Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (2): 387-400.doi: 10.3864/j.issn.0578-1752.2025.02.012

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Post-Freezing Quality and Targeted Lipidomics Analysis of Rongchang Pig Spermatozoa with Different Freezing Tolerance

GAO XiaoPing1,4(), PAN HongMei1,4, GUO ZongYi1,4, ZHANG JunJie2, LIN Yan3(), ZHANG Liang1,4()   

  1. 1 Chongqing Academic of Animal Science, Chongqing 402460
    2 College of Life Sciences, Sichuan Agricultural University, Ya’an 625000, Sichuan
    3 Department of Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 215500
    4 Pig Technology Innovation Center (Chongqing), Chongqing 402460
  • Received:2024-09-30 Accepted:2024-11-20 Online:2025-01-21 Published:2025-01-21
  • Contact: LIN Yan, ZHANG Liang

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Abstract:

【Background】Rongchang pig is one of the excellent local pig breeds in China, but the number of purebred Rongchang pigs is decreasing with the introduction of foreign-bred pigs as well as the shift of the market demand, and this trend seriously threatens the diversity of the genetic resources of pigs in China and the sustainable development of the pig breeding industry. In the process of frozen semen production in local pigs, the differences in post-freezing quality of spermatozoa from different boars are all related to their freezing tolerances, while the sperm lipid composition is one of the key factors affecting their freezing tolerance. 【Objective】The aim of this study was deeply analyze the quality characteristics of spermatozoa of Rongchang pigs with different freezing tolerances and their lipid compositions, and to screen out the candidate markers of sperm freezing tolerance. 【Method】Fourteen Rongchang pig semen were collected for freezing and preservation, and their post-freezing motilities were detected. Relative Motility (post-freezing motility / pre-freezing motility) was used as the criterion for freezing tolerance, and Rongchang pig spermatozoa were screened and grouped into groups for high and low freezing tolerance. Plasma membrane integrity, mitochondrial membrane potential, reactive oxygen species level, apoptosis level and in vitro oocyte penetration ability of spermatozoa from different freezing tolerance groups were detected after freezing, and the extent of freezing damages on the surface and internal microstructure of spermatozoa were observed by scanning electron microscopy and transmission electron microscopy. The medium- and long-chain fatty acid composition of fresh spermatozoa from different freeze-resistant groups was detected by targeted lipidomics technology, and differential fatty acids were screened out. 【Result】5 Rongchang pigs with high freezing tolerance spermatozoa and 5 Rongchang pigs with low freezing tolerance spermatozoa were screened by relative motility; the plasma membrane integrity, mitochondrial activity and in vitro oocyte penetration rate of spermatozoa in the high freezing tolerance group were significantly higher than that of spermatozoa in the low freezing tolerance group after freezing (P<0.05); the apoptosis rate of spermatozoa in the high freezing tolerance group after freezing was significantly lower than that of spermatozoa in the low freezing tolerance group (P<0.05). The surface and internal structural integrity of spermatozoa in the high tolerance group was higher than that of spermatozoa in the low tolerance group after freezing. Targeted lipidomics showed that at least 36 fatty acids were present in Rongchang pig spermatozoa. The content of 11 fatty acids, including palmitic acid and pentadecanoic acid, were significantly different between the sperm of the high and low freezing tolerance groups (P<0.05), and all of them were more abundant in the high freezing tolerance group. 【Conclusion】The spermatozoa of Rongchang pigs with high freezing tolerance were significantly better than those of low freezing tolerance in terms of physiological status, morphology and structure, and in vitro oocyte penetration function, and there were significant differences in fatty acid composition. The differential fatty acids screened in this study, such as palmitic acid, pentadecanoic acid, and arachidonic acid, could be used as candidate markers of freezing tolerance in Rongchang pig spermatozoa. These findings not only provided a new perspective for an in-depth understanding of the biological mechanism of sperm cryoinjury, but also laid a theoretical foundation for the development of efficient and safe cryoprotectants.

Key words: Rongchang pig, sperm freezing tolerance, targeted lipidomics

Fig. 1

Test flow chart"

Fig. 2

Relative motility screening for high and low freeze resistant semen"

Table 1

Comparison of sperm motility between GFE and PFE groups"

分组 Cluster 冻前活力 Pre-freeze motility (%) 解冻后活力 Post-thaw motility (%) 相对活力 Relative motility (%)
GFE 83.00±6.64 44.10±3.54a 0.53±0.02a
PFE 84.35±6.31 15.74±4.74b 0.19±0.06b

Table 2

Comparison of sperm quality after freezing in GFE and PFE groups"

分组
Cluster		 质膜完整性
Plasma membrane integrity（%）		 线粒体膜电位
Mitochondrial membrane potential（%）		 凋亡率
Apoptosis rate
（%）		 活性氧水平
Activated oxygen levels（%）		 穿卵率
Penetration rate
（%）
GFE 36.25±5.58a 43.37±5.88a 34.86±0.67b 27.70±1.96 37.61±2.11a
PFE 21.74±5.14b 20.57±7.68b 44.44±3.38a 26.69±2.54 16.14±1.36b

Fig. 3

Different fertilization results of oocytes A1-A2: Status of oocytes before and after washing; A3: Fluorescence map of unpenetrating oocytes; B1-B3: Bright field and fluorescence images of sperm embedded in the transparent zone of oocytes; C1-C3: Light field and fluorescence images of sperm entering the cytoplasm of oocytes"

Fig. 4

Scanning electron microscopic observation of spermatozoa in GFE and PFE groups A, B: Scanning electron micrographs of spermatozoa in the GFE group; C, D: Scanning electron micrographs of spermatozoa in the PFE group"

Fig. 5

Morphological classification of porcine spermatozoa by post-freezing transmission electron microscopy A: Longitudinal plane of sperm head; B: Longitudinal cross-section of sperm neck; C: Sperm tail longitudinal section; D: Cross section of sperm tail"

Fig. 6

Statistical analysis of different grades of spermatozoa in GFE and PFE groups A: Statistics of spermatozoa of different head types; B: Statistics of spermatozoa of different neck types; C: Statistics of spermatozoa of different mid-tail types; D: Statistics of spermatozoa of different tail cross sections"

Fig. 7

Bar chart of the proportion of fatty acid classification in Rongchang pig sperm"

Fig. 8

Detection and analysis of long-chain fatty acids in sperm of GFE and PFE groups A: the results of principal component analysis show that the red dots represent the GFE group and the blue dots represent the PFE group; B: fatty acid clustering heatmap"

Fig. 9

Significantly different fatty acids in spermatozoa of GFE and PFE group"

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[1] TONG ShiFeng,REN ZhiBin,LIN Fei,GE YuZhu,TAO JingLi,LIU Yang. Proteomic Analysis of Sperm with Different Freezing Tolerance in Erhualian Boar [J]. Scientia Agricultura Sinica, 2022, 55(23): 4743-4752.
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