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

Abstract

Piglet survival is a major challenge in the first few days postpartum and interventions during this period may improve survival and growth. This study investigated the effects of palmitoleic acid (C16:1n-7; PA) supplementation on growth performance, body temperature, fatty acid (FA), and energy metabolism in milk-replacer-fed piglets. Forty-eight piglets were stratified by body weight and randomly assigned to one of four dietary treatments (0%, 1%, 2%, and 3% PA supplementation as a percent of milk replacer) and given the diet through an orogastric tube. They were fed dietary treatments every 2 h for 4 d in the first week postpartum and all were sacrificed at the end of the experiment. The piglets were weighed daily, and half in each dietary treatment group, the same piglets each day, were exposed daily to a lower temperature for 2 h. Plasma samples were collected immediately before sacrifice for analyses of FA and other plasma metabolites. The weight of organs and empty body weight were determined after sacrifice. Liver and semimembranosus muscle tissue samples were collected and analyzed for FA content. Contents of C16:1n-7 and C18:1n-7 in both plasma and liver (P < 0.001), and C16:1n-7 in semimembranosus muscle (P < 0.001) increased linearly as PA supplementation increased. Most plasma FA levels (except C16:1n-7, C16:1n-9, and C22:5n-3) were lower in piglets exposed to lower temperatures 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 glycogen pool, liver weight, and gallbladder weight increased linearly (P < 0.05, P < 0.01, P < 0.05, and P < 0.001, respectively), but lung weight, liver nitrogen content, and body temperature drop decreased linearly (P < 0.01, P < 0.001, and P < 0.05, respectively) with PA supplementation. 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. In conclusion, this study demonstrated the importance of PA on the growth performance of the piglets by increasing their average daily gain and decreasing a drop in body temperature upon cold exposure, most likely due to a modified energy metabolism.

This study investigated the effects of palmitoleic acid supplementation on the growth and energy metabolism of artificially fed piglets. The results demonstrated that the supplementation of palmitoleic acid in piglets’ diet resulted in enhanced growth, better maintenance of body temperature in cold environments, and an increased liver glycogen pool; indicating the beneficial impact of palmitoleic acid supplementation on the overall growth performance of the piglets.

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 (Burns et al., 2012). Additionally, PA supplementation increased adipocyte lipolysis and the content of major lipases in a mice study (Bolsoni-Lopes et al., 2013). The survival of piglets during the first days of life is a major challenge as they are born with very limited amounts of energy reserves in the body (Le Dividich and Noblet, 1983; Theil et al., 2011) and lack brown adipose tissue unlike most other mammals (Trayhurn et al., 1989). Moreover, piglets are born into a colder environment than the intrauterine condition, which greatly challenges their thermogenic capacity and thus, increases the risk of death due to hypothermia. Sow’s milk, in contrast to most other mammals like ruminants, humans, rodents, etc. has a high content of C16:1n-7 (8 to 11 g/100 g FA) (Csapó et al., 1996) compared to 1 to 2 g/100 g FA in cow and human milk (Laws et al., 2009). Plant-based fats commonly used in sow’s diets and sow’s milk replacers are not rich sources of PA. Thus, sows synthesize PA from palmitic acid by the action of Δ9 desaturase (Kloareg et al., 2007) and accumulate it in the milk (Csapó et al., 1996). This implies that PA may play a critical role in the survival and growth of the piglets, thus warranting an investigation. Supplementation of PA 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 would result in the maintenance of the body temperature of cold-exposed piglets through modified energy metabolism and thereby improve growth. Therefore, this study investigated the effects of varying levels of PA supplementation on growth, energy metabolism, and the body temperature of milk-replacer-fed piglets during the first week postpartum.

Materials and Methods

The animal experiment procedures and care of animals under study were carried out in accordance with the Ministry of Food, Agriculture and Fisheries, The Danish Veterinary and Food Administration under Act 474 of May 15, 2014, and Executive Order 2029 of December 14, 2020. Rearing, housing, and sampling were in accordance with Danish laws for the care and use of animals for research purposes. The Danish Animal Experimentation Inspectorate approved the study protocol and supervised the experiment (License number: 2018-15-0201-01484).

Selection of piglets and intubation of orogastric tube

Piglets born from 6 sows (DanBred Landrace × DanBred Yorkshire) were allowed to suckle colostrum during the first 12 h postpartum. The sows gave birth to 22 piglets on average. Afterward, litters were weighed and ranked according to body weight (BW), and eight piglets around the median weight from each sow were ear-tagged and separated from their dam. The initial BW was used to calculate the dose of anesthesia for sedating the piglets, for stratifying the piglets to the dietary treatments, and later as a covariate in data analysis. 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 (10 mg/mL). Additionally, xylocaine was injected at the site of the puncture on the right side of the cheek. The puncture was made using an intraflon2 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 carefully inserted from the puncture site in the piglets’ cheek into their stomachs, with ~17 to 18 cm of the tube extending into the stomach of the piglets for feeding the piglets during the experimental period. 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 inside (32 × 60 cm), were used to house the piglets individually from immediately after orogastric intubation until the end of the experiment. The trial was conducted in two blocks, with 24 piglets per block. The three smaller cages within the portable metabolic cage were separated by a 70-cm tall transparent flexi-glass 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 with the piglets were kept at a room temperature of 34 °C during the experiment to match the critical body temperature of the piglets at an early age (Mount, 1959). However, half of the piglets in each treatment group were predetermined to be subjected to a low-temperature challenge once a day for 2 h during the experimental period. The same piglets were challenged each day. To make the handling of piglets easier during the temperature challenge, temperature-challenged piglets were placed in the same metabolic cage within their individual pen to be moved together. During the temperature challenge, the piglets were moved to a separate room set at 24 °C and challenged for 2 h at night. On the last day of the experiment, the piglets were immediately sacrificed after the temperature challenge. In the present study, we determined the duration of cold exposure through our subjective judgment, intending to induce shivering thermogenesis only for a short period. We believed that subjecting the neonatal piglets to cold exposure for more than 2 h could potentially result in unexpected adverse effects. Observations were made during the temperature challenge to verify if this temperature challenge triggered shivering thermogenesis in the piglets, and this goal was effectively attained during the practical experiment (T.F., personal observation during the experiment). The rectal temperature of the piglets was measured before and at the end of temperature challenges to record the potential drop in body temperature when exposed to the low temperature. Irrespective of the temperature challenge, piglets were maintained in their individual pen throughout the experiment.

Diet and feeding

Forty-eight piglets, 12 piglets per treatment (2 piglets per litter per dietary treatment), were stratified 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, Videbæk, 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 a protein source (20%), with soya and coconut oil as fat source (17%) (Table 1). The PA was added at 0%, 1%, 2%, and 3% of milk replacer to create four dietary treatments, calculated to provide 0.0, 0.61, 1.22, and 1.84 g/d of PA, respectively. These levels were chosen based on the assumption that the suckling piglets ingest 1.08 g/d of PA from sow’s milk; which contains 10% PA in the milk fat (Laws et al., 2018) and piglets ingest on average 750 g of milk in the first week of lactation (Hojgaard et al., 2020). Thus, if any, a potential breakpoint was expected to be observed between the 1% and 2% PA-supplemented groups, 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 the 0% PA-supplemented group, 2% in the 1% PA-supplemented group, etc.). The PA and/or palm oil in the respective treatments were added to a powder milk replacer and blended for 20 min using a kitchen blender to create a homogeneous mixture and stored at 4 °C until feeding.

Table 1.

Calculated and analyzed compositions of the milk replacer¹ (g/kg DM unless otherwise stated)

Item	Calculated	Analyzed
DM, g/kg	970	969
Crude protein	206	203
Lactose	412
Crude fat	175	175
Crude ash	58.8	45.4
Starch		36.1
Lysine	16.5	18.9
Methionine	4.64	4.48
Leucine		20.6
Threonine		13.3
Valine		13.1
Isoleucine		12.1
Phenylalanine		8.16
Arginine		6.12
Histidine		4.53
Tryptophan		3.12
Calcium	6.19
Phosphorus	4.64
Sodium	3.51
Zinc, mg/kg feed	103
Copper, mg/kg feed	134
Iron, mg/kg feed	155
Iodine, mg/kg feed	2.06
α-Tocopherol, mg/kg feed	134
Vitamin A, IE/kg feed	26,289
Vitamin D3, IE/kg feed	2,629
Metabolizable energy, MJ/kg	16.1

¹DanMilk Supreme 1.0 AB NEO A/S, Videbæk, Denmark.

The diets in the respective treatments were dissolved in warm water (~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). To minimize variation among piglets, the meal allowance was decided to be fed per kg BW. The daily meal allowance was set at 25 mL/kg BW at each feeding and was increased by 1 mL/kg BW each day of the experimental period. The daily meal allowance was determined based on a previously published work from Copenhagen University (Henriksen et al., 2022). Feeding began when the piglets were observed standing on their feet after orogastric intubation and they were fed every 2 h afterward 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 reflexes during the feeding to avoid overfilling 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 record their BW changes and to adjust their daily meal allowance accordingly.

Plasma sampling sacrificing 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 immediately before sacrificing 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. Afterward, piglets were euthanized using the blunt force trauma culling method, which 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 flat concrete floor. Once the piglets were observed to be in a recumbent position, the main blood vessels in the neck were severed to make sure that the piglets were completely dead, and blood drainage was monitored until it ceased to ensure uniformity in blood removal among the piglets. 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 its bile were collected and weighed separately. However, the weight of the gastrointestinal tract and its content was not weighed, thus discarded during the slaughter. After removing the internal organs, the empty BW was weighed. Approximately 3 g of liver and semimembranosus muscle 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 final 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 final BW at sacrifice. 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 (Theil et al., 2011).

Analyses for dry matter, crude protein, crude fat, crude ash, and starch (EC 152/2009) in the milk replacer were conducted by Eurofins Steins Laboratorium A/S (Vejen, Denmark) according to the Official Journal of the European Union (European-Commission, 2009). Nitrogen content in the liver and semimembranosus muscle was analyzed on a freeze-dried sample of the respective tissue according to the Dumas method (Hansen, 1989) using the vario MAX cube CN analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany), with L-glutamic acid used as a calibration 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 by Jensen et al. (2006).

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). Nonesterified 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, Tarrytown, NY, USA). 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. (1985). All analyses were carried out using an autoanalyzer, ADVIA 1800 Chemistry System (Siemens Medical Solutions). The free amino groups were analyzed based on the method of Larsen and Fernandez (2017).

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 modified method of Bligh and Dyer (1959) as previously reported by Jensen (2008). Lipids were extracted with 1.5 mL of distilled water, 3.0 mL of chloroform, 3.0 mL of 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 according to Petersen and Jensen (2014). The tubes were filled with argon and transferred to an oven for 20 min at 100 °C. After cooling 1.0 mL of boron trifluoride reagent was added, filled with argon, and placed in an oven for 45 min at 100 °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 FA as FA 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 a film thickness of 0.25 µm (Omegawax 320; Supelco 4-293-415, Sigma-Aldrich), and a flame ionization detector. 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 final target of 220 °C. Each peak was identified through a comparison of retention time with the external standard (GLC 68C, Nu-Prep-Check, Elysian, MN, USA). Total fat content was determined by gravimetry by removing, drying, and weighing 1.5 mL of the chloroform phase. Then, the total fat was calculated taking into consideration the contribution of the internal standard.

Statistical analyses

The experiment was designed as a completely randomized design, in which piglets were stratified based on BW and litter, and randomly assigned to one of four dietary treatments. The initial BW of the piglets was included as a covariate for the analyses of piglets’ performance. Block was included as a random effect in the model. Two piglets, each from the 0% and 3% PA supplemented group, lost the orogastric tubes right after the first day of feeding, thus excluded from the experiment. All statistical analyses were performed using SAS software (version 9.3, SAS Institute Inc., Cary, NC). Average daily nutrient intake, final BW, ADG, plasma metabolites, and FA compositions in the liver, semimembranosus muscle, 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 fixed effects. Unexpectedly, liver nitrogen content linearly decreased with increasing PA supplementation, which might be due to an increased liver glycogen pool. Consequently, 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 as repeated measure (days 1, 2, 3, and 4), and PA × days of challenge as fixed effects. In cases where the interaction between PA supplementation and the temperature challenge was statistically significant in all of the above models, we divided it into linear, quadratic, and cubic forms, and the corresponding P-values for the interactions were presented in parentheses next to the respective linear, quadratic, and cubic P-values for the contrasts. The partial power covariance function was used to account for the correlation between repeated measures of 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 coefficients of the orthogonal polynomial contrasts were generated using the IML procedure with the expected PA supplementation levels. The results are presented as the least squared means and the largest SEM. A statistical difference was considered significant at P < 0.05.

Results

Diet compositions

The fat contents of the dietary treatments ranged from 19.1% to 19.5% (Table 2). As the level of PA increased, the composition of C16:0, C18:0, and C18:1n-9 decreased (% of total FA), while that of C16:1n-7 increased. The PA was expected to contain 60% to 70% of C16:1n-7 in total FA according to the manufacturer information and the analyzed result verified this expectation (62%).

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

	Palmitoleic acid (PA) supplement1				Fat supplements
Fatty acid	0%	1%	2%	3%	Palmitoleic acid	Palm oil
C4:0	0.65	0.64	0.67	0.68	0.00	0.02
C8:0	2.59	2.55	2.58	2.64	0.00	0.11
C10:0	2.05	2.05	2.08	2.12	0.00	0.08
C12:0	13.0	12.9	13.1	13.4	0.00	0.72
C14:0	5.95	6.00	6.08	6.19	0.78	0.81
C16:0	26.0	24.7	23.3	21.8	8.01	38.1
C16:1n-7	0.28	3.78	7.35	10.7	87.0	0.12
C16:1n-9	0.04	0.12	0.23	0.33	1.61	0.04
C18:0	4.74	4.56	4.32	4.09	0.08	5.32
C18:1n-7	0.58	0.57	0.53	0.53	0.27	0.63
C18:1n-9	32.2	30.4	28.5	26.7	0.27	43.5
C18:2n-6	9.78	9.39	8.95	8.59	0.05	9.30
C18:3n-3	0.67	0.66	0.64	0.64	0.00	0.32
SAFA2	55.7	57.3	52.9	51.7	9.02	45.8
MUFA2	33.4	35.2	36.9	38.5	89.3	44.5
PUFA2	10.7	10.3	9.82	9.45	0.18	9.65
Fat percentage	19.1	19.4	19.5	19.1	98.3	96.6
Sum of fatty acids/100 g sample	18.1	17.7	17.8	17.0	71.1	74.0
Fatty acids as a percentage of total fat	94.9	91.5	91.1	88.8	72.2	76.6

¹0% = milk replacer mixed with 3% palm oil and 0% palmitoleic acid; 1% = milk replacer mixed with 2% palm oil and 1% palmitoleic acid; 2% = milk replacer mixed with 1% palm oil and 2% palmitoleic acid; 3% = milk replacer mixed with 0% palm oil and 3% palmitoleic acid.

²SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly unsaturated fatty acids.

Performance

Piglets supplemented with 3% PA had greater final BW and ADG compared to the unsupplemented (P = 0.01) and 1% PA supplemented (P = 0.02) groups. There was a linear increase (P < 0.01) in these traits with increasing PA supplementation in the diet (Table 3). The linear effect of dietary treatment was observed in piglets subjected to temperature challenge, in which body temperature drop decreased linearly with increasing PA supplementation (P < 0.05). The average daily intake of dry matter and other nutrients increased linearly with increasing PA supplementation (P < 0.05). The percentage weight of the gallbladder with its bile (P < 0.001) and liver (P = 0.02) were greatest at 2% and 3% PA supplementation, respectively, compared to the rest of the groups. Both gallbladder with bile (P < 0.001) and liver (P < 0.05) weight demonstrated a linear increase with increasing PA supplementation. The liver nitrogen content was lower (P < 0.001), and the liver glycogen pool was greater (P = 0.002) at 3% PA supplementation compared to the other groups.

Table 3.

Impact of C16:1n-7 supplementation on nutrient intake and growth performance of the piglets fed the dietary treatments during the first week postpartum

	Palmitoleic acid (PA) supplement1						Challenge (Ch)2				ANOVA			Contrast
	0%	1%	2%	3%	SEM		No	Yes	SEM		PA	Ch	PA*Ch	Linear	Quadratic	Cubic
n	11	12	12	11			23	23
Initial BW, g	1,331	1,337	1,344	1,268	52.8		1,282	1,358	40.1		0.65	0.13	0.21	0.39	0.38	0.67
Final BW, g	1,639b	1,644b	1,684ab	1,728a	31.4		1,664	1,683	25.8		<0.05	0.53	0.72	<0.01	0.44	0.79
ADG, g	75.4b	76.8b	86.7ab	97.8a	7.84		81.8	86.5	6.46		<0.05	0.53	0.72	<0.01	0.43	0.78
DMI, g/day	48.8	50.1	50.0	52.5	1.21		49.7	51.0	0.89		0.17	0.28	0.97	<0.05	0.59	0.41
CP intake, g/d	9.93	10.0	10.2	10.7	0.26		10.0	10.4	0.18		0.16	0.20	0.95	<0.05	0.39	0.73
Crude fat intake, g/day	9.60	9.86	10.1	10.4	0.25		9.82	10.1	0.18		0.17	0.20	0.94	<0.05	0.93	0.93
Starch intake, g/day	1.76	1.78	1.81	1.90	0.05		1.78	1.84	0.03		0.16	0.20	0.95	<0.05	0.39	0.73
Ash intake, g/day	2.22	2.24	2.27	2.39	0.06		2.24	2.32	0.04		0.16	0.20	0.95	<0.05	0.39	0.73
Semimembranosus muscle nitrogen, %	10.4	10.4	10.4	10.3	0.20		10.2	10.5	0.17		0.88	0.07	0.60	0.56	0.61	0.92
Liver nitrogen, %	5.98a	4.47b	5.30b	4.66c	0.23		5.15	5.55	0.20		<0.001	<0.05	0.77	<0.001	0.72	0.31
Liver glycogen pool, g	5.33b	5.39b	5.57b	6.40a	0.27		5.75	5.59	0.21		<0.01	0.46	0.77	<0.01	0.10	0.59
Empty BW,3 %	80.0ab	80.4a	78.4c	78.7bc	0.57		79.3	79.4	0.41		<0.05	0.77	0.28	<0.05	0.96	0.06
Liver,3 %	3.38b	3.40b	3.43b	3.85a	0.17		3.58	3.44	0.15		<0.05	0.29	0.52	<0.05	0.14	0.52
Lung,3 %	1.50a	1.38ab	1.38ab	1.25b	0.05		1.41	1.35	0.04		<0.05	0.21	0.55	<0.01	0.97	0.24
Heart,3 %	0.80	0.76	0.77	0.75	0.02		0.78	0.76	0.01		0.39	0.48	0.44	0.16	0.67	0.35
Kidney,3 %	0.66	0.65	0.68	0.64	0.03		0.68	0.63	0.02		0.23	<0.01	0.38	0.83	0.34	0.06
Spleen,3 %	0.14	0.14	0.14	0.13	0.004		0.14	0.14	0.003		0.34	0.56	0.37	0.19	0.18	0.69
Gallbladder,3 %	0.040b	0.046b	0.064a	0.062a	0.004		0.05	0.05	0.003		<0.001	0.81	0.17	<0.001	0.30	<0.05
Palmitoleic acid supplement (PA)						Days					ANOVA			Contrast
	0%	1%	2%	3%	SEM	1	2	3	4	SEM	Trt	Days	Trt*Days	Linear	Quadratic	Cubic
Temperature drop, °C4	1.07	1.00	0.75	0.71	0.17	1.16	0.87	1.09	0.41	0.16	0.14	<0.001	0.82	<0.05	0.90	0.45

^a,b,cMeans within a row with different superscripts differ (P < 0.05).

¹0% = milk replacer mixed with 3% palm oil and 0% palmitoleic acid; 1% = milk replacer mixed with 2% palm oil and 1% palmitoleic acid; 2% = milk replacer mixed with 1% palm oil and 2% palmitoleic acid; 3% = milk replacer mixed with 0% palm oil and 3% palmitoleic acid.

²Piglets were challenged at a lower room temperature (24 °C) for 2 h once daily during the experimental period.

³Empty body weight and organs’ weight were expressed as a percentage of final body weight at sacrifice.

⁴The difference in rectal temperature of the piglets before and after the end of the temperature challenge at 24 °C room temperature once per day for 2 h during the experimental period.

Plasma metabolites

The plasma concentration of glucose, triglycerides, and lactate dehydrogenase increased linearly with increased PA supplementation (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% PA supplementation compared to the other groups. Temperature-challenged piglets had lower plasma concentrations of lactate (P < 0.001) but had greater concentration of lactate dehydrogenase (P < 0.001) compared to the unchallenged group.

Table 4.

Impact of C16:1n-7 supplementation on plasma metabolites of the piglets at sacrifice

	Palmitoleic acid (PA) supplement1					Challenge (Ch)2			ANOVA			Contrast
	0%	1%	2%	3%	SEM	No	Yes	SEM	PA	Ch	PA*Ch	Linear	Quadratic	Cubic
n	11	12	12	11		23	23
Insulin, mU/L	1.65	1.71	2.06	2.05	0.33	2.13	1.60	0.23	0.70	0.10	0.64	0.30	0.92	0.66
Glucose, mM/L	5.37b	5.58ab	5.87a	6.02a	0.18	5.60	5.82	0.15	<0.01	0.08	0.17	<0.001	0.82	0.69
Triglycerides, mM/L	0.63c	0.88b	0.88b	1.02a	0.06	0.84	0.86	0.05	<0.001	0.56	0.07	<0.001	0.25	0.07
Nonesterified fatty acids, µM/L	98.8	94.6	94.2	92.3	5.15	93.7	96.2	3.91	0.84	0.64	0.20	0.34	0.80	0.79
Lactate, mM/L	7.97	8.23	8.31	9.27	0.81	9.47	7.42	0.73	0.24	<0.001	0.16	0.07	0.47	0.61
Urea, mM/L	0.93	0.93	0.91	0.99	0.04	0.90	0.97	0.04	0.54	0.11	0.10	0.40	0.36	0.52
β-hydroxybutyrate, mM/L	28.9	28.3	27.6	30.4	3.58	27.3	30.3	2.890	0.90	0.31	0.94	0.77	0.55	0.77
Total cholesterol, mM/L	2.18	2.09	2.19	2.25	0.10	2.25	2.11	0.08	0.49	0.08	0.40	0.38	0.34	0.54
Lactate dehydrogenase, U/L	554bc	512c	588b	656a	39.3	531	624	35.8	<0.001	<0.001	0.11	<0.001	0.02	0.20
Alanine aminotransferase, U/L	32.8	31.1	31.5	32.6	1.87	31.8	32.2	1.71	0.51	0.66	0.38	0.96	0.16	0.73
Albumin, g/L	12.1	11.6	11.7	11.7	0.37	11.8	11.8	0.34	0.43	0.83	0.06	0.19	0.31	0.55
Free NH2 group, µ equ. glutamate	1,681	1,694	1,789	1,806	78.4	1,798	1,687	59.9	0.52	0.14	0.23	0.15	0.98	0.62
Total protein, g/L	47.7	45.6	48.3	47.8	2.78	48.1	46.6	2.20	0.84	0.49	0.30	0.76	0.72	0.42
α-Tocopherol, µg/mL	0.58	0.48	0.57	0.59	0.05	0.56	0.55	0.04	0.06	0.61	0.29	0.39	0.07	0.10

^a,b,cMeans within a row with different superscripts differ (P < 0.05).

¹0% = milk replacer mixed with 3% palm oil and 0% palmitoleic acid; 1% = milk replacer mixed with 2% palm oil and 1% palmitoleic acid; 2% = milk replacer mixed with 1% palm oil and 2% palmitoleic acid; 3% = milk replacer mixed with 0% palm oil and 3% palmitoleic acid.

²Piglets were challenged at a lower room temperature (24 °C) for 2 h once daily during the experimental period.

Liver, semimembranosus muscle, and plasma fatty acid compositions

The majority of FA concentrations in the liver were affected by PA supplementation and demonstrated cubic response with increasing PA supplementation (Table 5). As expected, the concentration of C16:1n-7 and C18:1n-7 increased linearly with increasing PA supplementation (P < 0.001). For all FA that showed a cubic response, greater concentration was found in the unsupplemented, and 2% supplemented groups compared to the other groups. In contrast, the composition of FA in semimembranosus muscle was not affected by PA supplementation (Table 6), except for a linear increase in C16:1n-7 (P < 0.001) with increasing PA supplementation. Only C16:1n-7 (P < 0.001), C18:0 (P < 0.01), and C18:1n-7 (P < 0.001) showed a linear increase in the plasma of the piglets with increasing PA supplementation (Table 7). The concentrations of most FA in plasma were lower in temperature-challenged piglets compared to unchallenged piglets, except for C16:1n-7 (P = 0.52) and C16:1n-9 (P = 0.11).

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 first week of age

	Palmitoleic acid (PA) supplement1					Challenge (Ch)2			ANOVA			Contrast
Fatty acids	0%	1%	2%	3%	SEM	No	Yes	SEM	PA	Ch	PA*Ch	Lin	Quad	Cubic
n	11	12	12	11		23	23
C12:0	0.17	0.15	0.17	0.15	0.02	0.17	0.16	0.02	0.61	0.53	0.90	0.77	0.98	0.18
C14:0	0.34	0.28	0.36	0.30	0.03	0.33	0.31	0.02	0.28	0.50	0.43	0.73	0.90	<0.05
C16:0	2.94	2.41	2.76	2.33	0.19	2.61	2.61	0.13	0.06	0.98	0.29	0.09	0.76	<0.05
C16:1n-7	0.20b	0.27b	0.51a	0.61a	0.05	0.42	0.37	0.04	<0.001	0.31	0.36	<0.001	0.77	0.11
C16:1n-9	0.14	0.11	0.13	0.11	0.01	0.13	0.12	0.01	0.32	0.44	0.54	0.36	0.48	0.12
C18:0	1.83a	1.54b	1.87a	1.66b	0.10	1.73	1.72	0.08	<0.05	0.81	0.07	0.63	0.58	<0.001
C18:1n-7	0.27b	0.28b	0.39a	0.44a	0.03	0.36	0.34	0.05	<0.001	0.40	0.35	<0.001	0.44	0.13
C18:1n-9	3.28a	2.63b	2.93ab	2.37b	0.24	2.84	2.77	0.17	<0.05	0.72	0.47	<0.05	0.82	<0.05
C18:2n-6	1.23a	1.05b	1.22a	1.07b	0.06	1.13	1.16	0.05	<0.01	0.51	0.21	0.21	0.77	<0.01
C20:4n-63	1.12a	0.94b	1.08a	1.01ab	0.06	1.05	1.03	0.05	<0.01	0.54	<0.05	0.33 (0.04)	0.16 (0.62)	<0.01 (0.15)
C22:5n-3	0.06a	0.05b	0.06a	0.06ab	0.004	0.06	0.06	0.003	<0.01	0.53	<0.05	0.91 (0.05)	0.09 (0.85)	<0.01 (0.09)
C22:6n-3	0.32a	0.26b	0.31a	0.30ab	0.02	0.29	0.29	0.02	<0.05	0.94	<0.01	0.83 (0.06)	0.07 (0.61)	<0.01 (0.08)
SAFA4	5.57a	4.60b	5.59a	4.62b	0.36	5.16	5.03	0.26	<0.05	0.63	0.18	0.24	0.99	<0.01
MUFA4	4.01	3.39	4.04	3.65	0.33	3.84	3.70	0.28	0.31	0.63	0.543	0.77	0.69	0.07
PUFA4	2.94a	2.48b	2.88a	2.68ab	0.16	2.76	2.72	0.13	<0.01	0.70	<0.01	0.48(0.02)	0.19(0.53)	<0.01(0.21)
TFA4	12.7ab	10.5c	12.9a	10.8bc	0.87	12.0	11.5	0.63	<0.05	0.49	0.19	0.42	0.97	<0.01

^a,b,cMeans within a row with different superscripts differ (P < 0.05).

¹0% = milk replacer mixed with 3% palm oil and 0% palmitoleic acid; 1% = milk replacer mixed with 2% palm oil and 1% palmitoleic acid; 2% = milk replacer mixed with 1% palm oil and 2% palmitoleic acid; 3% = milk replacer mixed with 0% palm oil and 3% palmitoleic acid.

²Piglets were challenged at a lower room temperature (24 °C) for 2 h once daily during the experimental period.

³Numbers in parentheses are P-values for the interaction of linear, quadratic, and cubic with temperature challenge.

⁴SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly unsaturated fatty acids; TFA, total fatty acids.

Table 6.

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

	Palmitoleic acid (PA) supplement1					Challenge (Ch)2			ANOVA			Contrast
Fatty acids	0%	1%	2%	3%	SEM	No	Yes	SEM	PA	Ch	PA*Ch	Lin	Quad	Cubic
n	11	12	12	11		23	23
C12:0	0.60	0.60	0.59	0.68	0.06	0.63	0.60	0.05	0.50	0.51	0.12	0.29	0.37	0.76
C14:0	0.63	0.68	0.64	0.71	0.06	0.68	0.65	0.05	0.73	0.60	0.08	0.44	0.82	0.45
C16:0	3.96	4.25	3.98	4.27	0.35	4.18	4.06	0.27	0.80	0.68	0.12	0.62	0.99	0.39
C16:1n-7	0.26c	0.53bc	0.67b	0.93a	0.07	0.61	0.59	0.05	<0.001	0.69	0.61	<0.001	0.95	0.33
C16:1n-9	0.19	0.22	0.19	0.23	0.02	0.21	0.21	0.02	0.17	0.90	0.17	0.17	0.94	0.06
C18:0	1.39	1.44	1.46	1.47	0.08	1.42	1.46	0.07	0.88	0.66	0.23	0.43	0.78	0.97
C18:1n-7	0.61	0.67	0.64	0.70	0.05	0.65	0.66	0.04	0.41	0.67	0.20	0.18	0.89	0.31
C18:1n-9	3.69	3.93	3.65	3.90	0.35	3.91	3.67	0.35	0.87	0.44	0.13	0.80	0.98	0.45
C18:2n-6	1.62	1.60	1.61	1.67	0.12	1.62	1.63	0.10	0.92	0.92	0.27	0.68	0.66	0.96
C20:4n-6	0.69	0.69	0.71	0.67	0.03	0.67	0.71	0.03	0.67	0.16	0.82	0.77	0.46	0.33
C22:5n-3	0.07	0.07	0.08	0.09	0.004	0.07	0.08	0.004	0.44	<0.05	0.13	0.18	0.87	0.38
C22:6n-3	0.09	0.10	0.10	0.10	0.005	0.09	0.10	0.004	0.41	<0.01	0.71	0.28	0.21	0.73
SAFA3	6.80	7.19	6.90	7.37	0.55	7.14	6.99	0.43	0.81	0.75	0.11	0.50	0.94	0.45
MUFA3	4.95	5.56	5.38	6.02	0.49	5.58	5.38	0.37	0.37	0.63	0.14	0.13	0.95	0.38
PUFA3	2.67	2.67	2.70	2.73	0.16	2.66	2.73	0.13	0.97	0.60	0.30	0.69	0.89	0.92
TFA3	14.4	15.4	15.0	16.1	1.18	15.4	15.1	0.92	0.67	0.77	0.13	0.31	0.94	0.49

^a,b,cMeans within a row with different superscripts differ (P < 0.05).

¹0% = milk replacer mixed with 3% palm oil and 0% palmitoleic acid; 1% = milk replacer mixed with 2% palm oil and 1% palmitoleic acid; 2% = milk replacer mixed with 1% palm oil and 2% palmitoleic acid; 3% = milk replacer mixed with 0% palm oil and 3% palmitoleic acid.

²Piglets were challenged at a lower room temperature (24 °C) for 2 h once daily during the experimental period.

³SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly unsaturated fatty acids; TFA, total fatty acids.

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 first week of age

	Palmitoleic acid (PA) supplement1					Challenge (Ch)2			ANOVA			Contrast
Fatty acids	0%	1%	2%	3%	SEM	No	Yes	SEM	PA	Ch	PA*Ch	Lin	Quad	Cubic
n	11	12	12	11		23	23
C12:0	0.03	0.05	0.04	0.05	0.006	0.05	0.04	0.005	0.06	<0.01	0.45	0.15	0.39	0.08
C14:0	0.03	0.04	0.04	0.04	0.005	0.04	0.03	0.004	0.51	<0.01	0.76	0.22	0.98	0.31
C16:0	0.38	0.41	0.39	0.41	0.03	0.43	0.37	0.03	0.81	<0.01	0.70	0.56	0.93	0.40
C16:1n-7	0.01d	0.04c	0.07b	0.10a	0.005	0.06	0.05	0.004	<0.001	0.52	0.54	<0.001	0.96	0.31
C16:1n-93	0.01	0.01	0.01	0.01	0.006	0.01	0.01	0.005	0.30	0.11	<0.05	0.16 (0.09)	0.31 (0.64)	0.98 (0.98)
C18:0	0.20	0.21	0.22	0.24	0.02	0.23	0.20	0.01	0.07	<0.01	0.63	<0.01	0.71	0.73
C18:1n-7	0.03c	0.03bc	0.04b	0.05a	0.003	0.04	0.03	0.002	<0.001	<0.05	0.80	<0.001	0.52	0.57
C18:1n-9	0.51	0.53	0.50	0.52	0.04	0.55	0.47	0.03	0.88	<0.05	0.57	0.84	0.97	0.45
C18:2n-6	0.37	0.39	0.40	0.43	0.03	0.42	0.37	0.03	0.47	<0.05	0.80	0.13	0.47	0.79
C20:4n-6	0.08	0.08	0.08	0.08	0.006	0.09	0.07	0.005	0.54	<0.01	0.47	0.88	0.25	0.43
C22:6n-3	0.02	0.018	0.018	0.02	0.001	0.021	0.018	0.001	0.33	<0.01	0.40	0.98	0.08	0.89
SAFA4	0.67	0.73	0.71	0.76	0.06	0.78	0.65	0.05	0.55	<0.01	0.75	0.21	0.96	0.42
MUFA4	0.58	0.63	0.62	0.69	0.05	0.68	0.58	0.04	0.27	<0.05	0.72	0.07	0.80	0.40
PUFA4	0.51	0.51	0.53	0.56	0.04	0.56	0.49	0.03	0.57	<0.05	0.70	0.20	0.58	0.91
TFA4	1.76	1.89	1.85	2.02	0.45	2.02	1.73	0.12	0.44	<0.01	0.71	0.13	0.80	0.51

^a,b,c,dMeans within a row with different superscripts differ (P < 0.05).

¹0% = milk replacer mixed with 3% palm oil and 0% palmitoleic acid; 1% = milk replacer mixed with 2% palm oil and 1% palmitoleic acid; 2% = milk replacer mixed with 1% palm oil and 2% palmitoleic acid; 3% = milk replacer mixed with 0% palm oil and 3% palmitoleic acid.

²Piglets were challenged at lower room temperature (24 °C) for 2 h once daily during the experimental period.

³Numbers in parentheses are P-value for the interactions of linear, quadratic, and cubic with temperature challenge.

⁴SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly unsaturated fatty acids; TFA, total fatty acids.

Discussion

Piglet mortality during the first few days after birth is a major challenge in the pig industry as the emphasis on large litter size negatively impacts piglet birth weight and colostrum intake. Piglets are born without brown adipose tissue (Trayhurn et al., 1989) and have limited amounts of glycogen and body fat reserves at birth (Theil et al., 2011), making them susceptible to dying from noninfectious hypothermia if piglets lack optimal intake of colostrum to sustain their thermogenesis. Therefore, feeding strategies with the potential to enhance piglets’ energy metabolism are 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 supplementation that maximizes piglet performance with the expectation of detecting a breakpoint. However, none of the key response variables showed a breakpoint 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 the performances of the piglets, although the mechanism behind these responses cannot be explicitly described. The final BW and ADG of the piglet increased linearly in response to PA supplementation. The ADG of the piglets was 15 and 30% lower in the 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 an 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 the lipolysis of adipocytes 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 (Bolsoni-Lopes et al., 2013). In addition, the latter authors reported that PA supplementation increased glucose uptake and GLUT4 content associated with AMP-activated protein kinase activation in adipocytes; thus, cellular glucose is utilized for energy production. The observation that piglets fed a 3% PA supplemented diet had greater ADG than those fed either the unsupplemented or 1% PA supplemented diet may indicate that PA supplementation improved the nutrient utilization efficiency of the piglets. Studies in mice (Pu et al., 2011; Rossmeisl et al., 2020) and humans (Tricò et al., 2020) 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 final BW and ADG, the percentage of empty BW decreased linearly with increasing PA supplementation. Measurement of the weight of the gastrointestinal tract was not included in this study, which could partly account for the discrepancy between live BW and the 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 (Widdowson et al., 1976; Xu et al., 1992). Therefore, we speculated that PA supplementation might preferentially increase the growth of the gastrointestinal tract of the piglets in this study, and the linear increase in liver and gallbladder weights could be the implication for the preferential effect of PA supplementation on organ development.

In a conventional farrowing unit, piglets are not as quick to locate the creep heating area on the first day postpartum as they instinctively locate the mammary glands within a few minutes after birth. As a result, piglets often huddle together around the udder on the first 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 to 35 °C) around birth (Mount, 1959). In this study, piglets were purposely exposed to low-temperature challenges to mimic conditions in the farrowing room and trigger shivering thermogenesis 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.F., personal observation during the experiment). Though we did not measure the extent of heat production by the piglets directly, the linear decrease in a rectal temperature drop of the piglets in response to increased PA supplementation justifies that PA supplementation enhanced heat production during low-temperature challenges. Mount (1959) challenged the piglets in their first 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 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 influence on the degree of body temperature drop of the piglets.

The present results reported striking impacts of PA supplementation in reducing body temperature loss and improving the growth performance of the piglets. These results have strong practical implications for the pig industry, and the study revealed a new avenue for improving the 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 and enable us to evaluate the impact of PA supplementation on long-term survival rate of the piglets. 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 profiles from sow’s milk. One of the peculiarities of sow’s milk is that it contains a high level of PA (8 to 11 g) compared to cow milk (1 to 2 g), soya oil (0.4 g), or coconut oil (0.01 g) per 100 g of total FA (Ayorinde et al., 2000; Bee, 2000; Liau et al., 2011). Therefore, the present study suggests that PA can be used as a dietary ingredient in milk supplement optimization to improve the survival and growth of piglets. In this study, PA was obtained from a fish oil-producing company, which may be perceived as expensive. However, some species of blue-green algae contain a significant amount of PA (39 to 45.8 g/100 g total FA) and can serve as a cheaper and sustainable source (Holton et al., 1968; Lang et al., 2011). It is also worth exploring if maternal supplementation of PA during late gestation could alter the FA metabolism of sows and thus influence the PA composition of the sow’s colostrum and milk, leading to improved survival and growth of piglets.

Lactate dehydrogenase is an enzyme that catalyzes the reversible reaction between lactate and pyruvate, and extreme environmental conditions and heavy muscle activities can increase the concentration of lactate dehydrogenase in the blood (Hesseldeheer, 1969). Hesseldeheer (1969) 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 efficiency of lactate dehydrogenase with an increased level of muscle activity (Battellino et al., 1971). In our study, piglets were challenged for 2 h at 10 °C below their normal thermoneutral zone and this could be considered an extreme environmental condition for neonate piglets, which increases skeletal muscle shivering and thereby also increases 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 molecules of pyruvate in the mitochondria can produce up to 30 moles of adenosine triphosphate, while anaerobic metabolism only produces 2 moles of adenosine triphosphate (Zangari et al., 2020). 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, thus cells can utilize nonesterified fatty acids as sources of energy instead of glucose when plasma concentration of triglycerides is high. Studies have shown that lactate plays a crucial role in the distribution of carbohydrate energy for oxidation and glucose production under aerobic conditions in humans (Miller et al., 2002; Todd, 2014). Miller et al. (2002) conducted a lactate clamp experiment during resting and moderate exercise in men, where blood lactate concentration was clamped at ~4 mM by exogenous lactate infusion and found that increased blood lactate during moderate exercise led to an increase in lactate oxidation and spare blood glucose. The increased shivering thermogenesis observed in the present study (T.F., personal observation during the experiment) during the 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. (2002) also showed that lactate gluconeogenesis could increase during moderate exercise compared to resting.

The liver nitrogen content decreased linearly with increasing PA supplementation, unlike the linearly increased liver glycogen pool. The observation cannot be solely attributed to the findings, but it could be related to the sparing effect of the liver favoring more glycogen deposition than nitrogen in the liver with increasing PA supplementation. The increase in liver glycogen represents 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 concentrations 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 supplementation increases liver glycogen, or in other words has 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, though increased insulin concertation could depress FA oxidation (Groop et al., 1992). 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 the involvement of bile acid in fat metabolism. Bile acid aids in fat digestion and maintaining body cholesterol homeostasis (Burrin et al., 2013). Although not statistically significant, 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 unchallenged piglets. This strongly indicates that more FA was oxidized for energy generation in temperature-challenged piglets. However, we did not observe a similar trend of FA profile in the semimembranosus muscle, most likely because the FA in the muscle of these young piglets are an essential part of cellular membranes. A linear increase in liver C16:1n-7 concentration and a concurrent decrease in C16:0 and C18:1n-9 was observed with increasing PA supplementation, reflecting the change in dietary intake. Fatty acids with a carbon chain length longer than 12 are solely taken up through the lymphatic system and carried in chylomicrons to the liver, where they are incorporated in the lipoproteins and transported to the peripheral tissue (Lauridsen, 2017). However, remnant particles of the chylomicrons are left in the liver and may partly explain the FA composition observed in the liver in the present study.

Conclusion and Suggestion

The present study has demonstrated the importance of PA supplementation on the growth performance of the piglets. This effect is most likely caused by a shift in energy metabolism towards higher FA oxidation, while sparing glycogen oxidation, leading to an increased ADG of the piglets and a reduction in their body temperature drop during cold exposure. Future research should focus on assessing the feasibility of incorporating PA into milk replacers or supplementing the diets of late gestating and lactating sows to increase mammary uptake of PA, ultimately resulting in elevated PA concentration in sow’s colostrum and milk.

Glossary

Abbreviations

ADG

average daily gain

BOHB

β-hydroxybutyrate

BW

body weight

FA

fatty acids

PA

palmitoleic acid

Conflict of interest statement.

The authors declare that there are no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.