Effects of High Ambient Temperature on Milk Protein Synthesis in Dairy Cows and Goats: Insights from the Molecular Mechanism Studies

Abstract

Milk protein is well accepted for nutritional value compared with other sources of protein. Detailed understanding of the natural factors that can determine milk protein subcomponent (i.e., casein) not only fulfill the knowledge of protein synthesis but also provide the potential idea to improve milk quality. The variation in milk protein content from dairy cows and goats fed in tropical areas may determine the added value of milk from this region. Under prolonged high ambient temperature (HTa), dairy cows and goats are at the stage of heat stress. This physiological condition produces a negative effect on dairy cows and goats, i.e., food intake and milk yield. However, the higher milk protein content during summer is demonstrated in dairy goats in our condition. Likewise, an increase in heat shock protein 70 (Hsp70) gene expression from mammary epithelium cells isolated from either in vivo (summer and winter periods) and in vitro conditions suggests the direct effect of HTa on mammary gland and perhaps on milk protein synthesis. The intracellular effect of Hsp70 on milk protein synthesis has been proposed in regard to the endoplasmic reticulum and Golgi apparatus protein transportation and with the subcomponent of casein micelle. The present information reveals the molecular mechanism of HTa on milk protein synthesis.

1. Introduction

High ambient temperature (HTa) is the natural environmental condition in the tropical area. Dairy animals fed in tropical countries are living under prolonged HTa conditions. A decrease in the lactation performance in dairy animals is one of the well-known effects of HTa [1, 2, 3, 4]. In dairy cows, we have shown that the average daily milk yield (MY) from summer cows was 17% lower than from winter cows [4, 5]. The effect of HTa on MY was also consistent in dairy goats [6]. Although, a decrease in MY is the prominent negative effect of HTa, however, change in major milk composition from dairy animals during HTa exposure is not conclusive. The current chapter aims at showing the evidence that HTa has the potential to change milk protein in dairy goats fed under tropical areas. We first demonstrate the natural ambient condition. The effect of HTa on lactation performance and the mechanism has been informed. In addition to MY, the evidence of HTa effect on milk protein and the putative molecular mechanism of this phenomenon has also been proposed.

2. The current condition of high ambient temperature in Southeast Asia

The tropical countries are the area that delimited between the tropic of Cancer in the north (23.43° S) and the tropic of Capricon in the south (23.43° S). Based on the seasonality of monthly air temperature and precipitation, the climatic classification of the mainland Southeast Asia countries including Thailand, Laos, Myanmar, Cambodia and Vietnam are mainly the tropical savannah (Aw). In addition, the climatic classification of the maritime Southeast Asia countries including Malaysia, Indonesia, Brunei and Philippine is the tropical monsoon (Am). Due to the global warming effect, the temperature and humidity index (THI) which has been reported currently is approximately 10 degrees higher than that has been reported 30 years ago [7]. The current annual THI in the central of Thailand was approximately 85 [4]. The high value of THI in Thailand currently comes mainly from the high degree of ambient temperature (Ta) throughout the three main seasons. Interestingly, the difference in Ta between the highest level during the afternoon and the lowest level during the early morning is more than 10°C (Figure 1). This Ta difference (Ta-diff) is mainly the environmental condition influencing the lactation performance and perhaps the direct effect of temperature on mammary gland function [6].

Figure 1.

The pattern of ambient temperature in the central area of Thailand represent the typical climatic condition of the tropical area at the present time.

3. The HTa effect on physiological responses and milk yield

The effect of HTa on whole-body responses and MY should be considered before discussing the HTa effect on milk protein synthesis. Dairy cows and goats fed under HTa conditions in the tropical area have 15–17% lower MY during summer than during winter [4, 5, 6, 8]. Both direct and indirect effect of HTa on lactation performance has been purposed.

The direct effect of HTa on mammary gland function has been demonstrated using both in vitro and in vivo systems. Short-term low degree HTa exposure (37 and 39°C, 1 h) could activate the expression of the heat shock protein 70 (Hsp70) gene in the primary mammary epithelial cell (MEC) culture. This condition, however, could not activate beta 1,4-galactosyltransferase1 (β-GALT1), alpha lactalbumin (α-LA) and phosphokinase B (or Akt) genes [9]. However, it has been shown that a higher degree of HTa exposure has been shown to decrease Akt phosphorylation [10]. In addition, Akt knockdown decreased β-GALT1 and lactose synthesis [11]. The information suggested that the direct effect of HTa on milk synthesis may in part be related to Hsp70 and the role of Akt/β-GALT1 under the natural HTa condition is unclear. This conclusion is supported by the study of the seasonal effect on gene expression from MEC isolated from fresh goat milk. The degree of Hsp 70 gene expression, but not Akt and β-GALT1 genes, from MEC isolated from summer goat milk, was significantly higher than that from winter goat milk. In this investigation, milk yield from the summer period was significantly lower than the winter period [6]. The effect of HTa on Hsp70 and MY from both in vitro and in vivio is in line with the lower in ratio of β-GALT1 and Hsp70 expression (Figure 2). With several mechanisms of Hsp70 on intracellular functions, the role of Hsp70 on the milk synthesis pathway that could influence MY remains to be investigated and the role of Hsp70 on milk protein synthesis will be purposed as well in this chapter.

Figure 2.

The ratio of β-GALT1 and Hsp70 gene expression from both in vitro and in vivio system. Under the in vivo or natural condition, the MEC from winter period (Ta = 30°C at 1300) represent the CTa condition and from summer period (Ta = 37°C at 1300) represent the HTa condition. Under the in vitro condition, the mammary epithelium cell (MEC) was treated 1 hour under control Ta (CTa) and high Ta (HTa) were 37 and 39°C, respectively. * the significant effect of HTa on the ratio of β-GALT1 and Hsp70 gene expression was detected under natural conditions.

The indirect effect of HTa on MY mediates by the effect of HTa on decreased food intake (FI) and nutrient partition to the mammary gland [6, 12, 13, 14]. Dairy goats in the summer months had significantly lower FI and MY than that in winter months. Because the concentration of plasma cortisol from summer months was not different from winter months [6], whether these effects of HTa are part of the chronic heat stress mechanism is not conclusive. When considering the effect of HTa on FI in laboratory rats, the low degree of HTa exposure that decreased FI earlier than the activated hypothalamic-pituitary axis implies that HTa could decrease FI without stress [15, 16]. The information of behavioral and physiological responses to daily fluctuation of HTa is crucial knowledge regarding this phenomenon.

4. Stress responses under HTa during daytime

Behavioral and physiological responses of HTa during daytime is a piece of crucial information to support the hypothesis that dairy goats and cows fed under natural ambient conditions are at the stage of heat stress. Early phase responses of HTa are all behavioral outcomes without the activation of the hypothalamic-pituitary axis (HPA axis) including seeking shade, inactivity and decrease in food intake, etc. Mild degree heat stress is the second phase is characterized by the physiological responses and the activation of HPA axis. This level of heat stress is reversible and not harmful. Heat dissipation mechanism including sweating or panting is the major physiological response during this phase. The third phase of heat stress is a severe irrevisible level or heat stroke. We have shown previously that there is around a 10°C difference in Ta from early morning to the afternoon (Figure 1). The significant increases in both respiratory rate (RR) and rectal temperature (Tr) could be detected in dairy goats fed under this ambient condition (Figure 3). Similar patterns of these responses have been demonstrated in dairy cow fed under natural HTa conditions [17]. Moreover, the plasma concentration of cortisol from the afternoon was significantly higher than that from the early morning (Figure 4). This information suggests that in dairy goats when the difference of Ta during morning and afternoon is around 10°C, both evaporative heat dissipation and the HPA axis were activated. Although the dairy goat had significant heat dissipation via panting, the core temperature (via Tr) was set to around 1°C above normal level in the morning. It should be noted at this point that the behavioral and physiological responses of HTa in dairy goats are comparable to those we have investigated in the laboratory rat. Short-term mild degree of HTa exposure in rats that could not activate physiological responses (e.g., Tr and pack cell volume) failed to activate the paraventricular nucleus (PVN) of the hypothalamus [15]. It is well known that PVN is the most upper hypothalamic nuclei of the stress axis (or HPA axis). Taken together, we conclude that during daytime dairy goat fed under HTa of the tropical area is at the second phase of heat stress.

Figure 3.

The effect of high ambient temperature (HTa) on behavioral and physiological response in dairy goats during daytime from 0700 h to 1300 h. In the morning (0700 h), the respiratory rate (RR, upper) as behavioral response and the rectal temperature (Tr, lower) as the physiological response is at normal value. In the afternoon (1300 h), both RR and Tr increase significantly to 127 breaths per min and 39.64°C, respectively.

Figure 4.

The effect of high ambient temperature (HTa) on hormonal response in dairy goat during daytime from 0700 h to 1300 h of summer and winter period. * the significant effect of time.

Although the THI from winter was lower than that from summer in Thailand, both winter and summer THIs during the afternoon were higher than the value of 80 [4, 6]. It is possible to think that in Thailand dairy goat from both winter and summer times is at the state of heat stress and that the concentration of plasma cortisol per se could not be used as the separation index at this stress level. Finally, from the meteorological and behavioral viewpoints, dairy goat during summer period confronted with higher degree of heat stress than during winter period.

5. The effect of HTa on milk protein synthesis

During the summer period, both dairy cows and goats decreased in lactation performance. An evidence that dairy goat fed under the tropical area of Thailand during the summer period has a higher degree of heat stress than the winter period drives one interesting hypothesis. This hypothesis is whether the major compositions of milk from the summer period is different from that of the winter period. The analysis of major goat milk compositions revealed that the concentration of milk protein, but not milk lactose and fat, from the summer period was higher than that of the winter period (Figure 5). It should be noted that the present effect of HTa on milk protein is in contrast with previous reports [18, 19, 20]. The possible explanation for this discrepancy is perhaps the degree and duration of HTa exposure that is typical high throughout the year in the current condition of tropical area. Furthermore, the effect of HTa on milk protein synthesis seems to be specific because HTa did not affect the concentration of lactose both in vivo and in vitro studies. Likewise, HTa failed to change the expression of both beta-galactosyltransferase and alpha-lactalbumin which are the protein component of lactose synthase condition [6, 9]. An evidence from Prasanpanich et al. [21] could support the fact that higher milk protein content from heat-stressed cows under tropical conditions. The value of protein contents from grazed cows under heat stress conditions and indoor cows were 3.2 and 2.9%, respectively.

Figure 5.

The effect of high ambient temperature (HTa) on goat milk composition between winter and summer period. * the significant effect of season.

Because casein is the major milk protein, this section will focus on the effect of HTa and the casein synthetic pathway that may be the major cause of this phenomenon. With an evidence that HTa could activate Hsp70 expression from our current experiment [9], increase casein synthesis may be supported by the action of Hsp70 (Figure 6). Among a wide range of Hsp70 functions and subtypes [22, 23], Hsp70-5 or glucose-regulated protein 78 (GRP78) which locate at the endoplasmic reticulum (ER) and regulate ER chaperone and transportation has been studied with milk protein synthesis. Overexpression of GRP78 in bovine mammary epithelial cells increased milk protein synthesis [24]. In addition, the role of the Mammalian target of rapamycin (mTOR) as the posttranscriptional regulation has been revealed regarding to milk protein synthesis [25]. Interestingly, mTOR has been shown in HeLa cells that could stimulate Hsp70 synthesis via heat shock transcription factor 1 (HSF1) [26]. The effect of HTa that increase milk protein may be related to the mTOR/HSF1/Hsp70 pathway that regulate the posttranslational process of casein. The casein subtype is another regulatory mechanism controlling the posttranslational casein synthesis pathway. Basically, casein is the milk protein complex known as casein micelle that is composed of 4 major subtypes; αS1-casein, αS2-casien, β-casein and κ-casein. Before the casein incorporation process that takes place at the Golgi apparatus, it is important that all casein subtype need to synthesize and transported from ER to the Golgi apparatus via the ER-Golgi transport route. It has been demonstrated in αS1-casein deficient goat that αS1-casein is required for the efficient transport of β-casein and κ-casein [27]. Furthermore, the membrane-associated form of αS1-casein at ER plays a key role during the early steps of casein transport. Whenever αS1-casein has been down-regulation, the transport rate of other caseins to Golgi apparatus is highly decreased [28]. Taken together, it is interesting at this point that HTa could activate mTOR/HSF1/Hsp70 pathway and subsequently influence ER-Golgi transport of the casein subtype.

Figure 6.

The diagram demonstrates a putative mechanism that high ambient temperature (HTa) increases casein synthesis. Under prolonged HTa conditions, heat shock protein 70 (Hsp70) is increased. The upstream pathway that activates Hsp70 may be related to the mammalian target of rapamycin (mTOR). An increase in Hsp70 chaperone of ER-Golgi transport of casein subtype is the putative target that enhances casein production. The transportation of casein by the ER-Golgi route requires coat protein complex (COP) machinery; COPII and COPI proteins which initiate at the ER exit site (ERES). The vesicular tubular cluster (VTC) is the final step that casein will be transported to Golgi.

6. Conclusion

In this chapter, we demonstrate that dairy goat and cow fed under tropical area were at the state of heat stress. In addition to the effect of HTa on the reduction in MY, we show the evidence that long-term HTa exposure apparently increased milk protein. The physiological mechanism that HTa could influence milk protein synthesis has been proposed in particular with the casein synthesis pathway. Specifically, long-term HTa exposure activates mTOR/HSF1/Hsp70 pathway and subsequently increases the posttranslation process of casein synthesis via ER-Golgi casein transportation.