Response Surface Optimization of Bovine Bone Peptide Extraction Process for Studying Reproductive Damage in Fatigued Beagle Dogs (Canis lupus familiaris)
XIE Liu-Wei1,2, LI Bao-An2, SONG Ming-Qiang2, FAN Bing-Feng2, XING Bao-Rui3, LIU Qing2, SUN-Ning2, WU Zhen-Long4,*
1 College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China; 2 Police Dog Technology College, China Criminal Police College, Shenyang 110854, China; 3 Key Laboratory of Oral Diseases and Tissue Regeneration, Shandong Provincial Medical and Health Commission, Jinan Stomatological Hospital, Jinan 250001, China; 4 College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
Abstract：As one of the high-workload functional dogs, police dogs (Canis lupus familiaris) typically perform demanding tasks during routine training and missions, often accompanied by issues such as decreased endurance and reproductive disorders. Therefore, this study employed response surface methodology to optimize the extraction of bovine bone peptide (BBP), subsequently investigated its effects on preventing fatigue-induced reproductive damage in fatigued Beagle dogs. Beagles were randomly divided into 4 groups: Normal (normal feeding, no training), Model (exhaustion training), BBP-L (exhaustion training + 5 g/kg BBP), and BBP-H (exhaustion training + 10 g/kg BBP). Beagles underwent 4 weeks of BBP supplementation combined with downhill treadmill training at a 20° incline and 30 m/min speed, for 30 minutes daily, 5 days per week. After 4 weeks, a 30-minute non-weight-bearing exhaustive swimming test was conducted. At the conclusion of the experiment, blood samples were collected from the dogs, and testes were removed to assess sperm quality within the epididymides. Serum levels of blood glucose, blood urea nitrogen (BUN), lactic acid (LA), and lactate dehydrogenase (LDH) were measured in the Beagles. Measure pyruvate kinase (PK), succinate dehydrogenase (SDH), malondialdehyde (MDA), and superoxide dismutase (SOD) levels in testicular homogenates; Detect glycogen content in liver and muscle tissues; Finally, perform hematoxylin and eosin (HE) staining and immunohistochemical staining on testicular tissues from experimental dogs to analyze the effects of BBP on testicular histology and zonula occludens-1 (ZO-1) expression. The highest extraction rate of BBP was achieved under the following conditions: Boiling for 5 h, pH 10 during enzymatic hydrolysis, hydrolysis temperature of 55 ℃, and hydrolysis time of 6 h, yielding a rate of (35.12±1.25)%. Sperm quality and oxidative stress indicators revealed that after exhaustion training, the total sperm count, sperm density, sperm motility, and SOD levels in the trained dogs were significantly reduced (P<0.01), while MDA levels were extremely significantly elevated (P<0.001). These indicators were reversed after BBP supplementation. Serum biochemical indicators revealed that after exhaustive training, blood glucose, liver glycogen, muscle glycogen, SDH, and PK levels significantly decreased (P<0.01), while BUN, LA, and LDH levels significantly increased (P<0.01). After supplementation with BBP, blood glucose, liver glycogen, muscle glycogen, SDH, and PK levels significantly increased (P<0.01), while BUN, LA, and LDH levels significantly decreased (P<0.01). Histopathological examination revealed enlarged intercellular spaces and loose tissue structure in testicular supporting cells, accompanied by seminiferous tubule abnormalities. ZO-1 expression was significantly downregulated (P<0.01), with marked improvement after BBP supplementation (P<0.01). This study possesses important application value for alleviating problems such as endurance decline and reproductive dysfunction in police dogs during duty operations and high-intensity training.
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