Seasonal heat stress alters the milk yield, antioxidative levels and serum metabolites in primiparous and 3 multiparous dairy cows


 This study aimed to explore the seasonal heat stress affects the milk yield, antioxidative levels and serum metabolites in primiparous and multiparous dairy cows during early lactation. A total of two hundred dairy cows were selected according to their calving months (June, temperature humidity index (THI) =66.72; July, THI=70.30; August, THI=69.32; September, THI=67.20; October, THI=59.45). Blood samples were collected on day 0, 21, 50, 80, 100 after calving for serum oxidative status analysis and milk yield was recorded every day. The lower average daily milk yield in cows that calved in June and July (P<0.05), and average daily milk yield of multiparous cows was higher than that of primiparous cows that calved in the same month (P<0.05), suggesting that seasonal (June) heat stress negatively affected milk yield in both primiparous and multiparous cows at early lactation. Besides, 15 and 11 serum metabolites were changed in heat stress (average THI = 70.30) group compared with non heat stress (average THI = 59.45) group in primiparous cows and multiparous cows, respectively. These metabolites were mainly involved in the pathways of aminoacyl-tRNA biosynthesis, glyoxylate and dicarboxylate metabolism, and the metabolism of glycine, serine and threonine. These data suggested that heat stress negatively affected the milk yield and thus caused the the elevation of 2 the serum oxidative and antioxidative index Metabolic biomarkers associated with the heat stress 29 in serum were found 0 which providing the basement of evaluating indicator between heat stress and non heat stress groups in primiparous and multiparous cows.


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The effects of heat stress on dairy cows are multifactorial and not only have great impact on the productivity of 58 cows, present hazards to animal health and the oxidative-antioxidative balance, and even negatively affect an animal's 59 offspring when they mature to adulthood (Dahl et al. 2017). It has been reported that THI is a major factor that can 60 decrease the milk production of dairy cows when it is higher than 72, and which usually considered the threshold of 61 heat stress (Du et al. 1996(a); Du et al. 1996(b)). Milk yield is closely related to heat stress especially for the 62 high-producing cows (Gantner et al. 2017). Ravagnolo et al. (2000) founded that milk yield declines by about 0.2 kg 63 when THI (> 72) increased one unit. According to Kadzere et al. (2002) and Igono and Johnson (1990), cows in early 64 lactation are more sensitive to heat stress than those in late lactation.THI has been used as an indicator of thermal 65 comfort for dairy cows (Savaliya et

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Metabolomics is a powerful analytical technology for understanding biological systems from hundreds of thousands of 71 low-molecular weight metabolites in animals, and gas chromatography mass spectrometry (GC-MS) has also been used 72 in metabolomics studies to provide a global understanding of the pathophysiological changes brought about by the 73 environment (Zhao et al. 2015). For example, Tian et al. (2016) used metabolomics to study the heat stress candidate 74 biomarkers in milk serum. We hypothesized that the damage degree via THI before and after calving and serum 75 metabolites associated with the heat stress on primiparous and multiparous cows would be different. Therefore, this 76 study investigated the effect of THI on the milk yield, oxidative status, and serum metabolites in primiparous and 2 multiparous cows during early lactation. Our findings may provide guidance for the management of heat stress in dairy 78 farms and draw people's more attention to the health of dairy cows.

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This study was performed from June to the following February (next year) at a commercial dairy farm in Shandong 82 Province, P.R. China. Primiparous Holstein cows (n=100) and multiparous (parity: 2.5±0.5) Holstein cows (n=100) 83 were selected according to their calving days in June, July, August, September and October, respectively. All the cows 84 suffered different THI before and after calving. All experimental cows were fed the total mixed ration (TMR) ad libitum 85 as described previously (Zhao et al. 2019). Air temperature and humidity at a height of 1.5 m above the ground at both 86 ends and in the middle of the cowshed were recorded three times per day. Average daily temperature humidity index 87 (THI) was calculated every day (NRC, 1971). The cows were managed according to the guidelines for the care and use   The maximum THI, minimum THI, and average THI by month during the experiment are shown in Fig. 1. Briefly, 122 the average THI decreased from July to January. Average THI values in July and August were higher than that in other 123 months (P < 0.05). During the calving time (from June to October), the THI in October was lower than in other months 124 (P < 0.05). The maximum THI values in June, July, and August were 74.78, 75.65, and 75.17, respectively. All of these 125 maximum THI values were higher than 72, which are in the range of heat stress for dairy cows (Armstrong 1994) and 126 recent data indicates that a THI of approximately 68 affected the milk yield of high-yielding dairy cows (Zimbelman et

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The effect of different calculated THI before and after calving on the cows as shown in Table 1. The cows that 133 calved in June and July suffered high calculated THI after calving. The average daily milk yield (from 1-100 days) and 134 average daily milk yield after peak milk to 100 days of the primiparous cows that calved in June and July were lower 135 5 than those calved in September and October (P < 0.05). No significant difference was found among multiparous cows 136 that calved in different months (P > 0.05). And average daily milk yield after peak milk to 100 days of multiparous 137 cows that calved in June were lower than those calved in the other months (P < 0.05). In the same calving month, the 138 average daily milk yield of multiparous cows over 100 days and average daily milk yield after peak milk to 100 days 139 were higher than that of primiparous cows (P < 0.05). When compared with the cows that calved in June, the average 140 daily milk yield of primiparous cows that calved in September and October increased by 19.15% and 13.83%, 141 respectively. However, the average daily milk yield of multiparous cows increased by 2.86% and 7.64%, respectively.

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Milk yield negatively correlated with THI in both primiparous cows and multiparous cows (P < 0.05).

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Different lowercase letters (a,b) means significant difference (P < 0.05) in the same row.

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Different capital letters (A, B) means average daily milk yield and average daily milk yield after peak milk to 100 days is significantly different (P < 0.05) in the same column. 148 ** Means correlation analysis between average THI and average daily milk yield is significantly different (P < 0.01).

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The lactation curve of primiparous and multiparous cows from days 1 to 100 is shown in Fig. 2

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Oxidative and antioxidative index

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Our results showed that all of the serum oxidative indexes showed different variation trends during the early 162 lactation period, however, the overall concentration of every index in primiparous cows was higher than that in 163 multiparous cows in the same month (Fig. 3). On days 50 and 100, all the indexes were relatively low compared to 164 other times. Although no significant difference in MDA, SOD, and T-AOC in serum was found among the groups in 165 primiparous dairy cows on the calving day (P > 0.05), but GSH-Px of primiparous cows that calved in August, 166 September, and October was significantly higher than that in June and July (P < 0.05). No significant differences were 167 found in the above serum oxidative indexes were found in multiparous dairy cows on calving day. On day 21 after 168 calving, SOD and GSH-PX values in cows that calved in September and October were significantly higher than in those 169 that calved in June and July (P < 0.05). On day 50 after calving, SOD in primiparous cows that calved in October was 8 highest and GSH-PX and T-AOC were lowest when compared with other groups (P < 0.05). GSH-PX in multiparous 171 cows that calved in September and October was significantly higher than in those that calved in June and July (P < 172 0.05). On 80 and 100 days after calving, MDA in primiparous cows that calved in June and July was significantly 173 higher than that in cows that calved in October, and T-AOC in primiparous cows that calved in October was highest on 174 day 80 (P < 0.05). SOD in multiparous cows that calved in September and October was significantly higher than that in 175 those that calved in June on day 80 (P < 0.05). On day 100 after calving, GSH-PX in primiparous cows that calved in 176 September and October was significantly higher than that in those that calved in June and July (P < 0.05). Positive

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Pearson correlation coefficients were observed among the serum oxidative indexes (MDA, SOD, GSH-PX and T-AOC) 178 in primiparous cows and multiparous cows (P < 0.01) whereas no significant difference was found between THI and 179 serum oxidative index (Fig. 4).    with the results of current study. In the present study, when compared with the cows that calved in June, the average 236 daily milk yield of primiparous cows that calved in September and October increased by 19.15% and 13.83%, 237 respectively. However, the average daily milk yield of multiparous cows increased by 2.86% and 7.64%, respectively.

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These results indicate that the effect of THI on milk yield may be cumulative and delayed, and the multiparous cows are 239 more susceptible to higher THI, which supported the hypothesis that the damage degree of calculated THI on dairy 240 cows was more severe after calving than that before calving, and the effect of THI on multiparous cows was more 241 severe than multiparous cows. In agreement with the present study by Bernabucci et al. (2014), milk yield started to 242 decline when THI ranged from 73 to 76, and multiparous cows were more susceptible to heat stress than primiparous were found in multiparous cows, this could be due to the multiparous cows were more sensitive to heat stress than 275 primiparous cows and the synthesis of antioxidant enzymes was restrained, which could explain that SOD, T-AOC and 276 GSH-Px was higher in primiparous cows than that in multiparous cows. 9,12-Octadecadienoic acid is one isomer of 277 conjugated linoleic acid (CLA) have antioxidant properties (Yurawecz et al. 1995). And the present study showed 278 9,12-Octadecadienoic acid in the serum of NHS group was higher when compared with HS group in primiparous cows.

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This study demonstrated that heat stress negatively affected the milk yield and thus caused the elevation of the 282 serum oxidative and antioxidative index. Metabolic biomarkers associated with the heat stress in serum were found 283 which providing the basis of evaluating indicator between heat stress and non heat stress groups in primiparous and 284 multiparous cows. Moreover, we found that the cows were more susceptible to the heat stress after calving than that 285 before calving, and the multiparous cows were more sensitive to heat stress than the primiparous cows, and discover the 286 candidate biomarkers in serum.

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The study was supported by the National Natural Science Foundation of China (31902185) and the Preferential