Evaluation of the Congjiang Xiang Pig’s Sperm Quality by Flow Cytometry

Using high-quality male gamete is vital for ensuring the production of valuable offspring, which is economical in the animal industry, especially in livestock breeding and aquaculture. Sperm quality is one of the best discriminators for fertilization potential (Hadi et al., 2021; Aksoy et al., 2012). In many new technologies, semen quality is the most important criterion to measure the suitability of the method. In the study of developing cryopreservation methods/procedures (Li et al., 2021) and freezing extenders (Guo et al., 2021), freezing methods and so on, the sperm quality after thawing determines whether the formula and method are available. Semen analysis is an integral part of the work up for studying the breeding ability of different species, such as Holstein Friesian crossbred breeding bulls (Nag et al., 2021), Saanen goats (Lopes et al., 2021) and Coho salmon (Sandoval et al., 2021).

Conventional semen analysis recommended by the World Health Organization (WHO) includes the measurements of sperm morphology, motility, vitality and concentration, which are the main indexes to evaluate the properties of male fertility. This analysis is time/labor-consuming and a subjective scoring method. Although WHO had been proposed the guidelines for standardizing the procedure, the huge distinction of the result obtained by different technician or laboratory have been reported (Franken, 2015). The conventional semen analysis result only can explain a fraction of the total variation in fertility and other semen characteristics are essential to explain the remaining variation. Flow cytometry (FCM) can reduce the disadvantage of conventional sperm evaluation and it has been used to analyzing attributes of sperm, such as sex-sorted (Holden et al., 2020) and assessment of chilling injury in hypothermic stored boar spermatozoa (Jäkel et al.,  2021). Broekhuijse et al., (2012) used FCM to analyze the mitochondrial membrane potential, the integrity of plasma membrane and acrosome of boar spermatozoa under different feeding environments because this technique can provide statistically reliable data, after analyzing several thousand cells at a single-cell level within a few minutes. The spermatozoa which can fertilize an oocyte in vivo must have intact plasma membranes, acrosomes and sufficient cell metabolism. Propidium Iodide (PI) is a dead/moribund sperm-specific fluorescent dye that can distinguish dead or alive sperm. Mitochondria provide the energy required for sperm motility (Rebelo et al., 2021; Freitas et al., 2017). Rh123 was the most widely used mitochondrial-specific probe, which could identify the sperm with an abnormal mitochondrial membrane potential (Vašíček et al.,  2022). The integrity of the mammalian sperm plasma membrane and acrosome is important for the fertilizing rate. The spermatozoon must retain a normal acrosome so that the acrosome reaction can occur to facilitate fertilization. Fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) can combine with the damaged acrosome and fluoresces green.

The Congjiang Xiang pig is known for its early sexual maturity, mini size. It also has the physiology and other traits which was similarity to humans (Meng et al., 2020). Although they display promising potential as an experimental model and top-grade pork source, there are limited studies on the reproductive profile of the boars, particularly on sperm quality. Increasing litter size is a goal of pig breeders and producers in many countries, semen quality is the key factor in determining the fertilization rate and the litter size of sows (Meng et al., 2020; Gong et al., 2021) and few studies have described the FCM method for evaluating semen quality in pigs. Therefore, in this study, FCM was applied to detect the integrity of plasma membrane, acrosomal and mitochondrial function of adult Congjiang Xiang pigs.

Semen specimens collection and classification
 
The experiment period was from 2022 February to November. Three boars per breed of Congjiang Xiang pigs and Large White pigs were used in this study. A total number of 18 ejaculates (3 ejaculates per boar) were collected from six boars (aged 3 years) and sexually active at sampling, the same trained technician used the hand-glove method once a week. The spermatozoa-rich fraction was retained in a sterile thermal bottle and transported back to Guizhou provincial key laboratory of animal genetics, breeding and reproduction at Guizhou University at 37°C. The Guiyang Lvshengyuan Animal Husbandry Technology Development Co., Ltd. (SCXK: 81 20160007, SYXK (Qian) 2018-0010, Guiyang,China) provided the semen samples of the Congjiang Xiang pigs and the Large White pigs. The seminal fluid was lique fied for 30 min at 37°C.

Semen analysis
 
Each sample was divided in to two parts. One part was used for routine detection and the other part was used for FCM. Preliminary semen characteristics (color, smell, state and pH) of the samples were obtained. Per sample would be recorded and calculated the concentration, vitality, motility and abnormality under the microscope (Hadi and Ali, 2021). All samples were evaluated by the sophisticated observers.
 
Single staining and FCM examination
 
Integrity of plasma membrane
 
Before staining, the spermatozoa was washed and adjusted to 5×10⁶ sperms/mL by DPBS. Each sample (1 mL) was incubated with 5 μL PI dye solutions (1 mg/mL) for 15 min in the dark at 37°C. For eliminating the excess dye, the spermatozoa must wash by DPBS and then centrifuged. In FCM analysis, a flow rate of 2000 cells/s was used to analyze 10,000 sperms in each sample by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The application of forward scatter (FSC) and side scatter (SSC) was amplified and the FSC was obtained at 400 mW power output. The SSC was obtained at 365 mW power output. The logarithmic amplification was performed for fluorescence channel Fluorescence detector 2 (FL2).
 
Sperm mitochondrial function
 
RH123 (Beyotime, Shanghai, China) was diluted to 1 mg/mL in dimethyl sulfoxide (DMSO: Solarbio, Beijing, China). 2 μL Rh123 dye solution were added to 1 mL sample at 37°C in the dark for 15 min. In FCM analysis, the flow rate and the power of FSC and SSC were set as described in PI staining. The logarithmic amplification was performed for fluorescence channel Fluorescence detector 1 (FL1).
 
Acrosome integrity
 
FITC-PNA (Thermo Fisher Scientific, Waltham, MA, USA) dyes were adjusted to 1 mg/mL by DMSO. 1 mL sperm sample was stained with 2 μL FITC-PNA dye solution in the dark 15 min and washed with DPBS to remove background staining (Zhu et al., 2019). A total of 10,000 sperms were analyzed using FCM with fluorescence channel FL1.
 
Rh123/PI double staining and FCM examination
 
In Rh123/PI double staining, 1 mL sample was stained with 10 μL RH123 (1 μg/μL) for 15 min, at 37°C in the dark and mixed with 10 μL PI (1 μg/μL) for 5 min at the same condition. After the double staining, the sample was washed twice times and resuspended by 1 mL DPBS. The sample was analyzed by FCM immediately. Fluorescent measurements were compensated to minimize spillover fluorescence between the red and green spectra. The compensation of green fluorescent was 24 and the compensation of red fluorescent was 0.
 
Fluorescence microscope observation
 
After staining, 10 μL of the sperm suspension was placed on a clean slide to detect the sperm fluorescence staining by using the Nikon ECLIPSE-Ni+DS-Ri2 fluorescence microscope (Nikon Instruments Inc., Tokyo, Japan) with NIS-Elements BR acquisition and analysis software version 5.01 (Nikon Instruments Inc., Tokyo, Japan).
 
Statistical analysis
 
All data were expressed as mean±standard deviation (mean±SD). One-way analysis of variance (ANOVA) was used to determine the results of conventional semen parameters and FCM. All statistical data analys was performed with SPSS 13.0 software (IBM Corporation, Armonk, NY, USA) and P<0.05 was considered statistically significant.

Sperm function assessment in a semen sample is essential to analyze potential animal fertility. Laboratory semen features are correlated to farm animals’ farrowing rates and litter sizes (Singh et al., 2019; Mcpherson et al., 2014). According to previous studies, the result of routine semen evaluation is subjective and insensitive to monitor the testicular function because routine semen evaluation only can detecte the samples with inferior quality (Andrade et al., 2010). FCM has been developed, which is recognized as a valuable method for assessing sperm quality (acrosomal integrity, mitochondrial function and sperm plasma membrane integrity) in reproductive toxicology, veterinary science and clinical andrology. The Congjiang Xiang pig is one kind of Chinese minipig breed with high economic value (Meng et al., 2020; Gong et al., 2021). However, their specific male reproductive characteristics were less studied. The present study provides useful information on the sperm function of Congjiang Xiang pigs for their potential fertility via the conventional method and FCM evaluation.
 
Routine detection of semen quality
 
Both semen of Congjiang Xiang pigs and Large White pigs smelled slightly fishy. The state of semen was not different between the two breeds, but the color of the Congjiang Xiang pigs was milky white and that of the Large White pigs were light gray. The pH of the Large White pigs (pH=7.3) was lower than the Congjiang Xiang pigs (pH=7.5). There was no significant difference in sperm vitality, motility and abnormality (P>0.05, data not shown).The sperm deformation rate of Congjiang Xiang pigs (8.83±2.50) was expected and acceptable in healthy boars (Wolf, 2009).

The differences in boar fertility are mainly due to genetics (Wolf, 2009). Smital and Lopez analyzed the semen quality of different breeds using the semen routine analysis method and the sperm concentration of different breeds was significantly different (Lopez Rodriguez et al.,  2017). Compared with the Congjiang Xiang pigs, the sperm concentration of the Large White pigs was significantly higher (101.25±4.32 vs 70.38±5.09´106 sperm/mL, P<0.01). Different sperm concentrations and sperm numbers would result in different litter sizes of boar (Kommisrud et al., 2002; Johnson et al., 2000). Sperm concentration plays an important role in artificial insemination and the fertilization process because artificial insemination centers tend to dilute the ejaculates to introduce superior genes. From the sperm concentration perspective, the Congjiang Xiang pig is likely to have a weaker reproductive ability than the Large White pig.
 
Sperm membrane integrity
 
The integrity of the sperm membrane is one of the most important indexes in assessing sperm quality for predicting male fertility (Wysokińska et al.,  2021). As shown in Fig 1, the sperm membrane integrity of the Congjiang Xiang pigs was 85.01±1.7%, which showed no substantial difference from that of the Large White pigs (80.81±1.93%, P>0.05).


 
Sperm mitochondrial activity
 
As shown in Fig 2, the sperm mitochondrial activity of the Congjiang Xiang pigs was 75.17±2.38% (Fig 2B) and the sperm mitochondrial activity of the Large White pigs was 70.76±1.89% (Fig 2C). Mitochondria play a decisive factor in providing the energy required for sperm motility (Freitas et al., 2017). They are very important to the whole process of gamete production and reproduction, such as affecting reactive oxygen species (ROS) level (Murphy, 2016). However, there were no significant differences between Congjiang Xiang pigs and Large White pigs in this study (P>0.05).


 
Sperm acrosome integrity
 
As shown in Fig 3A, the sperms having intact acrosome displayed without green or with only faint green fluorescence in the head (FITC-PNA^-) and sperms with damaged acrosome exhibited green head (FITC-PNA⁺). The intact sperm acrosome proportion of the Congjiang Xiang pigs was 67.15±2.03%, which is lower than that of the Large White pigs (76.83±2.68%, P<0.05). The integrity of the sperm acrosome is the key to successful fertilization and proteolytic enzymes (acrosin) exist at the top of the sperm acrosome is closely related to the fertilization process (Sironen et al., 2010), which participates in the whole process of fertilization. Acrosome integrity was one of the sperm attributes which was used to distinguish between high and low-fertility bulls and sperm acrosome integrity could serve as a fertility biomarker (Bernecic et al., 2021; Gonzalez et al., 2020). The low intact sperm acrosome proportion may be one of the reasons that the reproductive ability of the Congjiang Xiang pigs was weaker than the Large White pigs.



There were no significant correlations between the FCM results and sperm forward motion and sperm vitality results of the two breeds. There was a positive correlation between the plasma membrane integrity and mitochondrial activity in the Congjiang Xiang pigs. An insignificant correlation between the plasma membrane integrity, mitochondrial activity and acrosome integrity was observed in the Large White pigs (date not shown).
 
Sperm Rh123/PI double staining
 
Rh123/PIdouble staining can detect the integrity of plasma membrane and mitochondrial function of sperm at the same time. Under the fluorescence microscope, after Rh123/PIdouble staining, sperms exhibited different states (Fig 4). By FCM, it was found that there were significant differences in the percentage of Rh123⁺/PI^- and Rh123^-/PI^- sperm between Congjiang Xiang pigs and the Large White pigs (P<0.05) (Fig 4A, B). The percentage of Rh123⁺/PI^- from Congjiang Xiang pigs was 70.27±1.76%, which is higher than that from the Large White pigs (61.32±0.86%, P<0.05). The percentage of Rh123-/PI^- from the Congjiang Xiang pigs was 17.81±0.53%, which is lower than that from the Large White pigs (24.45±1.42%, P<0.05). There were no significant differences between PI^- single staining and Rh123/PI- double staining in detecting sperm membrane quality (P>0.05). There were no significant differences between single Rh123⁺ and double Rh123⁺/PI in detecting sperm mitochondrial activity (P>0.05). Unlike single staining results, the Rh123/PI double staining appears to provide new evidence that there is an advantage of Congjiang Xiang pigs in the rate of plasma membrane integrity with normal sperm mitochondrial function compared with Large White pigs. The Rh123/PI double staining offers a more accurate and specific readout of the magnitude of the membrane integrity and mitochondrial activity in the boar sperm providing a method of reducing the sample needed for detecting boar sperm quality. The JC-1/PI double staining was used to detect sperm mitochondrial function in goats and bovine has been described before. However, this double staining has defects. In JC-1/PI double staining, the FCM cannot depart the red fluorescence of dead sperm and the red-orange of the sperm with high MMP affects the accuracy of the FCM result (Chai et al., 2011). Therefore, the Rh123/PI double staining was selected to improve the accuracy of sperm evaluation.According to previous studies, the Rh123/PI double staining can detect the bulls sperm mitochondrial function and plasma membrane integrity simultaneously(Johnson et al., 2020). It provides a simpler method for analyzing the relationship between sperm mitochondrial function, plasma membrane integrity and oligoasthenozoospermia (Zou et al., 2010).

Our study suggests that the semen of the Congjiang Xiang pigs is less rich in sperm concentration and sperm with acrosome integrity than that collected from the Large White pigs. The rate of live sperm with the normal mitochondrial function of Congjiang Xiang pigs was significantly higher and the rate of necrotic sperms was lower than those of the Large White pigs. Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the sperm quality of boar and reveal differences in MMP. This study first investigated the semen characteristics of the Congjiang Xiang pigs with FCM. These findings can guide nucleus herds in developing the standards of semen processing in Congjiang Xiang pigs and give new insights into their conservation and utilization.

All authors declare that they have no conflict of interest.