Morphological Characteristics of the Blood Cells and Spermatozoa in Chinese MonalsABSTRACT
The aim of this study is to investigate the morphological characteristics of blood cells and spermatozoa of Chinese monals (Lophophorus lhuysii) and to provide a basis for artificial insemination and physiological research. In this study, blood samples were collected by the wing vein puncture method, and semen was obtained by the massage technique. Blood cells were stained using Wright-Giemsa stain, while sperm were stained with Wright-Giemsa and modified Papanicolaou stain. The morphological and ultrastructural features of blood cells and sperm were observed using optical microscopy and scanning electron microscopy. The results indicate that mature red blood cells of the Chinese monal are oval with a smooth surface, measuring 12.43±0.89μm in major axis length. Immature red blood cells are larger and spherical, with a major axis length of 10.79±0.91μm. Sperm are elongated and rod-shaped, featuring a flagellum, with an average total length of 63.28±1.74μm, including a head length of 16.98±0.41μm and a tail length of 46.30±1.74μm. Scanning electron microscopy reveals a rough sperm surface with clear distinctions between the acrosome, head, and flagellum. Optical microscopy also shows a certain proportion of abnormal spermatozoa. This study elucidates the size and structural characteristics of blood cells and sperm in the Chinese Monal, providing a foundational basis for research in hematophysiology, fertilization, and population breeding, and offers technical support for the construction of genetic resource banks and the expansion of artificial populations.
Keywords:
Chinese monal; blood cells; sperm; morphological characteristics
INTRODUCTION
The Chinese Monal (Lophophorus lhuysii), a terrestrial bird endemic to China, belongs to the class Aves, order Galliformes, family Phasianidae, and genus Lophophorus. Chinese Monal is a first-class protected species, is classified as Vulnerable on the IUCN Red List, and is also one of the 36 precious and rare species targeted for conservation in the “14th Five-Year Plan for the Development of Forestry and Grassland Protection” (2021-2025) (CITES, 2023; BirdLife, 2024; IUCN, 2024). Chinese Monal is very infrequent, so it has significant conservation value.
More and more researchers have been conducting studies on the Chinese monal. Ecological niche modeling has been used to predict the potential suitable habitats for the Chinese Monal in the Qionglai Mountains, revealing that the habitat is highly fragmented and leading to the proposal of establishing corridors to facilitate species migration (Wang et al., 2017). Another study predicted the changes in the distribution range of the Chinese Monal under climate change conditions and suggested expanding existing protected areas and accelerating the research on artificial breeding (Xu et al., 2020). In addition, the intestinal microbiota and metabolites between wild and captive populations of the Chinese Monal were compared, revealing that the wild populations had higher alpha and beta diversity than the captive ones (Jiang et al., 2017). A study on the different seasons and ages of the intestinal microbiota found that the beta diversity had distinct seasonal variations, with the abundance of potential pathogens being higher in spring and summer (Huang et al., 2024). Some other researchers have conducted studies on the Chinese Monal from aspects such as mitochondrial genomes and microsatellites with the aim of increasing research materials on the Chinese Monal and providing a basis for its conservation (Ma et al., 2010; Xu et al., 2023). However, there have been no reports on the morphological structure of blood cells and sperm. Hemocytological observations can provide valuable information for disease diagnosis by examining the shape of blood cells supporting the determination of blood abnormalities (Luisa et al., 2022). Studies have shown that, when infected with Pseudomonas putida or Saprolegnia parasitica, red blood cells exhibit deformed nuclei, as well as acanthocytes, erythrocytes with vacuolated cytoplasm, and microcytes, all of which significantly increase, while the red blood cell count significantly decreases (Zaki et al., 2010; Fouad et al., 2025). Disease is one of the main factors limiting the population expansion of the Chinese Monal, and studying blood cell morphology is crucial for disease diagnosis. Sperm morphology research is a crucial aspect in assessing semen quality, revealing the mechanisms of spermatogenesis, and conducting fertilization biology studies (Oehninger S., 2020; Huang et al., 2024). The morphology of sperm directly affects their ability to penetrate the egg and the conception rate of the female, which is the best predictor of fertilized potentiality (Simmons et al., 2008). In feeding and breeding process, we found that the low fertility and fertilization rates of the Chinese Monal may be closely related to the sperm morphology. Therefore, observing the sperm morphology is important to select individuals with higher semen quality for breeding, increasing the success rate of artificial insemination, and increasing the population size of the captive Chinese Monal, with the ultimate goal of reintroduction into the wild.
Wright’s and Giemsa staining methods were utilized to stain red blood cells, microscopic examination of sperm was conducted using both Papanicolaou and Wright’s Giemsa staining to observe the microstructure, and scanning electron microscopy (SEM) was employed to examine the ultrastructure of the blood cells and sperm. Morphological parameters of the red blood cells and sperm were measured to provide foundational material for physiological research on the Chinese Monal, as well as to assist in disease diagnostics and artificial insemination.
MATERIALS AND METHODS
Sample Collection
Six adult male Chinese Monals from the Baoxing Fengtongzhai Nature Reserve Management Center in Sichuan Province (altitude: 2000 m; coordinates: 29°11’12.74”N, 102°09’36.15”W; approved for captive breeding in 1992) were selected in this study. All individuals were clinically healthy, with no evidence of disease and similar body weights. The animal study protocol was approved by the Ethics Committee of Sichuan Normal University (approval number: 2024LLSC0023). 1 ml of residual blood sample from routine health check-ups (collected from the wing vein) was stored in an EDTA-K2 anticoagulant tube (Livingstone, 2020). For semen collection, three adult male individuals that had been trained in artificial insemination and were accustomed to handling were selected. The males were restrained, their cloacal area cleaned and disinfected, and gentle massage was applied to induce semen release. A rubber bulb pipette was promptly employed to collect semen, which was subsequently diluted, stained, and promptly examined for sperm morphology (Kucera et al., 2025).
Blood Cell Morphology Observation
A drop of blood sample, stored in an EDTA-K2 anticoagulant tube, was taken for Wright’s and Giemsa staining using the EA36 Papanicolaou staining solution from Phygene Life Sciences Co., Ltd. Following the method described by Vives et al. (2004), the blood on the slide was fixed with methanol for 1 min, air-dried, stained with the stain solution, and then rinsed for 10 s. After drying for 30 mins, the samples were observed and photographed under a microscope (DM4B, Leica) using the Leica Application Suite X software. The morphology of red blood cells was examined at 400X magnification. For each animal, three blood smears were prepared. Each smear was observed under five different fields of view, with each field containing approximately 50 to 100 cells. The major and minor axes of the various cells were recorded, and the ViewPoint software (version 2021) was used for cell measurements, with results documented in Excel.
For the ultrastructural observation of blood cells, the blood from the anticoagulant tube was centrifuged at 5000 r/min for 10 mins using a D3024 benchtop high-speed microcentrifuge from Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd. The supernatant was discarded, and the lower layer of blood cells was resuspended in 0.1 mol/ml PBS (pH=7.2) and centrifuged. The cells were fixed in a 3% glutaraldehyde solution (111-30-8 AR grade, Nanjing Chemical Reagent Co., Ltd.) at 4oC, washed with ultrapure water (UPR-II-15TNZ ultrapure water system, Sichuan ULUPURE Ultrapure Technology Co., Ltd.) for 10 mins each time, and then fixed with 1% osmium tetroxide solution (13-24-33 Beijing Zhongjing Keyi Technology Co.,Ltd.) for 1 h. After the washing with ultrapure water, the samples were dehydrated through a graded series of ethanol (100092683 anhydrous ethanol, Sinopharm Chemical Reagent Co.Ltd.). The samples were then dropped on the glass slides, dried using a critical point dryer (K850, Quorum), and attached to the specimen stage. Gold coating was applied using an ion sputter (Smart Coater, JEOL, Japan), and the samples were observed and photographed under a scanning electron microscope (JSM-IT700HR, JEOL, Japan). The morphology of the blood cells was recorded during observation.
Sperm Morphology Observation
The collected semen was diluted at a ratio of 1:10 with prewarmed semen diluent at 38oC and divided into three parts. One part was subjected to Wright’s and Giemsa staining (using the same method described for the blood cells), another part was prepared for Papanicolaou staining by spreading the diluted semen into a thin smear on a slide, and the remaining part was processed for scanning electron microscopy. The diluted semen was spread into a thin film on a glass slide. For Papanicolaou staining, a drop of the diluted semen was placed on a slide and spread into a thin film. After fixation with alcohol, the sample was stained with hematoxylin. Following the removal of excess hematoxylin with acid alcohol, the sample was stained with EA50 stain solution. After clearing with dimethyl benzene, the sample was ready for observation. The morphology of the sperm was observed under a microscope at 400X magnification. Sperm with normal morphology had their head and tail lengths recorded, and abnormal sperm were also observed and documented (Annelisse et al., 2021).
For the ultrastructural observation, the diluted sperm was mixed with glutaraldehyde at a ratio of 1:1. The samples were then fixed in 3% glutaraldehyde at 4oC for 2 hours, followed by washing with ultrapure water and post-fixation with 1% osmium tetroxide for 1 hour. After dehydration through a graded ethanol series and critical point drying, the sperm were mounted, gold-coated, and observed under a scanning electron microscope to document their ultrastructure.
Data Processing
The experimental results were statistically analyzed using SPSS 26.0, and data were presented as mean ± standard deviation (Mean ± SD). One-way analysis of variance (One-Way ANOVA) was performed on the long diameter of different types of red blood cells, and pairwise comparisons between groups were conducted using LSD multiple comparisons. The differences between various types of cells were assessed with a significance level of α=0.05. If p<0.05, the difference was considered significant; if p>0.05, the difference was not significant. For sperm length measurements, box plots were first used to identify and exclude outliers (values below Q1 - 1.5 IQR and above Q3 + 1.5 IQR). Normality tests were then performed, and the average length was calculated.
RESULTS
Morphological Characteristics of the Blood Cells
Morphology of the Red Blood Cell
The mature red blood cells of the Chinese monal are oval, with an oval nucleus that is stained dark purple. The chromatin within the nucleus is dense and uniform. The nucleus is centrally located within the cell, and the cytoplasm is stained light red (Figure 1A, cell types indicated by arrows in the figure are all labeled in this manner). Under scanning electron microscopy (SEM), the cell boundaries are well-defined, and the cell membranes are smooth (Figure 1B). In contrast, immature red blood cells are round, with a round nucleus stained purple. The cytoplasm of immature cells has a loose structure, with rough and uneven boundaries and relatively large cells. The cytoplasm is stained light purple to transparent, distinct from the nuclear staining (Figure 1C). The morphological parameters of Chinese monal red blood cells are as follows: for mature red blood cells, the long diameter of the nucleus is 5.74 ± 0.67 μm, and the short diameter is 3.24 ± 0.43 μm. The long diameter of the cell is 12.43 ± 0.89 μm, and the short diameter is 7.70 ± 0.91 μm. For immature red blood cells, the long diameter of the nucleus is 5.00 ± 0.54 μm, and the short diameter is 3.65 ± 0.18 μm. The long diameter of the cell is 10.79 ± 0.91 μm, and the short diameter is 8.84 ± 0.88 μm (Table 1).
The microscope’s image of red blood cells. (A) Mature RBCs (400×) (→); (B) Mature RBCs(SEM, 8000×) (→); (C) Immature RBCs (400×) (→).
Morphology of the Granulocyte
Granulocytes are classified into neutrophils, eosinophils, and basophils based on the composition and function of their granules. Neutrophils are round, with a nucleus divided into two (commonly) or more lobes, stained purple, and symmetrically distributed. The neutrophilic granules are often located on top of the nucleus, and the cytoplasm is semi-transparent with coarse, visible, purple-stained neutrophilic granules (Figure 2A). Eosinophils are rare, round, and have an indistinctly lobed nucleus. The cell is dark blue, and the nucleus is often not visible under a light microscope due to the presence of numerous eosinophilic granules that are uniformly and delicately stained (Figure 2B). Basophils are relatively few, round, with a nucleus typically divided into two lobes, stained dark purple to black. The cytoplasm is stained light purple and contains many small granules (Figure 2C). The morphological parameters of these granulocytes are as follows: for neutrophils, the long diameter is 11.95 ± 0.74μm, and the short diameter is 11.13 ± 0.66μm. For eosinophils, the long diameter is 7.92 ± 0.54μm, and the short diameter is 7.27 ± 0.68μm. Finally, for basophils, the long diameter is 11.07 ± 0.4μm, and the short diameter is 10.37 ± 0.59μm (Table 2).
The morphology of granulocytes. (A) Neutrophils(400×)(→); (B) Eosinophils(400×) (→); (C) Basophils(400×) (→).
Morphology of the Agranulocyte
White blood cells are classified into granulocytes and agranulocytes based on the presence or absence of granules. Agranulocytes include large and small lymphocytes, as well as monocytes. In the Chinese monal, lymphocytes can be divided into large and small lymphocytes. Small lymphocytes are more numerous, round or nearly round, with rough and uneven cell boundaries. They are distributed in clusters or individually, stained purple, with a dark blue nucleus (Figure 3A). Large lymphocytes are round, distributed individually, with slightly rough cell boundaries. The nucleus is larger, stained dark purple, and the cytoplasm is stained light purple. The nuclear and cytoplasmic regions are distinguishable, and the nucleus is tangent to the cell membrane (Figure 3B). Monocytes are relatively large, with a large, non-lobed nucleus. The nucleus is kidney-shaped, horseshoe-shaped, dumbbell-shaped, or round, stained purple, and located in the center of the cytoplasm. The cytoplasm is stained light blue, containing fine sand-like chromatin granules (Figure 3C). Thrombocytes are distributed either individually or in clusters, exhibit a relatively small size, and have an oval shape. The nucleus occupies almost the entire cell, stained black-blue, and the cytoplasm is light blue (Figure 3D). The morphological parameters of these agranulocytes are as follows: for small lymphocytes, the long diameter is 6.86 ± 0.39 μm, and the short diameter is 5.04 ± 0.56 μm. For large lymphocytes, the long diameter is 8.48 ± 0.36 μm, and the short diameter is 7.47 ± 0.86 μm. For monocytes, the long diameter is 14.03 ± 1.06 μm, and the short diameter is 12.09 ± 1.04 μm. Lastly, for thrombocytes, the long diameter is 4.8 ± 0.41 μm, and the short diameter is 3.61 ± 0.51 μm (Table 3).
Morphology of Agranulocytes. (A) Small Lymphocytes(400×) (→); (B) Large Lymphocytes(400×) (→); (C) Monocytes(400×) (→); (D) Thrombocytes(400×) (→).
Comparison of the Blood Cell Size
The cells’ long diameter can represent the blood cells’ sizes. Monocytes have the largest size, while coagulation cells are the smallest. The long diameter of most cells differs significantly (p<0.05), and cell types can be differentiated based on their size. However, there is no significant difference in size between mature red blood cells and basophils, neutrophils and immature red blood cells, or large lymphocytes and eosinophils (Figure 4). Therefore, these cell types cannot be distinguished by size alone, and must be differentiated based on intracellular morphology.
Long Axis Lengths of Different Types of Blood Cells. Different letters indicate significant differences (p<0.05).
Morphology of the Sperm
As shown in Figures 5A and 5B, the Wright-Giemsa stain and modified Papanicolaou stain were used to observe the sperm morphology of the Chinese monal. Normal sperm consists of a head and a tail, with the tail further divided into the neck, middle, principal, and end segments. The Wright-Giemsa stain clearly distinguishes the head and tail of the sperm, although the boundary at the tail’s end is difficult to discern. The modified Papanicolaou stain provides a clearer view of the sperm tail than Wright-Giemsa. At the same time, Wright-Giemsa fails to reveal the acrosome portion of the head, which is visible but unclear with Papanicolaou staining. Scanning electron microscopy allows for clear observation of the boundary between the acrosome and the head and the boundary between the head and tail (Figure 5C and 5D). The sperm cell wall is relatively rough, and some sperm exhibit spherical protrusions on the cell wall. The flagellum shows small branches, as seen in Figures 5E and 5F. As shown in Figures 5A-5F, normal sperm exhibit normal morphology, with similar proportions of each part and smooth lines without severe bending or disconnection. In contrast, abnormal sperm display various defects, such as head-tail disconnection, severe angular bending or shortened and swollen heads, and overlapping of the head and tail. In Figure 5G, the sperm head is shortened and enlarged; in Figure 5H, the head is bent and twisted; in Figure 5I, the head and tail are folded; and in Figure 5J, the head and tail are disconnected.
The Morphology of the spermatid. (A) Swiss Giemsa staining for sperm analysis (HE for head, TA for tail, 400×); (B) Modified Papanicolaou Stain for Sperm Cytology (400×); (C) Shortened and swollen sperm head (400×); (D) Curved sperm head (400×); (E) Sperm head-tail folding (400×); (F) Fractured sperm tail (400×); (G) Overall sperm morphology (SEM, 4000×); (H) Head morphology of sperm (SEM, 8000×); (I) Bifurcated sperm flagellum (SEM, 20000×); (J) Globular protrusions on sperm surface (SEM, 70000×).
The size measurements of normal sperm in the Chinese monal are shown in Table 4. The average length of normal sperm in the Chinese monal is approximately 63.28 ± 1.74 μm, with the tail being the longest part, measuring 46.30 ± 1.74 μm, which accounts for about 7/10 of the total sperm length. The ratio of the head to the tail is approximately 1:2.73.
The overall shape of the sperm is elongated and rod-like, with a flagellum. In this study, some sperm exhibited abnormal morphology, including shortened and enlarged heads, bending, head-tail folding, and tail breaks, which affected the sperm’s motility. Figures 5G, H, I, and J show the deformities in the sperm head and tail. Among these deformities, as shown in Table 5, the three individuals had similar sperm deformity rates, around 44%. The highest rate of deformity was observed in the sperm head, at approximately 36%.
DISCUSSION
Blood plays a crucial role in maintaining the normal metabolism and can objectively reflect an animal’s physiological functions and metabolic state, serving as an important indicator for disease diagnosis (Peng et al., 2018). Establishing a reference system for blood parameters of endangered species is of significant practical importance for their breeding, conservation, and protection. Studies have shown that in a diseased state, the morphology and structure of cells undergo changes (Gu et al., 2024). As an essential part of the blood parameter reference system, morphological observation of blood cells is indispensable. In this study, we found that the blood cells of the Chinese Monal can be categorized into eight types: red blood cells, neutrophils, eosinophils, basophils, lymphocytes, monocytes, and thrombocytes.
Characterization of Red Blood Cells
Red blood cells vary among different animals. Mammalian red blood cells typically lack a nucleus with biconcave disc shape, which reduces their own respiration, decreases consumption, and enhances the efficiency of oxygen transport. Birds, amphibians, reptiles, and fish, being less evolutionarily advanced, mostly have oval-shaped red blood cells with nuclei. In this study we found that the red blood cells of the Chinese Monal are oval, and possess nuclei, being similar to those of other birds.
It is well know that red blood cells are species-specific: the broad-breasted white turkey has red blood cells ranging from 13.26-14.10 μm in length and 7.91-8.19 μm in width, the broad-breasted bronze turkey has red blood cells ranging from 14.41-14.75 μm in length and 8.74-9.17 μm in width (Bhattacherjee et al., 2017), the Coturnix japonica has red blood cells ranging from 10.62-12.42 μm in length and 6.57-7.00 μm in width (Tadjalli et al., 2003), the Common Kestrel has red blood cells ranging from 12.52-13.88 μm in length and 6.51-8.09 μm in width (Sheni et al., 2008), and the eastern sarus crane has red blood cells ranging from 13.4-16.09 μm in length and 7.73-8.33 μm in width (Narkkong et al., 2011). In addition, the volume of red blood cells is related to their oxygen transport capacity (Carlos et al., 2017). Research indicates that when chicken embryos are oxygen-deprived, the hematocrit in the blood increases due to an increase in the average red blood cell volume and red blood cell concentration (Tazawa et al., 2012). A meta-analysis found that red blood cell volume is closely related to altitude: as altitude increases, red blood cell volume and hemoglobin levels also increase to adapt to low-oxygen environments (Peter et al., 2013). In this study, we found that the Chinese Monal’s red blood cells range from 11.54-13.32 μm in length and 6.79-8.61 μm in width, which are smaller than those of the broad-breasted white turkey, broad-breasted bronze turkey, Common Kestrel, and eastern sarus crane, but larger than those of the Coturnix japonica. This suggests that the Chinese Monal may have stronger gas transport capabilities than the Coturnix japonica, but weaker than the other mentioned birds. The smaller red blood cell volume in captive Chinese Monals may be related to their adaptation to the low-altitude breeding environment, where they do not require high oxygen transport capacity to maintain vital activities. Additionally, when the body is diseased or inflamed, platelets become over-activated, with an increase in pseudopod formation and adhesion to the cell membrane of the red bloods, leading to aggregation and dispersion, which can cause red blood cell deformation (Pretorius et al., 2018). Inflammation leads to oxidative stress, resulting in the formation of acanthocytes, and changes in red blood cell shape and hemoglobin structure (Revin et al., 2019). Shape changes are caused by differential expansion of the two monolayers on the surface of the cell membrane, forming planocytes, stomatocytes, spherocytes, and echinocytes (Gerald et al., 2002). Another study has shown that animal infected with Salmonella experiencing an acute inflammatory response exhibit red blood cell membrane undulations, cell shrinkage, and membrane blistering or lysis through apoptosis (Universodade et al., 2019). In this study, no abnormalities were found in the red blood cells, but observing the blood cells of the Chinese Monal can help monitor their health status.
Characterization of White Blood Cells
White blood cells are an essential component of animals’ defense and protective mechanisms, with different types of white blood cells performing distinct functions, such as amoeboid movement, phagocytosis, and immune functions. The white blood cells of the Chinese Monal include large and small lymphocytes, monocytes, and granulocytes. Large activated T and B lymphocytes have active immune functions, capable of eliminating pathogenic factors (Chi et al., 2024), and small lymphocytes can respond to the body’s pathogens through immune surveillance, rapid response, and memory functions. Monocytes can phagocytize, enter damaged tissues through blood vessels, and transform into macrophages containing lysozyme, which engulf pathogenic microorganisms and the harmful chemicals they secrete (Shanze et al., 2024). In blood smears of the Chinese Monal, monocytes were less numerous, and neutrophils had fewer and more evenly distributed granules than basophils and eosinophils, staining lighter with a pale purple color. Among the white blood cells, monocytes were the largest, followed by neutrophils, basophils, large lymphocytes, eosinophils, and small lymphocytes. In blood observation, neutrophils were more numerous than eosinophils and basophils. Bacterial infections can lead to alterations in the proportions of white blood cells. When the body is infected with streptococci and an inflammatory response occurs, neutrophils mature under the influence of colony-stimulating factors. They migrate from the bone marrow to the bloodstream and spleen under the action of cytokines. Neutrophils eliminate streptococci through chemotaxis, apoptosis, phagocytic activation, degranulation, production of reactive oxygen species, and the formation of neutrophil extracellular traps. During this process, the percentage of neutrophils increases tenfold in the blood and threefold in the spleen, and monocytes also increase (Bleuzé et al., 2024). Besides, environmental factors are also closely related to white blood cells. Environmental pollution and changes in weather can activate inflammatory responses (Brook et al., 2010), which in turn lead to changes in white blood cell counts (van Eeden & Hogg, 2002). Studies have shown that when the body is exposed to PM2.5, the proportion of basophils decreases, while the proportion of neutrophils increases (Frampton et al., 2004). When the concentrations of NO2 and SO2 in the air increase, the proportion of total granulocytes is significantly reduced (Gao et al., 2019). The husbandry practices for Captive Chinese Monals included providing cooled boiled water, daily fecal removal, regular disinfection, and routine cage cleaning. The breeding site, surrounded by mountains, ensured strong air circulation. During the experimental period, no significant changes in white blood cell counts were observed, indicating satisfactory health status and effective management practices.
Characterization of Coagulation Cells
Mammalian platelets are irregular in shape, often appearing as circular, oval, or star-shaped structures, and are devoid of a nucleus. In contrast, thrombocytes in lower vertebrates are nucleated and typically exhibit a rounded, tear-drop, or spindle shape (Gerald et al., 2020). The cytoplasm of these thrombocytes contains clotting factors and procoagulant substances, which participate in the coagulation cascade, accelerating the formation of thrombin and subsequently promoting the cross-linking of fibrin and the formation of blood clots (Rose et al., 2010). Thrombocytes not only possess the capability to produce thrombin and thus fulfill their coagulation functions, but also exhibit properties of immune cells. During inflammation, these cells can induce a hypercoagulable state, leading to an increase in thrombocyte numbers and the phagocytic clearance of exogenous substances from the bloodstream (Fang et al., 2014). The thrombocytes of the Chinese Monal are the smallest among blood cells. Their small size enables them to rapidly circulate throughout the body to perform coagulation and immune functions.
Characterization of Sperm
The common methods for artificial insemination in birds include massage, artificial vagina, electrical stimulation, and epididymal sperm collection. The massage method is the most frequently used in the artificial insemination. In this study, the massage technique was employed to collect non-invasive semen samples.
Sperm morphology research is an important aspect of semen quality assessment, which can reveal the mechanisms of spermatogenesis, and conduct the fertilization biology studies. The morphology of sperm corresponds to their function, and sperm with normal fertilization capabilities must also have an intact morphological structure. The speed of sperm is the result of thrust and resistance, depending on the ratio between the length of the flagellum and the size of the head. Any changes that affect sperm morphology may lead to changes in sperm speed, thereby affecting the success rate of fertilization. An example of this is that the motility of abnormal sperm is inevitably reduced (Simmons et al., 2008). In this study, the sperm of the Chinese Monal was observed to have an elongated shape with a cap-like acrosome featuring a grooved structure, resembling the acrosomal morphology of non-passerine birds, which typically exhibit a cylindrical and conical acrosome, contrasting with the helically twisted acrosome often found in passerine birds (Aire AT, 2014). The ratio of the head length to the tail length of the Chinese Monal’s sperm is 1:2.73. The relatively large head-to-tail length ratio may result in greater resistance generated by the head compared to the propulsive force produced by the tail, potentially reducing sperm motility and affecting fertilization, which could be one of the reasons for its low fertility rate.
Sperm morphology is closely related to staining methods, and different species require different staining techniques (Xu et al., 2022). Under the Swiss Giemsa staining, the head and tail of the Chinese Monal’s sperm are more distinctly differentiated compared to the Papanicolaou staining, making it suitable for sperm morphology research. Under the Swiss Giemsa staining, it was found that the rate of abnormal sperm in the Chinese Monal is as high as 44%. The high rate of sperm abnormalities may be one of the reasons for the low fertilization rate in the Chinese Monal. The head of the sperm contains the nucleus, which includes highly condensed chromatin, and the acrosome is visible under a light microscope. The acrosome, derived from the Golgi apparatus, contains acrosomal enzymes that participate in the acrosomal reaction, but when the proteins which relate to acrosome development are abnormal, gamete fusion cannot occur (Khan et al., 2024). The proportion of abnormal sperm in the three individuals was similar, ranging from 42% to 45%. The head was the most common site of deformity in the Chinese Monal’s sperm, often characterized by shortening, swelling, and bending, accounting for 78% of all abnormalities. This may be due to the head being a storage site for energy, having a more fragile structure and less water content, thus being more susceptible to deformation (Sara et al., 2023). Abnormalities in the sperm tail accounted for 22% of the total number of deformities. These defects may have been caused by the abnormal migration of cytoplasmic droplets during spermatogenesis, leading to tail coiling and breakage. Such abnormalities may originate from the deletion or mutation of certain genes (Fumie et al., 2004; Zhang et al., 2020). However, the main types and phenotypes of sperm defects vary among other species. The primary defects in emu sperm are found in the head, while the main defects in ostrich sperm are located in the tail. In addition, the rate of sperm abnormalities also differs significantly among species. The average rate of sperm abnormalities is 17.3% in emus (Plessis et al., 2010), 17% in ostriches (Plessis et al., 2014), 25% in rock pigeons (Sontakke et al., 2004), and 17,9% in Northern Pintail ducks during the breeding season (Penfoid et al., 2000). The rate of abnormal sperm in the Chinese Monal, however, is much higher than that of these species, which may have an adverse impact on their reproduction. Abnormal sperm may lead to reduced vitality and penetrating power, decreasing the conception rate, and may also cause abnormal development of the embryo and deformities in the offspring, affecting their survival and health. The neck region of spermatozoa connects the head and tail segments, including the midpiece, principal piece, and endpiece, yet light microscopy cannot discern the sperm neck. Electron microscopy reveals spherical protrusions on the head and neck surface, which have been observed in both the Carib grackle and the cattle egret and are likely the mitochondrial helix (Aire et al., 2017; Roopnarine et al., 2021). The midpiece contains numerous mitochondria that supply energy for sperm motility. The principal piece is encased in a fibrous sheath that offers mechanical support for sperm undulation, with multiple nodal branches visible in the principal segment under scanning electron microscopy. The end piece is relatively slender and functions to propel the sperm forward (Lehti et al., 2017).
CONCLUSION
This study characterized the morphology of blood cells and sperm in the Chinese Monal, highlighting their diagnostic value for disease and fertilization capability. It lays the groundwork for further research on artificial insemination, sperm cryopreservation, and the conservation of genetic diversity in this rare species.
ACKNOWLEDGEMENTS
We would like to thank the staff of Feng Tongzhai National Nature Reserve Administrative Center for their assistance in conducting the research.
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FUNDING
This research was supported by the National Natural Science Foundation of China (Project no. 32200339), the Fundamental Research Funds of China West Normal University (Project no. 20A003), and the Innovation Team Funds of China West Normal University (grant no. KCXTD2024-5).
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DATA AVAILABILITY STATEMENT
Data will be available upon request.
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Data will be available upon request.
