Expression Patterns of Major Genes in Fatty Acid Synthesis, Inammation and Oxidative Stress Related Pathways and Quality Parameters During the Transition From Colostrum to Milk in Dairy Goats

Colostrum is quietly different from mature milk. The molecular regulation of milk secretion and quality in the transition period from colostrum to milk in goats is largely unknown. The present study, mammary gland secretion of goats was collected in 0 th , 4 th , 7 th , 14 th and 28 th days after parturition. In addition to composition and fatty acid prole of colostrum or milk, FASN, SCD, ACACA, COX-2, NRF2, TLR2, NF-kB, LTF and PTX3 genes expression patterns were determined from milk somatic cells. While somatic cell count (SCC), malondialdehyde (MDA), fat, fat-free dry matter (FFDM), protein and lactose were highest as expression levels of the oxidative and inammatory genes (P<0.05), freezing point and electrical conductivity were lowest in colostrum. With the continuation of lactation, most of the fatty acids, n3 ratio, and odour index had increased, on the other hand, C14:0 and C16:0 had decreased. In addition, FASN was upregulated almost 3 folds in 14 th day (P<0.05). While SCD was similar, ACACA was upregulated more than 5 folds in 7 th and 14 th days (P<0.05). Furthermore, signicant correlations were determined between studied genes. Although colostrum is important for offspring health in terms of inammation and oxidative stress related pathways, further studies are needed on complex molecular pathways including fatty acid synthesis on goat mammary gland.


Introduction
The world human population is expected to be around 10 billion by 2050. Animal production targets are rising in response to the increasing world population. Goat is an important farm animal for the production of dairy products, as well as for the supply of healthy, high-quality and valuable food, especially for the nutrition of children, the elderly and those with food allergies 1,2 . With the increasing demand for goat milk in recent years, goat breeding has gained importance and the amount of milk production has increased with the number of goats 3,4 .
Milk, an important animal product, is a vital biological secretion that begins with the birth. First secretion of mammary tissue, colostrum, has quietly different from mature milk. Besides its immunological function, colostrum is pivotal for the initial intake of nutrients in terms of the survival and sustainable health of the newborn 5

. Regulation of mammary secretion is controlled by local and systemic factors.
Therefore, colostrum and milk components are regulated by different mechanisms 5 .
Goat colostrum is richer in somatic cells, fat, protein, FFDM, lactose and immunoglobulins 5,6 . The considered changes occur in composition and properties during the transition from colostrum to mature milk. It has been reported that goat colostrum turns into milk in 4-5 days after birth goats 6,7 . On the other hand, transition to mature milk secretion is a process rather than straight conversion.
As is well known, milk traits are affected by genetic and environmental factors such as breed type, age, and parity. In addition, unlike other farm animals the milk secretion type of goats is apocrine 1 . Several studies have deepened to improve the molecular basis of milk secretion in cattle, however what is known about the molecular basis in the regulation of milk secretion and quality particularly in the transition period from colostrum to milk in goats is limited 8,9 .
The aims of this study were as follows: 1. Determination of the changes in macromolecules (fat, protein, lactose etc.) and some milk parameters (pH, electrical conductivity, freezing point, FFDM etc.) of mammary secretion from parturition to 28th day in goats.
2. Investigation of fatty acid pro le of the mammary secretion during colostrum to mature milk in Damascus goats.
3. Expression analysis of the genes related with fatty acid pro le, in ammation and oxidative stress related pathways in milk somatic cells of goats. 4. Reveal the correlations between the mentioned genes in milk somatic cells of goats.

Results
The properties of colostrum or milk obtained have presented in Figure 1. The SCC was around 5 million on the 0 th day, then it decreased dramatically on the 4 th day and was around 1 million by the 7 th day.
Following the lactation, the SCC was around 400 x 10 3 /mL in milk (P<0.01). While pH value was increased day by day, the levels of fat (%), FFDM (%), protein (%), and lactose (%) were decreased with the varying levels of signi cance (P<0.001). On the other hand, the freezing point and electrical conductivity of the samples increased on the 4 th day and remained at similar levels on the following days (P<0.001).
The level of MDA was highest with the 34.41±2.43 nmol/mL values in the 0 th day (P<0.001). However, it was 3 times lower on the following days and there were no signi cant differences between these days.
The marked downregulation was determined at oxidative stress and in ammation related genes. COX-2 was downregulated more than 2 folds in 4 th and 7 th days (P<0.05). On the other hand, NRF2 and TLR2 were approximately 3 folds downregulated in all days with the different signi cances (P<0.05 and P<0.01). Therewithal, NF-kB was downregulated on all days, while PTX3 was downregulated close to 4 folds in 14 th and 28 th days (P<0.05) ( Figure 3).

Discussion
Somatic cell count is one of the most important parameter that gives an idea about mammary health and milk quality in ruminants. On the other hand, SCC of milk may be high even if the animal is healthy because of the secretion type of milk in goats 1 . Although high SCC in milk is not desired in general, Rainard et al. (2018) 10 has stated that low SCC might also be a risk as it may increase susceptibility to mastitis. Colostrum, which is the rst secretion of the mammary gland, contains quite high SCC compared to mature milk as well as its difference in composition 5,11 .
Similar to the study conducted in goats by Moreno-Indias et al. (2012) 5 , SCC, which was initially about 5000 × 10 3 /mL in colostrum, tended to gradually decrease during the transition to milk in this study.
Sanches-Macias et al. (2014) 6 reported that from the 0th day to the 4th day, SCC had decreased from approximately 9000 × 10 3 /mL to 3000 × 10 3 /mL levels, and after the 15th day it had remained below 1000 × 10 3 /mL as this studies results. High SCC in colostrum was thought to be physiological and the possible cause, as reported in other studies, might be due to the leaky tight junctions between mammary epithelial cells 6,12 . In addition, breed type of goat was thought to be an important factor for differences in terms of SCC during transition colostrum to mature milk. While pH of samples was lower on 0th day, it had gradually increased during transition similar to other studies [13][14][15] . It was reported that high pH could prevent bacterial contamination 6 . It was possible to evaluate this situation as one of the physiological self-protection mechanisms of the mammary gland during the transition period.
In milk transition period, milk composition and properties change in ruminants 7,16 . While higher fat percentages have continued until 7th day, lactose has found highest on 0th day dramatically decreased on 4th day and stayed stable. The possible reason why the initial sample lactose content is higher that lactose is a source of energy that may be digested faster than fat and required for newborn 5 . While most of the milk quality parameter has dramatically decreased during transition, freezing point and electrical conductivity has increased since 4th day of lactation. Expectative reason of increases on these parameters are decreased percentages of parameters such as fat, protein, FFDM of secretion in addition to other compounds as minerals. Also, it has been reported that genetic background of breeds affected the composition of colostrum and milk in goats 7 .
MDA is primary biomarker for determination the lipid peroxidation and oxidative stress in tissues. Moreover, it has been reported that lactation period strongly related with milk MDA levels and it has higher values in early lactation stage 17 . While signi cantly higher values have found in colostrum, MDA has dramatically decreased with the continuation of lactation. In a study, it has been reported that composition and yield of milk is signi cantly correlated with MDA levels in cow milk 17 . According to correlation results in our study, high SCC has also associated with increased MDA levels in mammary secretion. Although milk MDA levels are understandably dependent on milk composition, more exploration at the molecular levels is needed to elucidate the mechanism of MDA in colostrum and milk.
There is a limited number of study about fatty acid pro le of colostrum and transition milk in goats 18,19 .
It is essential to explore the milk fatty acid pro le at different lactation stages to achieve the optimal bene ts from milk. Goat milk is richer from short-chain fatty acids. It has been determined that shortchain fatty acids responsible for the odour index in milk has tended to increase during the transition to mature milk. Beside short-chain fatty acids, medium-chain fatty acids have also increased during transition. It has reported that medium-chain fatty acids (C8:0, C10:0 and C12:0) in milk have antimicrobial effects 20 . Goat kids are susceptible to infections and therefore medium-chain fatty acids in secretion may have increased for the protective effects on young goats 18 . While short and medium-chain fatty acids and some of the long-chain fatty acids have gradually increased in the transition from colostrum to milk, some of the long-chain fatty acids have stayed stable from 0th to 28th day. It has thought that the likely cause of these results is unchanged ration ingredients of animals. Just because of it is known that composition of ration has major effect on the fatty acid pro le of goat milk 21 .
To date, effects of many genes related to fatty acid synthesis have been investigated in mammals. Milk fatty acids have been used as biomarkers in the diagnosis of feed e ciency, lipid mobilization, and some of the speci c disorders 22 . PUFA, odour index, n6, and n3 have increased with the continuation of lactation, while n6/n3 ratio has decreased. Hence, milk has become better quality with the transition period 23 . Compared to early lactation, percentage of PUFA has increased in 28th day. It has reported to decrease towards the end of the lactation in different goat breeds 24 . However, it has increased in the transition processes from colostrum to mature milk in this study. Although it has known that nutrition has important effects on milk fatty acid pro le, studies show that the lactation period is another major point on this parameter 25,26 . While there have been controversial reports about fatty acid pro le of milk in different lactation stages 24,27 , it has been showed up fatty acid pro le of goat milk is one of the most variable components among the lactation period, together with milk yield and composition 26 .
The molecular regulation of lipid metabolism remains largely unknown in ruminants. Fatty acid biosynthesis is an intriguingly complex biological process and FASN, SCD, and ACACA are largely effective genes in lipid biosynthesis. FASN, one of the highly expressed gene in goat colostrum, has been reported required for the maintenance of lactation 25,28 . Therewithal, FASN has mostly responsible to synthesis of short and medium-chain fatty acids together with ACACA 25 . It has been reported that inhibition of FASN led to reduce of medium-chain fatty acids and downregulation of ACACA in mammary gland of goat 29 . In addition, FASN in goat mammary gland might synthesize three fatty acid forms (short, medium and long-chain fatty acids) 29 . Herewith in this study, the expression level of FASN and ACACA has tended to uctuated, although they have increased during the transition period. On the other hand, SCD has showed similar expression levels. SCD is major regulator of MUFA synthesis in the mammary gland and MUFA levels has also stayed stable in all sampled days 30 . It has been reported that FASN, ACACA, and SCD had regulated together from pregnancy to end of the lactation in bovine mammary gland 31 . The positive correlations found between these genes have shown that they interact in the formation of the fatty acid pro le of goat milk. It has been reported in recent studies, FASN, SCD and ACACA genes might be candidate genes for quali ed animal product from the point of fatty acid pro le 30,32−34 . While some researches have studied on expression patterns of FASN, SCD and ACACA genes in goats 28,30,34 , to our knowledge there is no report about the activities of these genes in colostrum and transitional milk somatic cells of goats. It has clearly understood that despite the differences in upregulation levels, regulation of fatty acid synthesis in goat colostrum and early milk requires coordinately expression of mentioned genes.
With the beginning of lactation, antioxidant and immunological mechanisms have showed abnormal regulation in mammary gland. Antioxidant status of colostrum is important for maternal physiology and offspring health 35 . COX-2 and NRF2 are the most related genes with the oxidative status of tissues and biological liquids and have tended to be upregulation in response to immunological activity 36,37 . It has been reported that NRF2 increases by triggering TLR2 in leukocytes and oxidative stress may decrease by overexpressing TLR2 in goats 38 . In transition period, both COX-2 and NRF2 genes have showed signi cant downregulation with the similar expression patterns of TLR2 and NF-kB in milk somatic cells.
In in ammation status, oxidative stress initiates the in ammatory response by activating NF-kB and increases the expression levels of target genes such as COX-2. It has been reported that response to oxidative stress is formed by cross-interaction between NRF2 and NF-kB 39 . Moreover, PTX3 gene has also shown downregulation with continuation of lactation. Hence, expected correlations have obtained between mentioned genes expression levels in goat milk somatic cells.
Together with the rapid decrease number of somatic cells, PTX3 downregulated more than 3 folds on the 14th and 28th days. PTX3, which expression level has decreased with the transition from colostrum to mature milk, is thought to be regulated for both kid and maternal health. PTX3 has known to be a gene regulated by the molecular mechanisms of innate resistance to respond to infectious agents 40,41 . There has been limited knowledge about the activity and expression patterns of PTX3 in healthy goats, while one of the most expression of PTX3 has been reported is mammary gland 42 . It has been stated that PTX3 gene might be upregulated by in ammation in mammary tissue and its activity might be used an early marker of mastitis 42,43 . It has advocated that whether there is a correlation between PTX3 and SCC should be investigated in goat milk 42 . The results obtained from our study show that in addition to relations with the oxidative and in ammatory genes such as TLR2 and NF-kB, there is crucial correlation between PTX3 and SCC in early lactation of goats.
LTF has showed similar expression patterns during the transition. While negative correlation has been found between LTF and SCC as expected, it has positively correlated with FASN, SCD, and ACACA. In addition to the antimicrobial activities of medium-length fatty acids synthesized by the activities of FASN, SCD and ACACA, LTF has also antimicrobial activity in mammary gland 44 . However, what is known about the relation between fatty acid synthesis and LTF activity is quiet limited. Therefore, more research is needed on direct and indirect relations between LTF and fatty acid biosynthesis in mammary gland.
Indicator of oxidative stress and cytokines have known transferred from mother with colostrum and milk, may affecting the health of offspring 45 . On a study it has reported that it is appropriate to weaning of lambs at 28 days 46 . In addition to health of kids and goats, transition and mature milk should be investigated at the molecular levels for milk quality parameters.
In conclusion, variable interactions between genes have showed that the regulation of goat milk are intriguingly reorganized in transition process. To our knowledge this is the rst study to deeply explore the changes at the molecular levels of mammary secretion of goats from birth to 28th day of lactation. In addition, the additive effects of studied genes have signi cant roles on properties of colostrum, transition and mature milk in goats. To elucidate the underlying molecular mechanism of the goat mammary secretion, more comprehensive study is needed in this eld.  (Table 2).

Methods
Following parturitions, approximately 150 mL colostrum or morning milk samples were collected to nuclease free falcon tubes on the 0 th , 4 th , 7 th , 14 th , and 28 th days. Prior to sampling, udders and teats of goats were cleaned with sterile cotton gauzes. and each sample was studied as duplicated. The reaction was arranged 10 min at 95 °C, followed by 15 sec at 95 °C, 60 sec at 60 °C, and 40 cycles in RT-qPCR. On the other hand, ACTB and G6PD reference genes were used as internal control. Forward and reverse sequences of primers were shown in Table 3.  Descriptive statistics for each variable were calculated and presented as "Mean ± Standard Error of Mean". The Pearson correlation coe cient was used to determine the correlation between gene expression levels, somatic cell count and MDA levels. To determine the effect of time of sampling on milk quality and milk fatty acid parameters, linear mixed model was used. The following model with repeated measures design: Y ijk = µ + T j + e ijk . Where, Y ijk , dependent variable; µ, overall mean; T j , effect of time of sampling (j = Day of parturition, after parturition 4, 7, 14 and 28 d) and e ijk , residual error. Animals were assessed as a random effect, while the time of sampling was assessed as xed effect. When a signi cant difference was revealed, any signi cant terms was compared by simple effect analysis with Bonferroni adjustment. P<0.05 was considered as signi cant in all analyses. All data were analyzed using IBM SPSS Statistics software (Version 23.0) 51 .
Expression levels of genes were calculated by the 2 -ΔΔCt method and geometric mean of reference genes Ct values was used for gene expression analyzes 52 . The results were determined with comparing to 0 th day and presented as fold changes.