Supplementation of palmitoleic acid improved piglet growth and reduced body temperature drop upon a cold exposure

Background Survival of piglets poses a signi�cant challenge in the initial days after birth because piglets are lacking readily oxidizable brown adipose tissue and born with limited amount of body reserves, which in turn limited theirthermogenic capacity. This study investigated the effects of palmitoleic acid (PA) supplementation on growth performance, maintenance of body temperature, muscle fatty acid (FA) compositions, and energy metabolism in milk replacer fed piglets. Forty-eight piglets were strati�ed by body weight and randomly assigned to one of four dietary treatments (0%, 1%, 2%, and 3% PA supplementation as percent of milk replacer). Piglets were weighed daily, and half in each dietary treatment groups were exposed daily to low temperature for 2 h. Plasma and tissue samples were collected at the end of the experiment for further analyses. Results Contents of C16:1n-7 and C18:1n-7 in both plasma and liver (P < 0.001), and C16:1n-7 (P < 0.001) in semimembranosus increased linearly as PA levels increased. Most plasma FA levels (except C16:1n-7, C16:1n-9 and C22:5n-3) were lower in piglets exposed to low temperature than those that were not. Plasma glucose, triglycerides and lactate dehydrogenase levels increased linearly with PA supplementation (P< 0.001). Piglets’ average daily gain, liver weight, liver glycogen pools, and gallbladder increased linearly with PA supplementation (P < 0.05, P < 0.01, P < 0.05, and P < 0.001, respectively), but lung weight, liver nitrogen content, and body temperature drop at cold exposure decreased linearly with PA supplementation (P < 0.01, P < 0.001, and P < 0.05, respectively). Piglets exposed to low temperature had greater liver nitrogen (P < 0.05) and lactate dehydrogenase (P < 0.001) contents, but had lower liver weight (P < 0.01) and plasma lactate concentration (P< 0.05) than those that were not.


Introduction
Certain fatty acids (FA) are known to be able to modulate energy metabolism in the body and especially palmitoleic acid (PA; C16:1n-7) has attracted attention due to its ability in in vitro culture experiments with bovine adipocytes to downregulate de novo lipogenesis and upregulate FA oxidation, so FA are directed toward energy expenditure and away from storage [1].Additionally, PA supplementation increased adipocyte lipolysis and the content of major lipases in a mice study [2].The survival of piglets during the rst days of life is a major challenge as they are born with very limited amounts of energy reserves in the body [3,4] and lack brown adipose tissue unlike most other mammals [5].Moreover, piglets are born into a colder environment than the intrauterine environment, which greatly challenges their thermogenic capacity; thus, increases the risk of death due to hypothermia and starvation.Sow milk, in contrast to most other mammals like ruminants, humans, rodents etc. has a high content of C16:1n-7 (8-11g/100 FA) [6] compared to 1-2 g/100 FA in cow and human milk [7].Plant-based fats commonly used in sow diets and sow milk replacer are not rich sources of PA.Thus, sows synthesize PA from palmitic acid by the action of Δ9 desaturase [8] and accumulate it into the milk [6].This implies that PA may play a critical role in the survival and growth of the piglets, thus warrants an investigation.Palmitoleic acid supplementation may affect the energy metabolism of newborn piglets and improve their survival and growth during the critical period of life.
Our hypothesis was that PA supplementation can maintain the body temperature of the piglets upon cold exposure through modi ed energy metabolism and thereby improve growth.Therefore, this study investigated the effect of varying levels of PA supplementation on growth, energy metabolism, and body temperature of milk replacer fed piglets during the rst week postpartum.

Selection of piglets and intubation of orogastric tube
Piglets born from six sows (DanBred Landrace × DanBred Yorkshire) were allowed to suckle colostrum for the rst 12 h postpartum.Afterward, litters were weighed, sorted according to body weight (BW), and eight piglets from each sow, in the second-third of the weight group, were ear tagged and separated from their dam.The initial BW was used to calculate the dose of anesthesia used for sedating the piglets and for stratifying the piglets to the dietary treatments.
General anesthesia was induced with intramuscular injection of Zoletil mixture (0.125 mL/kg BW).The Zoletil mixture was prepared by dissolving one bottle of Zoletil (125 mg tiletamine and 125 mg Zolazepam; Virbac Animal Health, Kolding, Denmark) in 1.25 mL Ketamine (Ketaminol Vet, 100 mg/mL; Intervet Denmark, Skovlunde, Denmark), 6.5 mL xylazine (Rompun, 20 mg/mL, Bayer Health Care AG, Leverkusen, Germany), 2.0 mL butorphanol (Torbugesic Vet, 10 mg/mL, Scan Vet Animal Health A/S, Fredensborg, Denmark), and 2.0 mL methadone (10mg/mL).Additionally, xylocaine was injected at the site of puncture on the right side of the cheek.The puncture was made using intra on2 cannula (12G-L.80 mm, 2.7 mm internal diameter, 122.27,VYGON-5 rue Adeline 95440 Ecouen, France).Then, the orogastric tube (Enteral feeding tube, 310.06 06Fr-L.40cm-PVC,VYGON-5 rue Adeline 95440 Ecouen, France) was inserted into the stomach for feeding the experimental diets.The orogastric tube, approximately 17 to 18 cm in length from the puncture spot in the cheek to the tip of the tube, was inserted into the stomach of the piglets.The orogastric tube was sutured to the skin of the piglet at three spots to prevent it from falling off as well as to facilitate feeding.

Housing of the piglets and temperature challenge
Eight portable metabolic cages (100 × 75 cm), each containing three smaller cages (32 × 60 cm) were used to house the piglets individually from immediately after orogastric intubation until the end of the experiment.The three smaller cages within the metabolic cage were separated by a 70 cm tall transparent exiglass to comply with the Danish regulations for individually housed experimental animals.Sawdust was used as a bedding material in the small cages.The portable metabolic cages containing the piglets were kept at a room temperature of 34°C during the experiment to match with the critical body temperature of the piglets at early age [9].However, half of the piglets in each treatment group were subjected to low temperature once a day during the experimental period.
To make the handling of piglets easier during the temperature challenge, temperature challenged piglets were placed in the same metabolic cage to be moved together.The piglets were moved to a separate room set at 24°C and were challenged for 2 h each day during nighttime.However, the last temperature challenge was performed during the day and piglets were immediately sacri ced after the end of this temperature challenge.Observation was made during temperature challenge to verify if this temperature triggered shivering thermogenesis in the piglets.The rectal temperature of the piglets was measured before and at the end of temperature challenges to record a drop in body temperature when exposed to low temperature.

Diet and Feeding
Forty-eight piglets, 12 piglets per treatment, were strati ed for BW during orogastric intubation and randomly assigned to one of four dietary treatments.The piglets were fed a milk replacer formula (DanMilk Supreme, AB.NEO A/S, Videbaek, Denmark) with varying levels of supplemented PA in a dose-response study.The milk replacer was mainly based on whey protein concentrate and sweet whey powder as protein source (20% crude protein [CP]), with soya and coconut oil as fat source (17%) (Table 1).The PA was added to the milk replacer at 0%, 1%, 2%, and 3% of milk replacer to create four dietary treatments (T1, T2, T3 and T4), calculated to provide 0.0, 0.61, 1.22 and 1.84 g/d of PA, respectively.The levels were chosen based on the assumption that a suckling piglet ingests 1.08 g/d of PA from sow's milk; which contains 10% PA in the milk fat [10] and piglets ingest on average 750 g of milk in the rst week of lactation [11].Thus, if any, a potential break-point response is expected to be observed between T2 and T3, which are closest to the estimated PA intake of naturally suckling piglets.To counterbalance the energy density of the diet due to PA supplementation, palm oil was added to the milk replacer in the reverse order (3% in T1, 2% in T2, etc.).The PA and/or palm oil in the respective treatments were added to powder milk replacer and blended for 20 min using a kitchen blender to create a homogeneous mixture and stored at 4°C until feeding.
The diets in the respective treatments were dissolved in warm water (approximately 35°C) right before feeding, in a ratio of 130 g mixed diet per liter of water, according to the manufacturer's guidelines (DanMilk Supreme, AB.NEO A/S, Videbaek, Denmark).To minimize variation among piglets, the meal allowance was decided to be fed per kg BW.The daily meal allowance was 25 mL/kg BW at each feeding and was increased by 1 mL/kg BW each day of the experimental period.Feeding began when the piglets were observed standing on their feet after orogastric intubation and they were fed every 2 h afterwards until the end of the experiment.At each feeding, the daily allowance of the meal was drawn into a syringe and the syringe with its content was weighed before and after feeding to record the daily intake of the meal.The piglets were monitored for any re exes during the feeding to avoid over lling of their stomach.
Usually, the piglets remained quiet during feeding, but if they showed a sudden movement or hiccups, it was assumed that they had reached their voluntary intake and feeding would end.The piglets were weighed daily to track their daily BW changes and their daily meal allowance was adjusted accordingly.
Plasma sampling, sacri cing and tissue sampling A single blood sample was collected from the jugular vein into a 4 mL heparinized vacutainer tube using a G22 × 1″ 0.7 × 25 mm needle at the end of the experiment prior to sacri cing the piglets.The blood sample was centrifuged at 1,558 × g for 10 min at 4°C and plasma aliquots were harvested and kept at -20°C until further analyses.Afterwards, piglets were euthanized using blunt force trauma culling method and was performed by an experienced person by holding the piglet by both of its hind legs and striking the top of the head against a at concrete oor.Once the piglet was observed to be in a recumbent position, the main blood vessels in the neck were cut to make sure that the piglet was completely dead.Then the piglets were placed on a working table in dorsal recumbency to open the body cavity up and collect the internal organs.The liver, lung, heart, kidney, spleen, and gallbladder with bile were collected and weighed separately.However, the gastrointestinal tract and its content was not registered.After removing the internal organs, the empty BW was recorded.Approximately 3 g of liver and semimembranosus tissue samples were collected for determination of nitrogen content and FA compositions.

Calculations and analytical procedures
The average daily gain (ADG) of the piglets was calculated as the difference between the nal and initial BW, divided by the number of feeding days.The empty BW and internal organs were calculated as a percentage of the piglet's nal BW at sacri ce.The liver glycogen pool was calculated by multiplying the wet weight of the liver by 9.64 g of glycogen per 100 g of liver wet weight [4].
Analyses for dry matter (DM), CP, crude fat, crude ash and starch (EC 152/2009) in the milk replacer were conducted by Euro ns Steins Laboratorium A/S (Vejen, Denmark) according to the O cial Journal of the European Union [12].Nitrogen content in the liver and semimembranosus was analyzed on a freeze dried sample of the respective tissue according to Dumas method [13] using the vario MAX cube CN analysis (Elementar Analysensysteme GmbH, Langenselbold, Germany), with L-glutamic acid used as a calibrating standard.Plasma concentration of insulin was analyzed by an enzyme immunoassay using a Mercodia Porcine Insulin ELISA kit (Mercodia AB, Uppsala, Sweden), whereas plasma concentration of α-tocopherol was determined following the method previously described [14].
Plasma concentrations of glucose, lactate, and triglycerides were determined according to standard procedures (Siemens Diagnostics Clinical Methods for ADVIA 1800) using an autoanalyzer, the ADVIA 1800 Chemistry System (Siemens Medical Solutions, Tarrytown, NY).Non-esteri ed fatty acids in plasma were determined using the Wako, NEFA C ACS-ACOD assay method using an autoanalyzer, ADVIA 1800 Chemistry System (Siemens Medical Solutions).Plasma concentrations of cholesterol, albumin, total protein, alanine aminotransferase and lactate dehydrogenase were determined following standard procedures (Siemens Diagnostics Clinical Methods for ADVIA 1800).The β-hydroxybutyrate (BOHB) was determined as an increase in absorbance at 340 nm due to the production of nicotinamide adenine dinucleotide at slightly alkaline pH in the presence of BOHB dehydrogenase.This method utilized oxamic acid in the media to inhibit lactate dehydrogenase, as proposed by Harano et al. [15].All analyses were carried out using an autoanalyzer, ADVIA 1800 Chemistry System (Siemens Medical Solutions, Tarrytown, NY 10591, USA).The free amino-groups were analyzed based on the method of Larsen and Fernandez [16].
For the analysis of FA in diets, fat supplements, tissue, and plasma samples, each sample was homogenized in twice the volume of methanol by an Ultra-Turrax homogenizer, while being kept on ice.Aliquots of the homogenates corresponding to 0.251 g for milk replacer and fat supplements, 0.340 g for tissue, and 0.797 mL for plasma were weighed out in culture tubes and extracted by the modi ed method of Bligh and Dyer [17] as previously reported [18].Lipids were extracted with 1.5 mL of distilled water, 3.0 mL of chloroform, 3.0 mL methanol, and 5.0 mg of C19:0 (nonadecanoic acid, Sigma-Aldrich, St. Louis, MO) as the internal standard.The extracts were centrifuged for 10 min at 1,558 × g.Precisely, 1.0 mL of chloroform phase was transferred to a new tube, evaporated under a nitrogen stream, and then methylated with 0.8 mL of NaOH (2%) in methanol as described earlier [19].The tubes were lled with argon and transferred to an oven for 20 min at 100 º C.After cooling 1.0 mL of boron tri uoride reagent was added, lled with argon, and placed in an oven for 45 min at 100 º C. Finally, FA methyl esters were extracted with 2.0 mL of heptane and 4 mL of a saturated NaCl solution, followed by centrifugation for 10 min at 1,558 × g.A gas chromatograph (Hewlett Packard 6890, Agilent Technologies, Palo Alto, CA, USA) was used for quantifying the fatty acids as fatty acid methyl esters.
The chromatograph was equipped with an auto-column injector (HP 7673), a capillary column of 60 m × 0.32 mm inner diameter and lm thickness of 0.25 µm (Omegawax 320; Supelco 4-293-415, Sigma-Aldrich), and a ame ionization.The initial temperature was set to 86°C and increased to 200°C at a rate of 2°C per min.The temperature was then maintained at 200°C for 5 min before increasing to the nal target of 220 º C. Each peak was identi ed through a comparison of retention time with the external standard (GLC 68C, Nu-Prep-Check, Elysian, MN, USA).To determine the total fat content by the gravimetry, 1.5 ml of the chloroform phase was taken out, dried and weighed.Then, the total fat was calculated taking into consideration the contribution of the internal standard.

Statistical analyses
The experiment was designed as a complete randomized design, in which piglets were strati ed based on their BW and randomly assigned to one of four dietary treatments.The initial BW of the piglets was included as a covariate for the analyses of nal BW, ADG, organ weights and nutrient intake.All the collected data have been used in the analysis.All statistical analyses were analyzed using the SAS procedure (version 9.3, SAS Institute Inc., Cary, NC).
Average daily nutrient intake, nal BW, ADG, plasma metabolites and FA compositions in the liver, semimembranosus and plasma were analyzed using the MIXED procedure, including PA supplementation (0%, 1%, 2% and 3%), temperature challenge (yes and no), and PA × temperature challenge as xed effects.Unexpectedly, liver nitrogen showed a linear decrease with increasing PA supplementation, so liver nitrogen was included as a covariate in the analysis of FA compositions in the liver.To analyze the body temperature drop of the piglets subjected to temperature challenge, the model included PA supplementation, days of challenge (day 1, 2, 3, and 4), and PA × days of challenge as xed effects.The partial power covariance function was used to account for the correlation between repeated measures on the piglets' body temperature during the temperature challenge.Orthogonal polynomial contracts were used to evaluate the linear, quadratic, and cubic effects of the diets.The coe cients of the orthogonal polynomial contrasts were generated using the IML procedure in SAS with PA supplementation levels.The results are presented as least squared means and the largest SEM.A statistical difference was considered signi cant at P < 0.05.

Performance
Piglets supplemented with 3% PA had greater nal BW and ADG compared to the unsupplemented (P = 0.01) and 1% supplemented (P = 0.02) groups.There was a linear increase (P < 0.01) in these traits with increasing PA inclusion in the diet (Table 3).Linear effect of dietary treatment was observed in piglets subjected to temperature challenge, in which body temperature drop decreased linearly with increasing PA inclusion (P < 0.05).The average daily intake of DM and other nutrients increased linearly with increasing PA inclusion (P < 0.05).Percentage weight of gallbladder (P < 0.001) and liver (P = 0.02) were greatest at 2% and 3% PA inclusion levels, respectively, compared to the rest of the groups.Both gallbladder (P < 0.001) and liver (P < 0.05) weight demonstrated a linear increase with increasing PA inclusion in the diet.The liver nitrogen content was lower (P < 0.001), and the liver glycogen pool was greater (P = 0.002) at 3% PA inclusion compared to the other groups. 1 T1 = milk replacer mixed with 3% palm oil; T2 = milk replacer mixed with 2% palm oil and 1% palmitoleic acid; T3 = milk replacer mixed with 1% palm oil and acid; T4 = milk replacer mixed with 3% palmitoleic acid. 2 Piglets were challenged at lower room temperature (24°C) for 2 h once daily during the experimental period. 3The difference in rectal temperature of the piglets before and after the end of temperature challenge in 24°C room temperature once per day for 2 h during th period.

Plasma metabolites
The plasma concentration of glucose, triglycerides, and lactate dehydrogenase increased linearly as the inclusion of PA in the diet increased (P < 0.001; Table 4).Plasma concentration of glucose was lower in the unsupplemented group compared to those supplemented with 2% (P = 0.01) and 3% (P = 0.002) PA. Plasma concentrations of both triglycerides and lactate dehydrogenase were greatest (P < 0.001) at 3% inclusion of PA compared to the other groups.
Temperature challenged piglets had lower plasma concentration of lactate (P < 0.001) but had greater concentration of lactate dehydrogenase (P < 0.001) compared to the unchallenged group. 1 T1 = milk replacer mixed with 3% palm oil; T2 = milk replacer mixed with 2% palm oil and 1% palmitoleic acid; T3 = milk replacer mixed with 1% palm oil and palmitoleic acid; T4 = milk replacer mixed with 3% palmitoleic acid. 2 Piglets were challenged at lower room temperature (24°C) for 2 h once daily during the experimental period.

Liver, semimembranosus and plasma fatty acid composition
The majority of FA concentrations in the liver were affected by PA supplementation and demonstrated cubic response with increasing levels of PA (Table 5).

Discussion
Piglet mortality during the rst few days after birth is a major challenge in the pig industry as the emphasis on large litter size negatively impact piglet birth weight and colostrum intake.Piglets are born without brown adipose tissue [5] and have limited amounts of glycogen and body fat reserves at birth [4], making them susceptible to die from non-infectious hypothermia if piglets are lacking optimal intake of colostrum to sustain their thermogenesis.Therefore, feeding strategies with a potential of enhancing piglets' energy metabolism is worth investigating.This study investigated the role of PA supplementation on growth performance, FA and energy metabolism of the piglets during their early life.The experiment was designed to reveal the optimal level of PA supplement that maximize piglet performance with the expectation to detect a break point.However, none of the key response variables showed a break point, but rather demonstrated either a linear increase or decrease with increasing levels of PA supplementation.
The present study unveils the positive impacts of PA supplementation on performances of the piglets, although the mechanism behind these responses cannot be explicitly described.The nal BW and ADG of the piglet increased linearly in response to PA supplementation.The ADG of the piglets were 15 and 30% lower in unsupplemented group compared to 2 and 3% PA supplemented groups, respectively.Furthermore, the drop in body temperature of piglets subjected to temperature challenge decreased linearly with PA supplementation and were 30 and 33% greater in unsupplemented group compared to 2 and 3% PA supplemented groups, respectively, which could be due to increased oxidation of both fatty acid and glucose.In support of our results, it has been shown that PA supplementation enhanced lipolysis of adipocyte by increasing the content of the adipocyte triacylglycerol lipase and hormone sensitive lipase, the two major lipases in adipocytes, through a peroxisome proliferator-activate receptor α-dependent mechanism [2].In addition, the latter authors reported that PA supplementation increased glucose uptake and glucose transporter protein 4 content associated with 5' adenosine monophosphate-activated protein kinase in adipocytes; thus, cellular glucose utilized for energy production.The observation that piglets fed a 3% PA supplemented diet had greater ADG than those fed both unsupplemented and 1% PA supplemented diet, when daily nutrients intake did not differ among the dietary treatments, may indicate that PA supplementation improved nutrient utilization e ciency of the piglets.Studies in mice [20,21] and human [22] have reported that PA is involved in different metabolic pathways, such as stimulating glucose uptake through the modulation of glucose transporter proteins and glucokinase.The linear increase in plasma glucose with increasing PA supplementation, which might be modulated by upregulated glucose transporter proteins, could partly explain the improved performance of the piglets observed in the present study.In contrast to the nal BW and ADG, percentage of empty BW decreased linearly with increasing PA supplementation.Measurement of weight of gastrointestinal tract was not included in this study, which could partly account for the discrepancy between live BW and percentage of empty BW.Previous studies have shown that the gastrointestinal tract grows disproportionately faster in weight and length than the body during the early life of the piglets [23,24].Therefore, we speculated that PA supplementation might preferentially increase growth of the gastrointestinal tract of the piglets in this study, and the linear increase in liver and gallbladder weights could support this.
In a conventional farrowing unit, piglets are not as quick to locate the creep heating area on the rst day postpartum as they instinctively locate the mammary glands within few minutes after birth.As a result, piglets often huddle together around the udder on the rst day after birth, naturally challenged by the low room temperature of the farrowing unit (22°C), which is much lower than the critical body temperature of the piglets (34-35°C) around birth [9].In this study, piglets were purposely subjected to low temperature challenge to mimic conditions in the farrowing room and trigger shivering thermogenesis in order to evaluate the impact of PA supplementation on heat production in the piglets.Challenging the piglets at 24°C during the experimental period was observed to successfully trigger shivering thermogenesis (T.Feyera, personal observation during the practical experiment).Though we did not measure the extent of heat production by the piglets directly, the linear decline in rectal temperature of the piglets in response to increased PA supplementation justify that PA supplementation enhanced heat production during low temperature challenges.Mount [9] challenged the piglets in their rst week of age for 45 min at 23°C and observed a 0.4°C drop in body temperature, whereas a range of 0.7°C to 1.1°C was observed in the present study when piglets were challenged for 2 h at 24°C.These two studies suggest that the length of low temperature challenge has a strong in uence of the degree of body temperature drop of the piglets.
The present study observed striking impacts of PA supplementation in reducing body temperature loss and improving growth performance of the piglets.These results have strong practical implications for the pig industry, and the study revealed a new avenue for improving survival and growth of the piglets by manipulating a single dietary component.It is worth noting that the study was carried out for a short period only; and if the feeding duration was extended beyond that of the present study, a larger difference among the dietary treatments would be expected.To cope with the steady increase in litter size that exceeds the number of functional mammary glands in modern sows, pig farmers often use either a nurse sow or a milk supplement to increase piglet survival and growth.However, milk supplement is commonly optimized using plant-based fat, such as soya and coconut oils, which have different FA pro les from sow milk.One of the peculiarities of sow milk is that it contains a high level of PA (8-11 g) compared to cow milk (1-2 g), soya oil (0.4 g), or coconut oil (0.01 g) per 100 g of total FA [25][26][27].Therefore, the present study suggests that PA can be used as dietary ingredient in milk supplement optimization to improve survival and growth of piglets.In this study, PA was obtained from a sh oil producing company, which may be perceived as expensive.However, some species of blue-green algae contain signi cant amount of PA (39-458.8g/100 g total FA) and can serve as a cheaper and sustainable source [28,29].This coheres the green transition strategy in Europe.It is also worth exploring if maternal supplementation of PA during late gestation could alter the FA metabolism of sows and thus in uence the PA composition of sow colostrum and milk, leading to improved survival and growth of piglets.
Lactate dehydrogenase is an enzyme that catalyzes the reverse reaction between lactate and pyruvate, and extreme environmental conditions and heavy muscle activities can increase the concentration of lactate dehydrogenase in the blood [30].Hesseldeheer [30] observed a 50%-point increase in lactate dehydrogenase concentration in plasma after forcing pigs to exercise for 5 min, compared to a non-forced exercise group.Moreover, a rat study showed an increased e ciency of lactate dehydrogenase with increased level of muscle activities [31].Challenging neonate piglets for 2 h at 10°C below their normal thermoneutral zone is considered as an extreme environmental condition which increases skeletal muscle shivering and, thereby also increase the lactate dehydrogenase concentration in the blood.This observation corroborates the greater concentration of lactate dehydrogenase detected in piglets exposed to low temperature in this study compared to unchallenged piglets.Lactate dehydrogenase catalyzes the reaction of lactate to pyruvate, enabling pyruvate to enter the mitochondria.The oxidation of two moles of pyruvate in the mitochondria can produce up to 30 adenosine triphosphate, while anaerobic metabolism only produces 2 adenosine triphosphate [32].In addition, the linear increase in triglycerides accompanied with no effect on BOHB support the lactate dehydrogenase results that fat was burning off under aerobic condition.Studies have shown that lactate plays a crucial role in the distribution of carbohydrate energy for oxidation and glucose production under aerobic condition in humans [33,34].Miller et al. [33] conducted a lactate clamp experiment during resting and moderate exercise in men, and found that increased blood lactate during moderate exercise led to an increase lactate oxidation, and spare blood glucose.In the present study increased muscle contractions during temperature challenge could resemble physical activity, and result in increased lactate oxidation, partly explaining the lower plasma concentration of lactate in temperature challenged piglets compared to unchallenged piglets.Miller et al.
[33] also showed that lactate gluconeogenesis could increase during moderate exercise compared to resting.
The liver nitrogen content decreased linearly with increasing PA supplementation, while the liver glycogen pool increased linearly.This increase in liver glycogen represent an energy reserve, which may have positive effects on energy metabolism, survival, and growth of piglets.The rise in plasma glucose level may partly account for the increase in liver glycogen content.Both liver glycogen and plasma glucose concentration increased linearly with PA supplementation.Glucose and glycogen interconvert during energy metabolism, but it is unclear in the present study whether high plasma glucose leads to high liver glycogen pool or vice versa.However, according to the liver weight, it seems plausible that increasing PA increase liver glycogen, or in other words have a sparing effect on liver glycogen.The numerical increase in insulin concentration with increasing PA supplementation may have driven glucose storage in the liver and led to an increase in liver size.However, the lower liver weight in temperature challenged piglets suggests that glycogen was utilized, thus reducing the liver weight.On the other hand, the linear increase in gallbladder weight in response to PA supplementation suggests involvement of bile acid in fat metabolism.Bile acid aids in fat digestion and maintaining body cholesterol homeostasis [35].Although not statistically signi cant, the decrease in free fatty acids concentration in piglet plasma and the stable total cholesterol concentration supports this thought.Piglets subjected to temperature challenge had lower plasma concentrations of almost all FA compared to non-challenged piglets.This strongly indicate that more FA was oxidized for energy generation in temperature challenged piglets.However, we did not observe a similar trend of FA pro le in semimembranosus muscle, most likely because the FA in the muscle of these young piglets are an essential part of cellular membranes.

Conclusion and suggestion
The present study shows that adding PA to the diet of milk replacer-fed piglets during their rst week of life improves their growth performance and has a positive effect on their ability to maintain body temperature during cold stress.The study shows that increasing supplementation of PA in the diet of piglets results in a linear increase in the concentrations of C16:1n-7 and C18:1n-7 in both liver and plasma.Additionally, there was an increase in glucose, triglycerides, and lactate dehydrogenase concentrations, as well as average daily gain of the piglets with increasing PA supplementation.Body temperature drop in piglets subjected to temperature challenge decreased linearly with increasing levels of PA.Piglets subjected to temperature challenge had lower concentrations of lactate and FA in their plasma compared to unchallenged piglets.However, further research is necessary to determine the long-term effects and optimal levels of PA supplementation on piglets' performance.

Table 2
Analyzed relative fatty acid compositions (%) of the dietary treatments and fat supplements, and fat content of the experimental diets and fat supplements (%) and intake of fatty acid (g/g fat) in the respective treatments

Table 3
Impact of C16:1n-7 supplementation on nutrient intake and growth performance of the piglets fed the dietary treatments during the rst week postp

Table 4
Impact of C16:1n-7 supplementation on plasma metabolites of the piglets at sacri ce

Table 5
Effect of C16:1n-7 supplementation on fatty acid composition (mg/g of liver) of the liver in piglet fed the dietary treatment during the rst week of age

Table 6
Effect of C16:1n-7 supplementation on fatty acid composition (mg/g muscle) of muscle in piglets fed the dietary treatment during the rst week of age

Table 7
Effect of C16:1n-7 supplementation on fatty acid compositions (mg/mL plasma) of plasma in piglets fed the dietary treatment during the rst week of age