upper and middle third of the style, base of the style and in the ovary; the dynamics of pollen tube growth through these parts of the pistil) was determined using the fluorescence microscopy.Main results: The parameters of pollen tube growth and fruit set were primarily dependent on the genotype and variants of pollination. All studied parameters were significantly higher in the open-pollination variant compared with the self-pollination in all cultivars. In the self-pollination variant, ʻLeskovackaʼ and ʻVranjskaʼ had the highest number of pollen tubes that penetrated the ovary (2.10 and 0.54 in average, respectively), as well as the largest percentage of pistils with the penetration of pollen tubes in the nucellus of ovules six days after pollination (40.09% and 14.74%). Also, they had the highest percentage of initial fruit set (17.01% and 28.52%) and final fruit set (9.32% and 9.86%). Based on this, ʻLeskovackaʼ and ʻVranjskaʼ can be classified as self-compatible cultivars, while the others are self-incompatible.Research highlights: The majority of quince cultivars were self-incompatible. When establishing new orchards with these cultivars, care should be taken about the choice of pollenisers in order to achieve high yields.

Staphylococcusaureus protein A as a means of assessing sperm penetrability in cervical mucus in vitro.

Objective The effectiveness of Staphylococcus protein A (SPA) in improving the penetration ability of sperm and reducing antisperm antibody (ASA) titers in immunologically infertile males was evaluated.Methods Seminal fluid samples were obtained from 15 infertile men, and ASA titers were assessed with the latex agglutination test. Identification of immunoglobulin (Ig) classes and characterization of the antigens involved in the immune response were performed using indirect immunofluorescence. Local ASAs typically present as a mixture of IgG and IgA classes. The capillary tube penetration method was used to assess the capability of spermatozoa to penetrate the cervical mucus (CM).Results ASAs associated with the neck region of sperm showed a significantly lower migration distance in the CM of infertile females than ASAs associated with the head or tail segments. ASA-positive seminal fluid exhibited significant increases in the mean migration distance (2.6 ± 1.4 cm vs. 1.54 ± 1.1 cm, respectively; p< 0.001) and sperm concentration (174 ± 121.0 × 10³/mL vs. 101 ± 93.7 × 10³/mL, respectively; p= 0.033) after treatment with SPA compared to pre-treated samples. A significant reduction (p< 0.01) in the recorded ASA titer was detected.Conclusion These results indicate that SPA can be used as a sorting regimen for insemination programs. However, further studies are warranted to assess its influence on pregnancy rate.