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The fertility of birds depends on their ability to store enough viable sperm for an extended period of time in the sperm storage tubules (SST). The exact mechanism by which spermatozoa enter, reside in, and leave the SST remains controversial. The sperm of sharkasi hens showed a high tendency to agglutination, forming mobile filamentous bundles containing many cells. Due to the difficulty of observing the motility and behavior of spermatozoa in an opaque fallopian tube, we used a microfluidic device with a microchannel cross-section similar to that of spermatozoa to study spermatozoa agglutination and motility. This study discusses how sperm bundles form, how they move, and their possible role in extending sperm residency in the SST. We investigated sperm velocity and rheological behavior when fluid flow was generated within a microfluidic channel by hydrostatic pressure (flow rate = 33 µm/s). The spermatozoa tend to swim against the current (positive rheology) and the velocity of the spermatozoon bundle is significantly reduced compared to single spermatozoa. Sperm bundles have been observed to move in a spiral and increase in length and thickness as more single sperm are recruited. Sperm bundles were observed approaching and adhering to the sidewalls of the microfluidic channels to avoid being swept with fluid flow velocity > 33 µm/s. Sperm bundles were observed approaching and adhering to the sidewalls of the microfluidic channels to avoid being swept with fluid flow velocity > 33 µm/s. Было замечено, что пучки сперматозоидов приближаются и прилипают к боковым стенкам микрофлюидных каналов, чтобы избежать сметания со скоростью потока жидкости> 33 мкм / с. Sperm bundles have been observed to approach and adhere to the side walls of the microfluidic channels to avoid being swept away at fluid flow rates >33 µm/s.观察到精子束接近并粘附在微流体通道的侧壁上，以避免被流体流速> 33 µm/s 扫过。 33 µm/s 扫过。 Было замечено, что пучки сперматозоидов приближаются и прилипают к боковым стенкам микрожидкостного канала, чтобы избежать сметания потоком жидкости со скоростью > 33 мкм/с. Sperm bundles have been observed to approach and adhere to the side walls of the microfluidic channel to avoid being swept away by fluid flow at >33 µm/s. Scanning and transmission electron microscopy revealed that the sperm bundles were supported by abundant dense material. The data obtained demonstrate the unique mobility of Sharkazi chicken spermatozoa, as well as the ability of spermatozoa to agglutinate and form mobile bundles, which contributes to a better understanding of the long-term storage of spermatozoa in SMT.
To achieve fertilization in humans and most animals, sperm and eggs must arrive at the site of fertilization at the right time. Therefore, mating must occur before or at the time of ovulation. On the other hand, some mammals, such as dogs, as well as non-mammalian species, such as insects, fish, reptiles, and birds, store sperm in their reproductive organs for an extended period of time until their eggs are ready for fertilization (asynchronous fertilization 1 ). Birds are able to maintain the viability of spermatozoa capable of fertilizing eggs for 2-10 weeks2.
This is a unique feature that distinguishes birds from other animals, as it provides a high probability of fertilization after a single insemination for several weeks without simultaneous mating and ovulation. The main sperm storage organ, called the sperm storage tubule (SST), is located in the internal mucosal folds at the uterovaginal junction. To date, the mechanisms by which sperm enter, reside, and exit the sperm bank are not fully understood. Based on previous studies, many hypotheses have been put forward, but none of them has been confirmed.
Forman4 hypothesized that spermatozoa maintain their residence in the SST cavity through continuous oscillatory movement against the direction of fluid flow through protein channels located on SST epithelial cells (rheology). ATP is depleted due to the constant flagellar activity needed to keep the sperm in the SST lumen and motility eventually declines until the sperm are carried out of the sperm bank by fluid flow and begin a new journey down the ascending fallopian tube to fertilize the sperm. Egg (Forman4). This model of sperm storage is supported by the detection by immunocytochemistry of aquaporins 2, 3 and 9 present in SST epithelial cells. To date, studies on chicken semen rheology and its role in SST storage, vaginal sperm selection, and sperm competition are lacking. In chickens, sperm enters the vagina after natural mating, but more than 80% of the spermatozoa are ejected from the vagina shortly after mating. This suggests that the vagina is the primary site for sperm selection in birds. In addition, it has been reported that less than 1% of spermatozoa fertilized in the vagina end up in SSTs2. In artificial insemination of chicks in the vagina, the number of spermatozoa reaching SST tends to increase 24 hours after insemination. So far, the mechanism of sperm selection during this process is unclear, and sperm motility may play an important role in SST sperm uptake. Due to the thick and opaque walls of the fallopian tubes, it is difficult to directly monitor sperm motility in the fallopian tubes of birds. Therefore, we lack basic knowledge of how spermatozoa transition to SST after fertilization.
Rheology has recently been recognized as an important factor controlling sperm transport in the mammalian genitalia. Based on the ability of motile spermatozoa to migrate countercurrently, Zaferani et al8 used a corra microfluidic system to passively isolate motile spermatozoa from penned semen samples. This type of semen sorting is essential for medical infertility treatment and clinical research, and is preferred over traditional methods that are time and labor intensive and can compromise sperm morphology and structural integrity. However, to date, no studies have been conducted on the effect of secretions from the genital organs of chickens on sperm motility.
Regardless of the mechanism that maintains sperm stored in the SST, many investigators have observed that resident spermatozoa agglutinate head-to-head in the SST of chickens 9, 10, quails 2, and turkeys 11 to form agglutinated sperm bundles. The authors suggest that there is a link between this agglutination and long-term storage of spermatozoa in the SST.
Tingari and Lake12 reported a strong association between spermatozoa in the sperm-receiving gland of the chicken and questioned whether avian spermatozoa agglutinate in the same way as mammalian spermatozoa. They believe that the deep connections between sperm in the vas deferens may be due to the stress caused by the presence of a large number of sperm in a small space.
When evaluating the behavior of spermatozoa on fresh hanging glass slides, transient signs of agglutination can be seen, especially at the edges of the semen droplets. However, agglutination was often disturbed by the rotational action associated with continuous movement, which explains the transient nature of this phenomenon. The researchers also noticed that when the diluent was added to the semen, elongated “thread-like” cell aggregates appeared.
Early attempts to mimic a spermatozoon were made by removing a thin wire from a hanging drop, which resulted in an elongated sperm-like vesicle protruding from the drop of semen. The spermatozoa immediately lined up in a parallel fashion within the vesicle, but the entire unit quickly disappeared due to the 3D limitation. Therefore, to study spermatozoa agglutination, it is necessary to observe the motility and behavior of spermatozoa directly in isolated sperm storage tubules, which is difficult to achieve. Therefore, it is necessary to develop an instrument that mimics spermatozoa to support studies of sperm motility and agglutination behavior. Brillard et al13 reported that the average length of sperm storage tubules in adult chicks is 400–600 µm, but some SSTs can be as long as 2000 µm. Mero and Ogasawara14 divided the seminiferous glands into enlarged and non-enlarged sperm storage tubules, both of which were the same in length (~500 µm) and neck width (~38 µm), but the mean lumen diameter of the tubules was 56.6 and 56.6 µm. . , respectively 11.2 μm, respectively. In the current study, we used a microfluidic device with a channel size of 200 µm × 20 µm (W × H), whose cross section is somewhat close to that of the amplified SST. In addition, we examined sperm motility and agglutination behavior in flowing fluid, which is consistent with Foreman’s hypothesis that fluid produced by SST epithelial cells keeps sperm in the lumen in a countercurrent (rheological) direction.
The aim of this study was to overcome the problems of observing spermatozoa motility in the fallopian tube and to avoid the difficulties of studying the rheology and behavior of spermatozoa in a dynamic environment. A microfluidic device was used that creates hydrostatic pressure to simulate sperm motility in the genitals of a chicken.
When a drop of a diluted sperm sample (1:40) was loaded into the microchannel device, two types of sperm motility could be identified (isolated sperm and bound sperm). In addition, spermatozoa tended to swim against the current (positive rheology; video 1, 2). Although sperm bundles had lower velocity than that of lonesome sperm (p < 0.001), they increased the percentage of sperm displaying positive rheotaxis (p < 0.001; Table 2). Although sperm bundles had lower velocity than that of lonesome sperm (p < 0.001), they increased the percentage of sperm displaying positive rheotaxis (p < 0.001; Table 2). Хотя пучки сперматозоидов имели более низкую скорость, чем у одиночных сперматозоидов (p < 0,001), они увеличивали процент сперматозоидов, демонстрирующих положительный реотаксис (p < 0,001; таблица 2). Although the spermatozoa bundles had a lower velocity than that of single spermatozoa (p < 0.001), they increased the percentage of spermatozoa showing positive rheotaxis (p < 0.001; Table 2).尽管精子束的速度低于孤独精子的速度（p < 0.001），但它们增加了显示阳性流变性的精子百分比（p < 0.001；表2）。尽管 精子束 的 速度 低于 孤独 的 速度 （p <0.001） ， 但 增加 了 显示 阳性 流变性 精子 百分比 （p <0.001 ； 2。。。。。。）））））） Хотя скорость пучков сперматозоидов была ниже, чем у одиночных сперматозоидов (p < 0,001), они увеличивали процент сперматозоидов с положительной реологией (p < 0,001; таблица 2). Although the speed of sperm bundles was lower than that of single spermatozoa (p < 0.001), they increased the percentage of spermatozoa with positive rheology (p < 0.001; Table 2). Positive rheology for single spermatozoa and tufts is estimated at approximately 53% and 85%, respectively.
It has been observed that the spermatozoa of sharkasi chickens immediately after ejaculation form linear bundles, consisting of dozens of individuals. These tufts increase in length and thickness over time and may remain in vitro for several hours before dissipating (video 3). These filamentous bundles are shaped like echidna spermatozoa that form at the end of the epididymis. Sharkashi hen semen has been found to have a high tendency to agglutinate and form a reticulate bundle in less than one minute after collection. These beams are dynamic and able to stick to any nearby walls or static objects. Although sperm bundles reduce the speed of sperm cells, it is clear that macroscopically they increase their linearity. The length of the bundles varies depending on the number of sperm collected in bundles. Two parts of the bundle were isolated: the initial part, including the free head of the agglutinated sperm, and the terminal part, including the tail and the entire distal end of the sperm. Using a high-speed camera (950 fps), free heads of agglutinated spermatozoa were observed in the initial part of the bundle, responsible for the movement of the bundle due to their oscillatory motion, dragging the remaining ones into the bundle with a helical motion (Video 4). However, in long tufts, it has been observed that some free sperm heads adhered to the body and the terminal portion of the tuft act as vanes to help propel the tuft.
While in a slow flow of fluid, the sperm bundles move parallel to each other, however, they begin to overlap and stick to everything that is still, so as not to be washed away by the current flow as the flow speed increases. The bundles form when a handful of sperm cells approach each other, they begin to move in synchrony and wrap around each other, and then stick to a sticky substance. Figures 1 and 2 show how the sperm approach each other, forming a junction as the tails wrap around each other.
The researchers applied hydrostatic pressure to create fluid flow in a microchannel to study sperm rheology. A microchannel with a size of 200 µm × 20 µm (W × H) and a length of 3.6 µm was used. Use microchannels between containers with syringes fitted at the ends. Food coloring was used to make the channels more visible.
Tie interconnect cables and accessories to the wall. The video was taken with a phase contrast microscope. With each image, phase contrast microscopy and mapping images are presented. (A) The connection between two streams resists the flow due to helical motion (red arrow). (B) The connection between the tube bundle and the channel wall (red arrows), at the same time they are connected to two other bundles (yellow arrows). (C) Sperm bundles in the microfluidic channel begin to connect with each other (red arrows), forming a mesh of sperm bundles. (D) Formation of a network of sperm bundles.
When a drop of diluted sperm was loaded into the microfluidic device and a flow was created, the sperm beam was observed to move against the direction of the flow. The bundles fit snugly against the walls of the microchannels, and the free heads in the initial part of the bundles fit snugly against them (video 5). They also stick to any stationary particles in their path, such as debris, to resist being swept away by the current. Over time, these tufts become long filaments trapping other single spermatozoa and shorter tufts (Video 6). As the flow begins to slow down, long lines of sperm begin to form a network of sperm lines (Video 7; Figure 2).
At high flow velocity (V > 33 µm/s), the spiral movements of threads are increased as an attempt to catch many individual sperm forming bundles better resist the drifting force of the flow. At high flow velocity (V > 33 µm/s), the spiral movements of threads are increased as an attempt to catch many individual sperm forming bundles better resist the drifting force of the flow. При высокой скорости потока (V > 33 мкм/с) спиралевидные движения нитей усиливаются, поскольку они пытаются поймать множество отдельных сперматозоидов, образующих пучки, которые лучше противостоят дрейфующей силе потока. At high flow rates (V > 33 µm/s), the helical movements of the strands increase as they try to catch many individual spermatozoa forming bundles that are better able to resist the drifting force of the flow.在高流速(V > 33 µm/s) 时，螺纹的螺旋运动增加，以试图捕捉许多形成束的单个精子，从而更好地抵抗流动的漂移力。在 高 流速 (v> 33 µm/s) 时 ， 的 螺旋 运动 增加 ， 以 试图 许多 形成 束 单 个 精子 ， 从而 更 地 抵抗 的 漂移力。。。。。。。。。。 При высоких скоростях потока (V > 33 мкм/с) спиральное движение нитей увеличивается в попытке захватить множество отдельных сперматозоидов, образующих пучки, чтобы лучше сопротивляться силам дрейфа потока. At high flow rates (V > 33 µm/s), the helical movement of the filaments increases in an attempt to capture many individual spermatozoa forming bundles to better resist the drift forces of the flow. They also tried to attach microchannels to the sidewalls.
Sperm bundles were identified as clusters of sperm heads and curling tails using light microscopy (LM). Sperm bundles with various aggregates have also been identified as twisted heads and flagellar aggregates, multiple fused sperm tails, sperm heads attached to a tail, and sperm heads with bent nuclei as multiple fused nuclei. transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that the sperm bundles were sheathed aggregates of sperm heads and the sperm aggregates showed an attached network of wrapped tails.
The morphology and ultrastructure of spermatozoa, the formation of spermatozoa bundles were studied using light microscopy (half section), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), sperm smears were stained with acridine orange and examined using epifluorescence microscopy.
Sperm smear staining with acridine orange (Fig. 3B) showed that the sperm heads were stuck together and covered with secretory material, which led to the formation of large tufts (Fig. 3D). The sperm bundles consisted of sperm aggregates with a network of attached tails (Fig. 4A-C). Sperm bundles are composed of the tails of many spermatozoa stuck together (Fig. 4D). Secrets (Fig. 4E,F) covered the heads of spermatozoa bundles.
Formation of the spermatozoa bundle Using phase contrast microscopy and sperm smears stained with acridine orange, showed that the heads of spermatozoa stick together. (A) Early sperm tuft formation begins with a sperm (white circle) and three sperm (yellow circle), with the spiral starting at the tail and ending at the head. (B) Photomicrograph of a sperm smear stained with acridine orange showing adherent sperm heads (arrows). The discharge covers the head(s). Magnification × 1000. (C) Development of a large beam transported by flow in a microfluidic channel (using a high speed camera at 950 fps). (D) Micrograph of a sperm smear stained with acridine orange showing large tufts (arrows). Magnification: ×200.
Scanning electron micrograph of a sperm beam and a sperm smear stained with acridine orange. (A, B, D, E) are digital color scanning electron micrographs of spermatozoa, and C and F are micrographs of acridine orange stained sperm smears showing attachment of multiple spermatozoa wrapping the caudal web. (AC) Sperm aggregates are shown as a network of attached tails (arrows). (D) Adhesion of several spermatozoa (with adhesive substance, pink outline, arrow) wrapping around the tail. (E and F) Sperm head aggregates (pointers) covered with adhesive material (pointers). The spermatozoa formed bundles with several vortex-like structures (F). (C) ×400 and (F) ×200 magnifications.
Using transmission electron microscopy, we found that sperm bundles had attached tails (Fig. 6A, C), heads attached to tails (Fig. 6B), or heads attached to tails (Fig. 6D). The heads of the spermatozoa in the bundle are curved, presenting in section two nuclear regions (Fig. 6D). In the incision bundle, the spermatozoa had a twisted head with two nuclear regions and multiple flagellar regions (Fig. 5A).
Digital color electron micrograph showing the connecting tails in the sperm bundle and the agglutinating material connecting the sperm heads. (A) Attached tail of a large number of spermatozoa. Notice how the tail looks in both portrait (arrow) and landscape (arrow) projections. (B) The head (arrow) of the sperm is connected to the tail (arrow). (C) Several sperm tails (arrows) are attached. (D) Agglutination material (AS, blue) connects four sperm heads (purple).
Scanning electron microscopy was used to detect sperm heads in sperm bundles covered with secretions or membranes (Figure 6B), indicating that the sperm bundles were anchored by extracellular material. The agglutinated material was concentrated in the sperm head (jellyfish head-like assembly; Fig. 5B) and expanded distally, giving a brilliant yellow appearance under fluorescence microscopy when stained with acridine orange (Fig. 6C). This substance is clearly visible under a scanning microscope and is considered a binder. Semi-thin sections (Fig. 5C) and sperm smears stained with acridine orange showed sperm bundles containing densely packed heads and curled tails (Fig. 5D).
Various photomicrographs showing aggregation of sperm heads and folded tails using various methods. (A) Cross-sectional digital color transmission electron micrograph of a sperm bundle showing a coiled sperm head with a two-part nucleus (blue) and several flagellar parts (green). (B) Digital color scanning electron micrograph showing a cluster of jellyfish-like sperm heads (arrows) that appear to be covered. (C) Semi-thin section showing aggregated sperm heads (arrows) and curled tails (arrows). (D) Micrograph of a sperm smear stained with acridine orange showing aggregates of sperm heads (arrows) and curled adherent tails (arrows). Note that a sticky substance (S) covers the head of the spermatozoon. (D) × 1000 magnification.
Using transmission electron microscopy (Fig. 7A), it was also noted that the sperm heads were twisted and the nuclei had a spiral shape, as confirmed by sperm smears stained with acridine orange and examined using fluorescence microscopy (Fig. 7B).
(A) Digital color transmission electron micrograph and (B) Acridine orange stained sperm smear showing coiled heads and attachment of sperm heads and tails (arrows). (B) × 1000 magnification.
An interesting finding is that Sharkazi’s sperm aggregates to form mobile filamentous bundles. The properties of these bundles allow us to understand their possible role in the absorption and storage of spermatozoa in the SST.
After mating, the sperm enter the vagina and undergo an intense selection process, resulting in only a limited number of sperm entering the SST15,16. To date, the mechanisms by which sperm enter and exit the SST are unclear. In poultry, spermatozoa are stored in the SST for an extended period of 2 to 10 weeks, depending on the species6. Controversy remains about the condition of the semen during storage in the SST. Are they in motion or at rest? In other words, how do sperm cells maintain their position in the SST for so long?
Forman4 suggested that SST residence and ejection could be explained in terms of sperm motility. The authors hypothesize that sperm maintain their position by swimming against the fluid flow created by the SST epithelium and that sperm are ejected from the SST when their velocity falls below the point at which they begin to move backward due to lack of energy. Zaniboni5 confirmed the presence of aquaporins 2, 3, and 9 in the apical portion of SST epithelial cells, which may indirectly support Foreman’s sperm storage model. In the current study, we found that almost half of Sharkashi’s spermatozoa show positive rheology in the flowing fluid, and that agglutinated sperm bundles increase the number of spermatozoa showing positive rheology, although agglutination slows them down. How sperm cells travel up the bird’s fallopian tube to the site of fertilization is not fully understood. In mammals, the follicular fluid chemoattracts spermatozoa. However, chemoattractants are believed to direct spermatozoa to approach long distances7. Therefore, other mechanisms are responsible for sperm transport. The ability of sperm to orientate and flow against the fallopian tube fluid released after mating has been reported to be a major factor in targeting sperm in mice. Parker 17 suggested that spermatozoa cross the oviducts by swimming against the ciliary current in birds and reptiles. Although it has not been experimentally demonstrated in birds, Adolphi18 was the first to find that avian sperm gives positive results when a thin layer of liquid between a coverslip and slide is created with a strip of filter paper. Rheology. Hino and Yanagimachi [19] placed a mouse ovary-tubal-uterine complex in a perfusion ring and injected 1 µl of ink into the isthmus to visualize fluid flow in the fallopian tubes. They noticed a very active movement of contraction and relaxation in the fallopian tube, in which all the ink balls were steadily moving towards the ampulla of the fallopian tube. The authors emphasize the importance of tubal fluid flow from the lower to the upper fallopian tubes for sperm uplift and fertilization. Brillard20 reported that in chickens and turkeys, spermatozoa migrate by active movement from the vaginal entrance, where they are stored, to the utero-vaginal junction, where they are stored. However, this movement is not required between the uterovaginal junction and infundibulum because the spermatozoa are transported by passive displacement. Knowing these previous recommendations and the results obtained in the current study, it can be assumed that the ability of spermatozoa to move upstream (rheology) is one of the properties on which the selection process is based. This determines the passage of spermatozoa through the vagina and their entry into the CCT for storage. As Forman4 suggested, this may also facilitate the process of sperm entering the SST and its habitat for a period of time and then exiting when their speed starts to slow down.
On the other hand, Matsuzaki and Sasanami 21 suggested that avian spermatozoa undergo motility changes from dormancy to motility in the male and female reproductive tracts. Inhibition of resident sperm motility in the SST has been proposed to explain the long storage time of sperm and then rejuvenation after leaving the SST. Under hypoxic conditions, Matsuzaki et al. 1 reported high production and release of lactate in the SST, which may lead to inhibition of resident sperm motility. In this case, the importance of sperm rheology is reflected in the selection and absorption of spermatozoa, and not in their storage.
The sperm agglutination pattern is considered a plausible explanation for the long storage period of sperm in the SST, as this is a common pattern of sperm retention in poultry2,22,23. Bakst et al. 2 observed that most spermatozoa adhered to each other, forming fascicular aggregates, and single spermatozoa were rarely found in quail CCM. On the other hand, Wen et al. 24 observed more scattered spermatozoa and fewer spermatozoa tufts in the SST lumen in chickens. Based on these observations, it can be assumed that the propensity for sperm agglutination differs between birds and between spermatozoa in the same ejaculate. In addition, Van Krey et al. 9 suggested that random dissociation of agglutinated spermatozoa is responsible for the gradual penetration of spermatozoa into the lumen of the fallopian tube. According to this hypothesis, spermatozoa with lower agglutination capacity should be expelled from the SST first. In this context, the ability of spermatozoa to agglutinate may be a factor influencing the outcome of sperm competition in dirty birds. In addition, the longer the agglutinated sperm dissociates, the longer fertility is maintained.
Although spermatozoa aggregation and aggregation into bundles have been observed in several studies2,22,24, they have not been described in detail due to the complexity of their kinematic observation within the SST. Several attempts have been made to study sperm agglutination in vitro. Extensive but transient aggregation was observed when the thin wire was removed from the dangling seed drop. This leads to the fact that an elongated bubble protrudes from the drop, imitating the seminal gland. Due to 3D limitations and short drip drying times, the entire block quickly fell into disrepair9. In the current study, using Sharkashi chickens and microfluidic chips, we were able to describe how these tufts form and how they move. Sperm bundles formed immediately after semen collection and were found to move in a spiral, showing positive rheology when present in the flow. Furthermore, when macroscopically viewed, sperm bundles have been observed to increase the linearity of motility compared to isolated spermatozoa. This suggests that sperm agglutination may occur prior to SST penetration and that sperm production is not limited to a small area due to stress as previously suggested (Tingari and Lake12). During tuft formation, the spermatozoa swim in synchrony until they form a junction, then their tails wrap around each other and the head of the spermatozoon remains free, but the tail and distal part of the spermatozoon stick together with a sticky substance. Therefore, the free head of the ligament is responsible for the movement, dragging the rest of the ligament. Scanning electron microscopy of the sperm bundles showed attached sperm heads covered with a lot of sticky material, suggesting that the sperm heads were attached in resting bundles, which may have occurred after reaching the storage site (SST).
When a sperm smear is stained with acridine orange, extracellular adhesive material around the sperm cells can be seen under a fluorescent microscope. This substance allows sperm bundles to adhere to and cling to any surrounding surfaces or particles so that they do not drift with the surrounding flow. Thus, our observations show the role of spermatozoa adhesion in the form of mobile bundles. Their ability to swim against the current and stick to nearby surfaces allows sperm to stay longer in the SST.
Rothschild25 used a hemocytometry camera to study the floating distribution of bovine semen in a drop of suspension, taking photomicrographs through a camera with both vertical and horizontal optical axis of the microscope. The results showed that the spermatozoa were attracted to the surface of the chamber. The authors suggest that there may be hydrodynamic interactions between the sperm and the surface. Taking this into account, along with the ability of Sharkashi chick semen to form sticky tufts, it may increase the likelihood that semen will adhere to the SST wall and be stored for long periods of time.
Bccetti and Afzeliu26 reported that the sperm glycocalyx is required for gamete recognition and agglutination. Forman10 observed that hydrolysis of α-glycosidic bonds in glycoprotein-glycolipid coatings by treating avian semen with neuraminidase resulted in reduced fertility without affecting sperm motility. The authors suggest that the effect of neuraminidase on the glycocalyx impairs sperm sequestration at the utero-vaginal junction, thereby reducing fertility. Their observations cannot ignore the possibility that neuraminidase treatment may reduce sperm and oocyte recognition. Forman and Engel10 found that fertility was reduced when hens were inseminated intravaginally with semen treated with neuraminidase. However, IVF with neuraminidase treated sperm did not affect fertility compared to control chickens. The authors concluded that changes in the glycoprotein-glycolipid coating around the sperm membrane reduced the ability of sperm to fertilize by impairing sequestration of sperm at the utero-vaginal junction, which in turn increased sperm loss due to the speed of the utero-vaginal junction, but not affects sperm and egg recognition.
In turkeys Bakst and Bauchan 11 found small vesicles and membrane fragments in the lumen of the SST and observed that some of these granules had fused with the sperm membrane. The authors suggest that these relationships may contribute to the long-term storage of spermatozoa in SST. However, the researchers did not specify the source of these particles, whether they are secreted by CCT epithelial cells, produced and secreted by the male reproductive system, or produced by the sperm itself. Also, these particles are responsible for agglutination. Grützner et al27 reported that epididymal epithelial cells produce and secrete a specific protein that is required for the formation of single-pore seminal tracts. The authors also report that the dispersion of these bundles depends on the interaction of epididymal proteins. Nixon et al28 found that the adnexa secrete a protein, the acidic cysteine-rich osteonectin; SPARC is involved in the formation of sperm tufts in short-beaked echidnas and platypuses. The scattering of these beams is associated with the loss of this protein.
In the current study, ultrastructural analysis using electron microscopy showed that the spermatozoa adhered to a large amount of dense material. These substances are thought to be responsible for the agglutination that condenses between and around the adherent heads, but at lower concentrations in the tail region. We assume that this agglutinating substance is excreted from the male reproductive system (epididymis or vas deferens) along with semen, since we often observe semen separating from lymph and seminal plasma during ejaculation. It has been reported that as avian spermatozoa pass through the epididymis and vas deferens, they undergo maturation-related changes that support their ability to bind proteins and acquire plasma lemma-associated glycoproteins. The persistence of these proteins on resident sperm membranes in the SST suggests that these proteins may influence the acquisition of sperm membrane stability 30 and determine their fertility 31 . Ahammad et al32 reported that spermatozoa obtained from various parts of the male reproductive system (from the testes to the distal vas deferens) showed a progressive increase in viability under liquid storage conditions, regardless of storage temperature, and viability in chickens also increases in the fallopian tubes after artificial insemination.
Sharkashi chicken sperm tufts have different characteristics and functions than other species such as echidnas, platypuses, wood mice, deer rats, and guinea pigs. In sharkasi chickens, the formation of spermatozoa bundles reduced their swimming speed compared to single spermatozoa. However, these bundles increased the percentage of rheologically positive spermatozoa and increased the ability of spermatozoa to stabilize themselves in a dynamic environment. Thus, our results confirm the previous suggestion that sperm agglutination in SST is associated with long-term sperm storage. We also hypothesize that the propensity of sperm to form tufts may control the rate of sperm loss in SST, which may alter the outcome of sperm competition. According to this assumption, spermatozoa with low agglutination capacity release SST first, while spermatozoa with high agglutination capacity produce most of the offspring. The formation of single-pore sperm bundles is beneficial and affects the parent-child ratio, but uses a different mechanism. In echidnas and platypuses, the spermatozoa are arranged parallel to each other to increase the forward speed of the beam. Bundles of echidnas move about three times faster than single spermatozoa. It is believed that the formation of such sperm tufts in echidnas is an evolutionary adaptation to maintain dominance, since females are promiscuous and usually mate with several males. Therefore, spermatozoa from different ejaculates compete fiercely for the fertilization of the egg.
Agglutinated spermatozoa of sharkasi chickens are easy to visualize using phase contrast microscopy, which is considered advantageous because it allows easy study of the behavior of spermatozoa in vitro. The mechanism by which sperm tuft formation promotes reproduction in sharkasi chickens is also different from that seen in some placental mammals representing cooperative sperm behavior such as wood mice, where some spermatozoa reach the eggs, helping other related individuals reach and damage their eggs. to prove yourself. altruistic behaviour. Self-fertilization 34. Another example of cooperative behavior in spermatozoa was found in deer mice, where spermatozoa were able to identify and combine with the most genetically related spermatozoa and form cooperative groups to increase their speed compared to unrelated spermatozoa35.
The results obtained in this study do not contradict Foman’s theory of long-term storage of spermatozoa in SWS. The researchers report that sperm cells continue to move in the flow of epithelial cells lining the SST for an extended period of time, and after a certain period of time, the energy stores of the sperm cells are depleted, resulting in a decrease in speed, which allows the expulsion of small molecular weight substances. energy of spermatozoa with the flow of fluid from the lumen of the SST The cavity of the fallopian tube. In the current study, we observed that half of the single sperm showed the ability to swim against flowing fluids, and their adhesion in the bundle increased their ability to show positive rheology. Furthermore, our data are consistent with those of Matsuzaki et al. 1 who reported that increased lactate secretion in SST may inhibit resident sperm motility. However, our results describe the formation of sperm motile ligaments and their rheological behavior in the presence of a dynamic environment within a microchannel in an attempt to elucidate their behavior in SST. Future research may focus on determining the chemical composition and origin of the agglutinating agent, which will undoubtedly help researchers develop new ways to store liquid semen and increase the duration of fertility.
Fifteen 30-week-old bare-necked male sharkasi (homozygous dominant; Na Na) were selected as sperm donors in the study. The birds were reared at the Research Poultry Farm of the Faculty of Agriculture, Ashit University, Ashit Governorate, Egypt. Birds were housed in individual cages (30 x 40 x 40 cm), subjected to a light program (16 hours of light and 8 hours of darkness) and fed a diet containing 160 g of crude protein, 2800 kcal of metabolizable energy, 35 g of calcium each. 5 grams of available phosphorus per kilogram of diet.
According to data 36, ​​37, semen was collected from males by abdominal massage. A total of 45 semen samples were collected from 15 men over 3 days. Semen (n = 15/day) was immediately diluted 1:1 (v:v) with Belsville Poultry Semen Diluent, which contains potassium diphosphate (1.27 g), monosodium glutamate monohydrate (0.867 g), fructose (0.5 d) anhydrous sodium. acetate (0.43 g), tris(hydroxymethyl)aminomethane (0.195 g), potassium citrate monohydrate (0.064 g), potassium monophosphate (0.065 g), magnesium chloride (0.034 g) and H2O (100 ml), pH = 7, 5, osmolarity 333 mOsm/kg38. Diluted semen samples were first examined under a light microscope to ensure good semen quality (moisture) and then stored in a water bath at 37°C until use within half an hour after collection.
The kinematics and rheology of spermatozoa are described using a system of microfluidic devices. Semen samples were further diluted to 1:40 in Beltsville Avian Semen Diluent, loaded into a microfluidic device (see below), and kinetic parameters were determined using a Computerized Semen Analysis (CASA) system previously developed for microfluidics characterization. on the mobility of spermatozoa in liquid media (Department of Mechanical Engineering, Faculty of Engineering, Assiut University, Egypt). The plugin can be downloaded at: http://www.assiutmicrofluidics.com/research/casa39. Curve velocity (VCL, μm/s), linear velocity (VSL, μm/s) and average trajectory velocity (VAP, μm/s) were measured. Videos of spermatozoa were taken using an inverted Optika XDS-3 phase contrast microscope (with 40x objective) connected to a Tucson ISH1000 camera at 30 fps for 3 s. Use the CASA software to study at least three areas and 500 sperm trajectories per sample. The recorded video was processed using a homemade CASA. The definition of motility in the CASA plug-in is based on the swimming speed of the sperm compared to the flow rate, and does not include other parameters such as side-to-side movement, as this has been found to be more reliable in fluid flow. Rheological motion is described as the movement of sperm cells against the direction of fluid flow. Spermatozoa with rheological properties were divided by the number of motile spermatozoa; spermatozoa that were at rest and convectively moving spermatozoa were excluded from the count.
All chemicals used were obtained from Elgomhoria Pharmaceuticals (Cairo, Egypt) unless otherwise noted. The device was manufactured as described by El-sherry et al. 40 with some modifications. The materials used to fabricate the microchannels included glass plates (Howard Glass, Worcester, MA), SU-8-25 negative resist (MicroChem, Newton, CA), diacetone alcohol (Sigma Aldrich, Steinheim, Germany), and polyacetone. -184, Dow Corning, Midland, Michigan). Microchannels are fabricated using soft lithography. First, a clear protective face mask with the desired microchannel design was printed on a high resolution printer (Prismatic, Cairo, Egypt and Pacific Arts and Design, Markham, ON). The masters were made using glass plates as substrates. The plates were cleaned in acetone, isopropanol and deionized water and then coated with a 20 µm layer of SU8-25 by spin coating (3000 rpm, 1 min). The SU-8 layers were then gently dried (65°C, 2 min and 95°C, 10 min) and exposed to UV radiation for 50 s. Post-exposure bake at 65°C and 95°C for 1 min and 4 min to crosslink exposed SU-8 layers, followed by development in diacetone alcohol for 6.5 min. Hard bake the waffles (200°C for 15 min) to further solidify the SU-8 layer.
PDMS was prepared by mixing the monomer and hardener in a weight ratio of 10:1, then degassed in a vacuum desiccator and poured onto the SU-8 main frame. The PDMS was cured in an oven (120°C, 30 min), then the channels were cut out, separated from the master, and perforated to allow tubes to be attached at the inlet and outlet of the microchannel. Finally, PDMS microchannels were permanently attached to microscope slides using a portable corona processor (Electro-Technic Products, Chicago, IL) as described elsewhere. The microchannel used in this study measures 200 µm × 20 µm (W × H) and is 3.6 cm long.
The fluid flow induced by hydrostatic pressure inside the microchannel is achieved by maintaining the fluid level in the inlet reservoir above the height difference Δh39 in the outlet reservoir (Fig. 1).
where f is the coefficient of friction, defined as f = C/Re for laminar flow in a rectangular channel, where C is a constant depending on the aspect ratio of the channel, L is the length of the microchannel, Vav is the average velocity inside the microchannel, Dh is the hydraulic diameter of the channel, g – acceleration of gravity. Using this equation, the average channel velocity can be calculated using the following equation: