The Study On Occurrence Law And Formation Mechanism Of Dairy Goats With Fleshy Udder

Dairy goat production is an important component of livestock production.Improving milk production and composition are of great significance for promoting the development of milk industry and improving the milk yield of dairy goats.Fleshy udder,as an important phenomenon affecting the functional lifespan and milk production of dairy goats,is common in various large-scale goat farms,but there is currently a lack of systematic research on their occurrence regularity,formation mechanism,and prevention.Based on this,we conducted an investigation on the occurrence situation and summarized the occurrence rules of dairy goats with fleshy udder in a large-scale farm in Shaanxi Province,and performed a systematic study on the abnormalities of fleshy udder in dairy goats at the histological and cellular levels.The following results were obtained:（1）It was found that dairy goats with fleshy udder were relatively common in the large-scale farms of Guanzhong dairy goats.Taking a large-scale dairy goats farm in Fuping County as an example,the annual incidence of Guanzhong dairy goats with fleshy udder was about 5%in 2019-2021.The appearance of dairy goats with fleshy udder during lactation period were normal,but compared to normal goats,there was a solid and drooping sensation,and the udder was still larger after lactating.The daily milking volume was significantly lower than that of normal goats（P<0.05）.After entering dry period,the udder of normal dairy goats degenerated more significantly than that with fleshy udder.In addition,most of the milk goats with fleshy udder have not experienced clinical mastitis,especially those born after the second pregnancy,with almost no history of clinical mastitis.（2）H&E staining of paraffin section of fleshy udder in dairy goats showed that during early lactation（within two months postpartum）,the relative alveolar density of fleshy udder was lower than normal mammary gland（P<0.05）,and the connective tissue between acini was more developed.Transmission electron microscope observation showed that in fleshy udder of dairy goats,there were relatively fewer lactoprotein particles in the acinar cavity and mammary epithelial cells（MECs）with regular and untight arrangement,and the reticular fiber filled with collagen fibers was proliferating irregularly.（3）Immunohistochemical staining of paraffin sections of fleshy udder in dairy goats showed that compared to normal dairy goats,the expression levels of estrogen receptor（ER）αand progesterone receptor（PR）in MECs of fleshy udder dairy goats during early lactation（within two months postpartum）were significantly higher,while prolactin receptor（PRLR）expression levels were significantly lower（P<0.05）.There is no significant difference in the expression of ERβ.ELISA results showed that E₂ and PROG levels in serum of dairy goats with fleshy udder during early lactation were both significantly lower than that of normal dairy goats（P<0.05 and P<0.01 respectively）,and the PRL level was significantly higher than that of normal dairy goats（P<0.01）,which was obviously contrary to the changes in hormone receptor levels.The subsequent results showed that the average daily milking volume of goats in the estradiol benzoate injection group was higher than that in the control group,but there was no significant difference（P>0.05）.This indicated that estrogen injection had a certain effect on preventing the appearance of fleshy udder and improving the lactation ability of dairy goats with low circulating E₂ levels in the late pregnancy without significance.（4）Transcriptome sequencing of fleshy udder was carried out,and it was found 307 up-regulated and 389 down-regulated genes among 696 differentially expressed genes.Through KEGG analysis,it was found that the transcription levels of PR and downstream RANKL/Cyclin D1 related to MECs proliferation were upregulated with varying degrees.In addition,gene expression of mammalian target of rapamycin（m TOR）signaling pathways related to milk protein synthesis,lipid metabolism,autophagy,and cell survival and proliferation,as well as MAPK,PI3K-AKT,AMPK,and Erb B signaling pathways directly related to m TOR signaling pathways,has undergone large-scale changes.Subsequently,the ELISA results showed the serum RANKL and OPG levels of dairy goats with fleshy udder were significantly higher than those of normal dairy goats（P<0.05）.The correlation analysis showed that there was a correlation of serum hormone levels and RANKL levels of dairy goats in early lactation,indicating that the occurrence of dairy goats with fleshy udder was closely related to the abnormal RANKL signaling pathway.（5）We conducted the co-culture of goat mammary epithelial cells（GMECs）and adipocyte-like cells induced by adipose derived stem cells（ADSCs）and RANKL treatment experiments on primary cells derived from mammary gland to explain the mammary gland structure and lactation ability decrease in fleshy udder at the cellular level.Co-cultured with adipocyte-like cells in different time,the results showed that from the beginning of co-culture to 7 d,the expression levels of RANKL protein in co-culture group were higher than those in control group,especially at 7 d,the expression of RANKL was the most significant（P<0.05）,indicating that by 7 d,co-cultured with adipocyte-like cells was beneficial for the RANKL expression of GMECs.The expression level of Elf5 protein in GMECs co-cultured with different days was higher than that in control group,especially after 7 d of co-culture,the expression level of Elf5 protein in GMECs co-cultured was significantly higher than that in control group（P<0.05）.Further immunofluorescence staining results also showed that after 7days of co culture,GMECs formed a large number of globules,while the control group had only a small number,indicating that from the beginning of co-culture to 11 d,co-cultured with adipocyte-like cells had been beneficial for GMECs forming a lactating acinar structure,and the impact was most significant at 7 d.Treating with high level of RANKL for 24 h,the significant up-regulated p-IκB and Cyclin D1 and down-regulated p-Stat5a in GMECs with lactation induction（P<0.05）indicated that IκB/p65/Cyclin D1 pathway was up-regulated significantly and lactoprotein synthesis was inhibited by RANKL,while there was no change without lactation induction and the treatment of bait receptor OPG could not reverse this effect.Besides,the Elf5 expression of GMECs was down-regulated without significant difference（P>0.05）with high level of RANKL for 24 h.And there was no significant change in mammospheres formation and Elf5 expression of GMECs under 3D conditions,indicating that high level of RANKL played a significant role in inhibiting the milk proteins expression of GMECs without significant effect on acinar formation,and OPG failed to reverse this effect.In summary,this study has determined the structure of fleshy udder and has discovered that the occurrence of dairy goats with fleshy udder is related to abnormal levels of E₂,PROG and PRL and up-regulated RANKL pathway.There is a significant positive correlation between E₂ level during lactation and early milk production.Co-culturing with adipocyte-like cells induced by ADSCs from mammary promote the RANKL expression and mammospheres formation of GMECs,while high levels of RANKL inhibit the synthesis of milk proteins in GMECs,which indicates that RANKL upregulation participate in the fleshy udder formation and the decrease in milk production.This study provides a theoretical basis for the prevention and lactation ability improvement of fleshy udder.