A total of 63 adult Tree Sparrows (11 females and 12 males in 2017, 40 males in 2018) captured with mist nets in May of 2017 and 2018 were used for this study. The sample area, Liujiaxia (35°56′N, 103°15′E) in a northwestern part of China, is a relatively unpolluted village and provides a suitable habitat for Tree Sparrows. Each bird captured was weighed and measured for their wing length and tarsus length, and also sexed based on the presence of a brood patch (Selander and Yang 1966). The body temperature of the adult captured in 2017 was also estimated. On completion, the females were ringed with a uniquely numbered metal band and then released. And males were protected from light and brought back alive to the laboratory, 48 (8 in 2017 and 40 in 2018) of them were used for evaluating sperm motility parameters under different conditions.
The male Tree Sparrows captured in this study were also used for the analysis of several physiological and biochemical indexes and for histology observations. Therefore, these birds were euthanized according to the “Animal Experimental Ethical Inspection Form” (see Additional file 1: Fig. S1), and their seminal glomera were collected conveniently. However, it is to be noted that a non-invasive method to obtain sperm by gently massaging the bird’s cloaca works well in passerine birds (Wolfson 1952), and it should be advocated whenever euthanasia is not absolutely necessary.
Sperm motility parameters were assessed using the WLJY-9000 (WEI-LI New Century Technical Development, China) device with the standardized 10 μm-depth slide chambers. This system, based on the WHO laboratory manual for the examination and processing of human semen, has a thermostat that can maintain the temperature from room temperature up to 70 °C. In addition, the maximum velocity of sperm movement detected by WLJY-9000 is 180 μm/s, up to 20 fields can be captured in each analysis. In each field, 4‒20 frames (20 frames in the present study) were tracked for sperm motility parameters assessment, and no more than 1000 spermatozoa can be identified.
Estimating body temperature
The body temperature of the captured 12 males and 11 females was immediately detected with a UT325 thermometer (Uni-Trend, China). A clean probe was slightly inserted into the cloaca approximately 1 cm deep and removed when the reading was stable.
The test results showed the body temperatures of adult Tree Sparrows were 40.13 ± 1.56 °C for male, and 40.38 ± 1.36 °C for female. We used these values as bases for exploring the optimal conditions of sperm motility parameters evaluation using the CASA system.
Sperm motility parameters detected by the CASA system
Male Tree Sparrows were acclimatized to the laboratory for 3 h before they were euthanized. Then, they were immediately dissected for a series of experiments, and their right seminal glomus was extracted for obtaining sperm suspension in preheated Hank’s Balanced Salt Solution (HBSS, an isotonic solution that can buffer pH and preserve the sample’s osmotic pressure). Sperm swam out from the seminal glomus into the surrounding media and diffused, forming a “cloud”, and in about 10 s the “cloud” diffused completely in the medium. The sperm suspension was then kept warm (at the corresponding analysis temperature) in a water bath until analysis. Next, 10 μL of diluted semen were dropped on the center of the pre-warmed slide chamber and a coverslip was placed over the sample. And the slide was put in the thermostat and sperm motility parameters were assessed by the WLJY-9000 CASA system at 100× magnification. For each analysis, sperm motility parameters were collected and recorded by the capture of at least 5 nonconsecutive fields (a total of at least 500 spermatozoa) within 30 s. The sperm in each field were selected by adjusting the grayscale threshold, and the selected debris and round cells were manually deleted prior to analysis. The following sperm motility parameters were determined: (1) Sperm motility: rapid progressive motility (the percentage of rapid progressive sperm with a linear velocity ≥ 25 μm/s), slow progressive motility (the percentage of slow progressive sperm with a linear velocity < 25 μm/s), non-progressive motility (all other patterns of motility with an absence of progression), immotility (the percentage of immotile sperm). In addition, progressive motility (the sum of rapid and slow progressive motility) is a vital indicator of ejaculated sperm to evaluate their swimming ability (Kathiravan et al. 2011), which was assessed in this study; (2) Sperm velocity: the curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) can be evaluated by the CASA system, and these three sperm velocity parameters were strongly associated with each other (Pearson’s r > 0.96, p < 0.001), thus the present study chose VCL, the velocity over the actual sperm trajectory, as a measure of sperm swimming speed (hereafter referred to as sperm velocity); (3) Sperm movement trajectory: path linearity (the linearity of actual sperm track, LIN = VSL/VCL), path wobble (departure of actual sperm track from average path, WOB = VAP/VCL), and path straightness (linearity of the average path, STR = VSL/VAP).
Effect of time since dilution and until analysis on sperm motility parameters
The right seminal glomus of 8 males brought back to the laboratory and euthanized in 2017 was removed and cut in half in 0.5 mL of HBSS, which could keep the sperm concentration between 2 and 50 × 106 sperm/mL (the recommended concentration for sperm motility parameters assessment in WHO laboratory manual for the examination and processing of human semen). The preheated temperature of HBSS was set to 40 °C (the body temperature of Tree Sparrows), and the pH was set to 7.5 (according to the semen pH of poultry) (Orunmuyi et al. 2013) and because weak alkaline pH has shown increased sperm movement (Holm and Wishart 1998). Next, a 10-μL sperm suspension was used for the assessment of the sperm motility parameters by the WLJY-9000 CASA system at 3, 5, 15, 20, and 30 min after dilution.
The initial experiment results showed that the percentage of progressive sperm decreased with time, which largely dropped after 15 min. Based on the results of this first experiment, in 2018, sperm samples of 8 other males were used to assess sperm motility parameters at 1 (the time sperm were suspended fully in medium and load the sperm suspension onto the microscope slides), 3, 5, 7, 9, 11, 13, and 15 min after dilution in HBSS with pH 7.5 at 40 °C.
Effect of temperature on sperm motility parameters
We evaluated the effect of the temperature on sperm motion characteristics using sperm samples from 8 males, whose right seminal glomus was divided into 3 equal portions with ophthalmic scissors in 2018. Then, the three pieces were put immediately into different Eppendorf tubes containing 0.2 mL of HBSS with pH 7.5 and placed in a water bath at either 38, 40 or 42 °C. Approximately 4 min after dilution, motility parameters of sperm from different Eppendorf tubes were analyzed by the WLJY-9000 CASA system at 38, 40 and 42 °C.
Effect of pH on sperm motility parameters
Based on the semen pH of poultry (Orunmuyi et al. 2013) and previous results in other species (Holm and Wishart 1998), the pH values of HBSS in this experiment were set to three groups (7.0, 7.5 and 8.0 in group 1, 6.0, 7.0 and 7.5 in group 2, and 7.5, 8.0 and 9.0 in group 3) to control the impact of individual variation on the comparison of sperm motility parameters between different pH levels. A total of 24 birds (7 in group 1, 8 in group 2 and 9 in group 3) were used in this experiment, the right seminal glomus of which was divided into 3 equal portions with ophthalmic scissors. Then, the 3 sections were immediately put into Eppendorf tubes containing 0.2 mL of 40 °C pre-warmed HBSS with different pH values. Approximately 4 min after dilution, sperm motility parameters were assessed by the WLJY-9000 CASA system at 40 °C.
Experimental data are expressed as mean values ± standard deviation (SD) or ratio. Statistical analyses were performed using SPSS 20.0 statistical software (IBM SPSS Inc., USA). Two-tailed Pearson correlation analysis was used to check for relationships between sperm velocity parameters. All proportion data including sperm motility and sperm movement trajectory were logit transformed prior to analysis.
The change of sperm motility parameters at different analysis time was investigated using the repeated measures analysis of general linear model (GLM). In order to validate the univariate F-test, the “Epsilon” values (when the Greenhouse–Geisser was > 0.7, the Huynh–Feldt correction should be used, and if not, the Greenhouse–Geisser correction should be used) would be used to calculate an appropriate adjustment to the degrees of freedom when the assumption of sphericity was not met (p < 0.05). Besides, curve fittings were performed using SigmaPlot 14.0 software (Systat Software Inc., USA) to simulate the association between analysis time (independent variable) and sperm motility parameters (dependent variables), and the equations that most closely fit the actual data were found (large coefficient of determination, and p < 0.05).
To investigate the effect of temperature and pH on sperm motility parameters, the univariate analysis of GLM was conducted. The sperm motility parameters entered as dependent variable, while temperature or pH entered as a fixed factor. Besides, male identity was entered as a random factor to control for individual variation, and the time since dilution and until analysis was a covariate.
The model assumptions were validated by testing the residuals’ normality, and pairwise comparisons were corrected by the Bonferroni method. Besides, SigmaPlot 14.0 software was also used for bar graphs, line charts and scatter plot.