ORIGINAL RESEARCH published: 11 February 2021 doi: 10.3389/fvets.2021.574250 Oral Meloxicam Administration in Sows at Farrowing and Its Effects on Piglet Immunity Transfer and Growth Elena Navarro 1*, Eva Mainau 1 , Ricardo de Miguel 2 , Déborah Temple 1 , Marina Salas 1 and Xavier Manteca 1 1 Department of Animal and Food Science, School of Veterinary Science, Universitat Autònoma de Barcelona, Bellaterra, Spain, 2 Department of Animal Pathology, University of Zaragoza, Zaragoza, Spain Many factors can lead to an inadequate development of piglets during their first days of life, including poor maternal behavior, which can be due to pain caused by farrowing, and reduced colostrum ingestion. This study investigates the action of meloxicam administered orally at farrowing on piglet weight gain and immunity transfer. Thirty-five multiparous sows were divided into two groups and treated with 0.4 mg/kg of oral meloxicam (oral meloxicam group; n = 18) or with a mock administration (control group; n = 17). A total of 382 piglets were individually weighed on the farrowing day (day 0), as well as on days +9 and +20. Immunoglobulin G (IgG) and A (IgA) concentrations in piglet serum and in sow’s saliva, colostrum and milk were measured. Additionally, Interleukin-2 Edited by: (IL-2), Interleukin-4 (IL-4) and Interferon gamma (IFN-g) in serum of piglets and in sow’s Edward Narayan, milk or colostrum were studied. All samples were obtained on days +1, +9, and +20. The University of Queensland, Australia Piglets from sows in the oral meloxicam group tended to grow faster from day +9 to Reviewed by: day +20 than did piglets from control sows (p = 0.059), and this difference was also Keelin Katherine Mary O’Driscoll, observed in piglets with low body weight (BW) at birth (p = 0.056). The oral meloxicam Teagasc, Ireland group sows tended to increase the colostrum levels of IgA and IgG, as compared with Peter Wynn, Charles Sturt University, Australia control sows on day +1 (p = 0.068 and p = 0.072, respectively). IgA levels in piglet *Correspondence: serum from the oral meloxicam group were significantly higher than in the control group Elena Navarro on day +1 and +9 (p = 0.019 and p = 0.011 respectively). Furthermore, IL-2 and IL- elena.navarro@uab.cat 4 levels in the serum of piglets from sows in the oral meloxicam group tended to be Specialty section: higher than that in the control group on day +9 (p = 0.078 and 0.056, respectively). This article was submitted to The administration of meloxicam orally at the beginning of farrowing in multiparous Animal Behavior and Welfare, sows increased immunoglobin and cytokine concentrations in colostrum, improving both a section of the journal Frontiers in Veterinary Science humoral and cellular immune response of piglets. Pre-weaning growth of piglets born with Received: 19 June 2020 a low BW improved in the meloxicam-treated group. Accepted: 18 January 2021 Keywords: pain, farrowing sow, piglet, immunity transfer, weight gain, meloxicam, immunoglobulin Published: 11 February 2021 Citation: Navarro E, Mainau E, de Miguel R, Temple D, Salas M and Manteca X INTRODUCTION (2021) Oral Meloxicam Administration in Sows at Farrowing and Its Effects Piglets are born agammaglobulinemic because of the epitheliochorial placentation of swine (1, 2). on Piglet Immunity Transfer and An early and sufficient intake of colostrum is crucial for piglet growth and survival (3, 4), as it Growth. Front. Vet. Sci. 8:574250. is the source of energy as well as active and passive immunity. Colostrum is a complex mammary doi: 10.3389/fvets.2021.574250 secretion released from the time of farrowing (early-colostrum) to 12 h (mid-colostrum) and up Frontiers in Veterinary Science | www.frontiersin.org 1 February 2021 | Volume 8 | Article 574250 Navarro et al. Oral Meloxicam Administration in Sows to 36 h post-farrowing (late-colostrum) (5). Early-colostrum is cytokines (Interleukins IL-2 and IL-4, and Interferon Gamma mostly produced before farrowing and contains up to 75% of IFN-g), taking into account the sow parity effect. Immunoglobulin G (IgG) and 20% of Immunoglobulin A (IgA), which are central elements of humoral immune responses. After farrowing, IgG concentration drastically drops, whereas IgA MATERIALS AND METHODS reduction during lactation is more gradual due to its role in the The experimental protocol described in this experiment was regulation of piglet intestinal microbiota, which is critical for the approved by the Institutional Animal Care and Use Committee prevention of digestive problems (5). of the Universitat Autònoma de Barcelona (CEEAH-1591) and Colostrum-associated cellular immunity has been overlooked the Generalitat de Catalunya (DMAH-6720). Written informed for a long time. It contains around 106 cells/mL, up to consent was obtained from the owners for the participation of 25% of them being lymphocytes (6, 7). Immune responses their animals in this study. are orchestrated via complex signaling pathways within cells mediated by cytokines, which are small proteins with a plethora of effects. IL-2, IL-4 and IFN-g are important cytokine mediators Animals, Housing and General of the adaptive immune response, thus their quantification Management allows for partial characterization of the immune response. Sample size was calculated by means of ENE 3.0. The sow was IL-2 is mainly produced by T lymphocytes and induces the the experimental unit. Based on two previous studies carried proliferation of T and B lymphocytes and the activation of out by Mainau et al. (18, 19), a reference mean average daily Natural Killer cells (NK) (8–10). IL-4 triggers differentiation gain (ADG) of 0.2 kg/day was established at sow level for the of T helper lymphocytes toward the Th2 subset, which is control group and an expected mean ADG of 0.225 kg/day related to humoral and anthelmintic responses (8, 10), while was considered for the treatment group. An overall standard IFN-g activates macrophages and elicits the differentiation of deviation of 0.025 kg/day (at sow level) was assumed with a power T helper lymphocytes toward the Th1 subset, thus favoring of 80% and confidence level of 95%. A prevision of 17 sows per cellular responses and boosting protection against intracellular group was predicted. microbes (10). The experimental procedure was carried out on a commercial Non-steroidal anti-inflammatory drugs (NSAIDs) have farm (Heura S.L.; Santa Perpètua de Mogoda, Barcelona, Spain), analgesic, anti-inflammatory, anti-endotoxic and anti-pyretic with 9 farrowing barns equipped with an evaporative cooling effects. It has been proven that NSAIDs administered to sows system each. From December 2017 to March 2018, a total of 35 help them recover from a painful situation such as lameness hybrid (Landrace x Duroc) multiparous sows from 2nd to 7th (11) or post-partum dysgalactia syndrome (12, 13). NSAIDs also parturition were randomly selected the day of farrowing. At least decrease the mortality rate at weaning in litters from healthy 5 replicates with 5 to 10 sows per replicate were studied. sows (14) and in sows with dysgalactia syndrome (13). However, On day 109 of gestation, sows were moved to the farrowing studies on the effect of NSAIDs, on sow welfare, piglet growth barn and were housed in individual farrowing crates (1.95 × and immunity transfer in healthy sows show discrepancies. 0.60 m) built with steel bars. Farrowing crates were centrally Meloxicam administered to healthy sows around farrowing located in farrowing pens (2.40 × 1.80 m) with fully metal-slatted improves post-farrowing sow recovery (15, 16) and enhances floors for sows and plastic-slatted floors for piglets. A metal pad piglet growth, especially at weaning (17, 18) and in piglets with ensured 36◦ C of heat for the piglets during the first week of life, low body weight (BW) at birth (18, 19). Nevertheless, other and heat lamps were placed over the metal pad the first day of studies administering NSAIDs to healthy sows around farrowing life. The temperature in the farrowing barn was kept constant did not find enhanced sow welfare and recovery post-farrowing at ∼20◦ C, and the light was on from 07:00 to 17:00 h every (20) or improve piglet growth (15, 21). day. Sows were offered 2.6 kg of feed per day, divided into two To our knowledge, only two studies have looked into the meals (07:00 and 15:00 h) and water was available ad libitum effects of NSAIDs administered to sows around farrowing from drinkers. and have assessed passive immunity transfer via colostrum Thirty days before farrowing, all sows were vaccinated with and immune system development in piglets (18, 20). Both Clostridium novyi (2 mLSuiseng R , Hipra SA; Girona, Spain). studies recorded IgG transfer measured in piglet serum without Sixteen days after farrowing, sows that were not expected to exploring immunoglobulins in sow colostrum or milk. These be culled (n = 31 sows) were vaccinated with Parvovirus and studies did not measure other relevant immune factors for piglet Erysipelothrix rhusiopathiae (2 mL Eryseng R parvo, Hipra SA; growth and survival, such as IgA or cytokines. Mainau et al. Girona, Spain) and with Leptospira spp (2 mL Autovacuna R syva, (18) demonstrated that the administration of meloxicam orally Syva SAU; León, Spain). On day 113 of gestation, farrowing was at the beginning of farrowing in multiparous sows increased hormonally induced with 2 mL of Planate R (Cloprostenol 0,092 the concentration of IgG in piglet serum and enhanced their mg/mL, MSD Animal Health; Friesoythe, Germany) divided into pre-weaning growth. two injections of 1 mL (07:00 h and 11:00 h). Only hormonally The present study aims to evaluate the effect of meloxicam induced farrowings that started on the morning of day 114 of administered orally to healthy sows at the beginning of farrowing gestation were included in the study. Lame sows or those with on piglet growth, also including the effect of sex and immune any kind of visible disease symptoms such as mastitis, diarrhea, transfer via colostrum of immunoglobulins (A and G) and fever, or respiratory problems were not included in the study. Frontiers in Veterinary Science | www.frontiersin.org 2 February 2021 | Volume 8 | Article 574250 Navarro et al. Oral Meloxicam Administration in Sows Treatments and manual interventions during farrowing sows on day +1 (colostrum) and on days +9 and +20 (milk). followed the usual routine of the farm and were performed Sows were injected with 0.7 mLof Oxytocin IM (Hormonipra R , by the same person. The following treatments were allowed Hipra SA; Girona, Spain), and 30 s later, 2 mL of colostrum during farrowing and were administered intramuscularly (IM) and milk were collected into sterile tubes. Colostrum and milk in the neck. When the time interval between the birth of samples were immediately frozen at −20◦ C until analysis. two piglets exceeded 1 h and the cervical canal was dilated, Each pig was individually weighed at farrowing (day 0), and on 1 mL of Oxytocin (Hormonipra R , HipraSA, Girona, Spain) was days +9 and +20. One day after farrowing, during one suckling injected. When the cervical canal was not sufficiently dilated, event, 3–4 piglets of each litter were selected for blood sampling. sows were treated with 200 mg of Vetrabutine hydrochloride Piglets were chosen so that at least one of them was suckling from (Monzal R , Boehringer Ingelheim España; SA, Barcelona, Spain). the sow’s craneal teats, another one from middle teats and yet When sows were very nervous around farrowing Carazolol another one from caudal teats. The same piglets from each litter (Suacron R , Divasa Farmavic SA; Barcelona, Spain) or Azaperone were blood sampled on days +1, +9, and +20. Blood samples (Stressnil R , Janssen Animal Health, Elanco; Brussels, Belgium) (1–2 mL) were collected into heparinized tubes from the anterior were administered. vena cava. Samples were centrifuged for 6 min at 2,058 × g and A total of 382 piglets, identified individually by a numeric plasma was stored in Eppendorf tubes at −80◦ C until analysis. ear tag, were included in the study. Piglets were weaned at All samples were analyzed at the Murcia University Veterinary 21 days of age, according to veterinary recommendations, and Hospital Laboratory. Immunoglobulin G (IgG) and A (IgA) moved to another barn of the farm equipped with conditioned concentrations in piglet serum and sow saliva, colostrum and infrastructures for very young piglets. milk were quantified by using two commercially available sandwich ELISAs (IgA and IgG ELISA Quantitation Kit; Bethyl Experimental Procedure Laboratories; Montgomery, TX, USA). Interleukin-2 (IL-2), In each replicate, sows were randomly allocated into two Interleukin-4 (IL-4) and Interferon gamma (IFN-g) in piglet homogeneous groups, regarding parity, and treated with either serum, sow saliva, milk or colostrum were analyzed using 0.4 mg/kg body weight of meloxicam (Metacam R 15 mg/mL Oral MILLIPEX R MAP Porcine Cytokine/Chemokine Panel Kit Suspension, Boehringer Ingelheim Vetmedica GmbH; Ingelheim, (EMD Millipore; Darmstadt, Germany). Rhein, Germany) or a mock administration with an empty syringe. Treatments were administered at the beginning of the Statistical Analysis farrowing, between the first and the third piglet. If any further Data were analyzed using the SAS software (SAS Institute Inc.; anti-inflammatory treatment was required, the sow was excluded Cary, NC, USA). The experimental unit for data analysis was the from the study. individual sow. All descriptive values in the Results section are Litter size was standardized at 11–12 piglets by cross-fostering shown as the mean ± standard error (SE). Significance was set at within 6–8 h post-farrowing. Cross-fostering was carried out p < 0.05, and tendency at p < 0.1, in all cases. within each treatment. Each treatment was identified with two The Mann-Whitney Wilcoxon test was used to test whether different colored cards in order to make the treatment blind to the performance values (other than piglet weight and average farm and laboratory staff. daily gain) obtained at the individual sow level were significantly different between treatments. Data Collection Normality tests of residuals were performed for each For each sow, the following parameters were registered during dependent variable. Weight of piglets and ADG (From birth to farrowing by direct observations: the duration of farrowing day+9, from day+9 to weaning and from birth to weaning) were (defined as the period of time between the first and the last normally distributed without data transformation. A general piglet born), the condition of each piglet at birth (born alive, linear mixed model (proc MIXED) for repeated measures was stillborn or mummified fetus), the piglet’s sex (male or female), used. Model included the fixed effects of treatment (control the number of treatments and manual interventions during vs. oral meloxicam), day (at birth, day +9 and at weaning), farrowing, and the number of piglets cross-fostered and weaned. sex (males vs. females) and their pair interactions. Day and The presence of placenta retention was also recorded. During piglet within sow were introduced as repeated effects. Weight lactation, piglet mortality was registered. The number of sows at birth was introduced as a covariate for the analysis of weight culled after weaning and the interval between farrowing and the at day +9 and at weaning, and litter size was introduced as a following fertile insemination were recorded. covariate in all the models. The residual maximum likelihood One and 9 days after farrowing (day +1 and day +9) and the was used as a method of estimation. Differences in least-square day before weaning (day +20), saliva samples were collected from means were investigated after using a Tukey adjustment for all sows using Salivette R tubes (Sarstedt; Nümbrecht, Germany). multiple comparisons. The same models were used to study the Each tube contained a cotton swab, which was clipped with a performance of piglets categorized by quintiles according to their Kocher clip, and sows were allowed to chew it for around 1 min. weight at birth: very light (from 0.670 to 1.294 Kg), light (from Then, the cotton swab was placed in the tube and centrifuged >1.294 to 1.492 Kg), mid (from >1.492 to 1.625 Kg), heavy (from at 6,048 × g for 13 min. Saliva samples (∼1–2 mL per cotton >1.625 to 1.878 Kg) and very heavy (from >1.878 to 2.427 Kg). swab) were stored in Eppendorf tubes and frozen at −80◦ C until IgG and IgA concentrations in piglet serum, sow saliva and analysis. Colostrum and milk samples were collected from all colostrum or milk, IL-2, IL-4 and IFN-g in colostrum or milk, Frontiers in Veterinary Science | www.frontiersin.org 3 February 2021 | Volume 8 | Article 574250 Navarro et al. Oral Meloxicam Administration in Sows TABLE 1 | Mean, standard error (SE), median (MED) and 95% confidence intervals for median (95% CI) of performance parameters and treatment records studied in the control and oral meloxicam groups during the whole trial period (from farrowing to weaning at 21 days). Control Oral meloxicam P-value (n = 17 sows) (n = 18 sows) Items Mean SEa MED 95%CI Mean SE MED 95%CI Parity 4.06 0.441 3 2–7 4.28 0.394 4 2–7 0.582 Piglets born at the moment of the treatment 1.70 0.143 2 1–3 1.78 0.117 2 1–3 0.856 Total duration of farrowing (h) 3.27 0.328 3.47 1.12–5.47 3.31 0.387 3.06 1.38–7.28 0.817 Total piglets born per litter 13.47 0.912 13 6–21 12.73 0.576 13 9–17 0.621 Live born per litter 11.88 0.624 12 6–18 11.36 0.584 11 8–16 0.489 Stillborn per litter 0.94 0.358 1 0–6 0.83 0.259 0.5 0–4 0.986 Mummified fetus per litter 0.65 0.226 0 0–2 0.55 0.217 0 0–3 0.815 Cross-fostered piglets per litter 11.18 0.231 11 10–13 10.67 0.256 10.5 8–12 0.209 Crushing deaths per litter 0.47 0.229 0 0–3 0.17 0.121 0 0–2 0.322 Total liveborn mortality 0.76 0.304 0 0–4 0.61 0.282 0 0–4 0.662 Total weaned piglets 10.41 0.193 10 9–12 10.06 0.338 10 6–12 0.569 Manual intervention per sow 0.47 0.174 0 0–2 0.38 0.230 0 0–4 0.422 Oxytocin treatment per sow 0.24 0.106 0 0–1 0.17 0.900 0 0–1 0.637 Total treatments per sow 0.24 0.106 0 0–1 0.33 0.140 0 0–2 0.731 A total of 35 sows and 354 piglets were included in the study. P-value from Mann-Whitney Wilcoxon test is shown. a SE, standard error. and IFN-g in serum of piglets were normally distributed after TABLE 2 | Mean and standard error (SE) of the piglet weight at birth, 9 days after a log transformation. IL-2 and IL-4 in piglet serum followed a farrowing (day + 9) and at weaning (day + 20) in Kilograms and the Average Daily normal distribution without data transformation. Additionally, Gain (ADG) of piglets from birth to day + 9 after farrowing, from birth to weaning extreme outliers detected by proc UNIVARIATE box plot and from day + 9 to weaning in Kilograms per day for 354 piglets regarding procedures were deleted. treatment (control vs. oral meloxicam). Immunity measurements in piglets and sows were analyzed Control Oral meloxicam using general linear mixed models (proc MIXED) for repeated measures. Models for immunity sow measurements included Mean SE Mean SE P-value the fixed effects of treatment (oral meloxicam vs. control), day Weight at birth (Kg) 1.510 0.065 1.600 0.063 0.996 (day +1, +9, and +20), parity (from 2nd to 7th) and their pair Weight at day +9 (Kg) 3.556 0.071 3.499 6.538 0.909 interactions. Day was introduced as a repeated effect. Models Weight at weaning (Kg) 6.479 0.109 6.538 0.103 0.644 for immunity piglet measurements included the fixed effects ADG from birth to day +9 (Kg/day) 0.218 0.010 0.217 0.010 0.981 of treatment (oral meloxicam vs. control), day (day +1, +9, ADG from birth to weaning (Kg/day) 0.243 0.006 0.251 0.006 0.275 and +20), sex (male vs. female), the position at the udder ADG from day +9 to weaning (Kg/day) 0.261b 0.007 0.275a 0.007 0.059 (anterior, middle and posterior teats) and their pair interactions. Day and piglet within sow were introduced as repeated effects. Different superscripts (a, b) in the same row indicate significant differences within each The residual maximum likelihood was used as a method of effect (p < 0.05). Tendency has been shown at p < 0.1. estimation. Differences in least-square means were investigated after using a Tukey adjustment for multiple comparisons. All general linear mixed models included replicate (from 1 to 5) and Treatment did not have an effect on the time interval between farrowing barn (from 1 to 9) as random effects. weaning and the following fertile insemination (4.769 ± 0.121 days in oral meloxicam group vs. 7.071 ± 1.811 days in the RESULTS control group; p = 0.893), or on the number of sows culled after weaning (0.111 ± 0.076 in the oral meloxicam group vs. 0.176 ± Performance Parameters and Treatment 0.095 in the control group; p = 0.608). Records Twenty-four piglets died during lactation, which represents Results on performance and treatment records at the individual 6.28% of mortality. Oral meloxicam treatment of sows did not sow level are summarized in Table 1. Both treatment groups significantly affect piglet mortality (6.84% from the control group (oral meloxicam vs. control) were similar when the experimental and 5.73% from oral meloxicam group; p = 0.661). procedure started in terms of performance data recorded The mean and standard error (SE) of weight at birth, during farrowing. weight on day +9 and weight at weaning are summarized in Frontiers in Veterinary Science | www.frontiersin.org 4 February 2021 | Volume 8 | Article 574250 Navarro et al. Oral Meloxicam Administration in Sows Table 2. Piglet weight at birth, on day +9 and at weaning weaning in the oral meloxicam group (267.93 ± 7.793 gr) than in was not different between the control and the oral meloxicam the control group (240.11 ± 9.207 gr) (p = 0.056). group. Piglet sex had a significant effect on the weight of the piglets, males being heavier than females at weaning (Table 1, Supplementary Material). Immunoglobulins G and A Concentrations Average daily gain (ADG) data are also summarized in in Saliva and Colostrum or Milk of Sows Table 2. Oral meloxicam treatment of sows tended to increase and in Piglet Serum piglet ADG from day +9 to weaning. Piglet sex had a significant Immunoglobulin G and A (IgG and IgA) concentrations in saliva, effect on the ADG, males growing faster than females from birth colostrum or milk of sows and in piglet serum by treatment effect to weaning and from day +9 to weaning. on days +1, +9, and +20 after farrowing are shown in Figure 1. Piglet weights (at birth, on day +9 and at weaning) and ADG IgG levels in sow saliva (µg/mL) were affected by day after (from birth to day +9, from day+9 to weaning and from birth farrowing (day +1: 44.89 ± 4.486; day +9: 9.08 ± 1.810 and to weaning) were not affected by treatment in piglets born with day +20: 3.28 ± 0.475; p < 0.001 in all pair comparisons), but light, mid, heavy or very heavy weight at birth. Piglets with very were not affected by treatment (p = 0.547) irrespectively of the light weight at birth tended to have a higher ADG from day+9 to day studied. FIGURE 1 | IgA and IgG at days +1, +9, and +20 after farrowing regarding treatment received by sows (control vs. oral meloxicam) in sow saliva (A,B), sow colostrum and milk (C,D) and piglet serum (E,F). Significant differences were established at p < 0.01(**) and tendency was set at p < 0.1(#). Boxes represent the interquartile range (IQ = Q3-Q1), horizontal lines inside the boxes represent the median and the cross (+) represents the mean values of the data. Whisker bars were calculated from the IQ (Upper: Q3 + 1.5 × IQ; lower: Q1 – 1.5 × IQ), and reflect the variability of the data outside Q1 and Q3. Points outside the box-and-whiskers plot represent extreme values of the population. Frontiers in Veterinary Science | www.frontiersin.org 5 February 2021 | Volume 8 | Article 574250 Navarro et al. Oral Meloxicam Administration in Sows IgA levels in sow saliva (µg/mL) were affected by day after IL-2, IL-4 and IFN-g concentration in colostrum or milk of farrowing (day +1: 239.64 ± 21.202, day +9: 118.477 ± 12.267 sows (ng/mL) were affected by day after farrowing (p < 0.001 and day +20: 60.81 ± 7.295; p < 0.001 in all pair comparisons), in all cases). IL-2, IL-4 and IFN-g in sow colostrum on day +1 but were not significantly affected by treatment; (p = 0.704) (IL-2: 1.51 ± 0.166; IL-4: 10.12 ± 1.289; IFN-g: 62.99 ± 5.505) irrespective of the day studied. showed higher concentrations than in sow milk on day +9 (IL-2: IgG levels in colostrum or milk of sows (mg/mL) were affected 0.50 ± 0.079; IL-4: 2.60 ± 0.540; IFN-g: 24.31 ± 3.827) and on by day after farrowing and were higher on day +1 (24.48 ± day +20 (IL-2: 0.73 ± 0.089; IL-4: 4.02 ± 0.641; IFN-g: 41.86 ± 1.484) than on days +9 (3.75 ± 0.953) and +20 (1.77 ± 0.298) 4.585), whereas concentrations on day +20 were higher than on (p < 0.001 in both cases). IgG levels in colostrum from the day +9. sows treated with oral meloxicam tended to be higher than in IL-2, IL-4, and IFN-g concentrations in colostrum or milk the control group on day +1 (p = 0.072). However, on days of sows were not significantly affected by treatment (p = 0.206, +9 and +20, IgG concentration in sow milk was not affected 0.142, and 0.322 respectively). by treatment. IL-2, IL-4 and IFN-g concentrations in colostrum or milk of IgA levels in colostrum or milk of sows (mg/mL) were affected sows were affected by parity (p = 0.010, p < 0.001 and p = 0.008 by day after farrowing and were higher on day +1 (7.48 ± 0.577) respectively). The general pattern was that sows in their second than on days +9 (4.41 ± 0.347; p < 0.001) and +20 (3.72 ± 0.315; parity showed lower levels of cytokines than did older sows (three p < 0.001). IgA concentration on day +9 and +20 were similar parturitions or more). Specifically, IL-2 levels in colostrum or (p = 0.246). Furthermore, IgA levels in colostrum of sows treated milk of sows in their second parity were lower than those in sows with oral meloxicam tended to be higher than in the control in their fourth (p = 0.016) and fifth parity (p = 0.023) and tended group on day +1 (p = 0.068), but on day +9 and +20, IgA levels to be lower than in sows in their seventh parity (p = 0.098). IL-4 in sow milk were not affected by treatment. levels in colostrum or milk of sows in their second parity were IgA and IgG concentrations in saliva and in colostrum or milk lower than those in sows in their third (p = 0.005), fourth (p were not affected by parity (saliva: p = 0.290 and p = 0.192, = 0.001), fifth (p = 0.001), sixth (p = 0.035) and seventh parity respectively, and colostrum or milk: p = 0.127 and p = 0.232). (p = 0.002). IFN-g levels in colostrum or milk of sows in their The interaction between treatment and parity was not significant second parity were lower than those in sows in their fourth parity (saliva IgA p = 0.113; IgG p = 0.925 and colostrum or milk IgA (p = 0.008) and tended to be lower than in sows in their sixth p = 0.239; IgG p = 0.112). parity (p = 0.070). IgG levels in piglet serum (mg/mL) were affected by day after IL-2 and IL-4 levels in piglet serum (ng/mL) were affected by farrowing (day +1: 29.93 ± 1.377; day +9: 20.26 ± 0.935 and day day after farrowing, and were higher on day +9 (IL-2: 2.40 ± +20: 11.48 ± 0.466; p < 0.001 in all pair comparisons). IgG levels 0.173; IL-4: 17.15 ± 1.442) than those on days +1 (IL-2: 1.33 in piglet serum were not significantly affected by treatment (p = ± 0.070; IL-4: 8.53 ± 0.526; p < 0.001 in both cases) and +20 0.963), sex (p = 0.189) or piglet position at the udder (p = 0.811) (IL-2: 1.55 ± 0.145; IL-4: 10.67 ± 1.156; p < 0.001 and p = irrespective of the day studied. 0.001, respectively). IgA levels in piglet serum (mg/mL) were affected by day after IL-2 and IL-4 levels in piglet serum were affected by treatment, farrowing (day +1: 20.63 ± 1.314, day +9: 1.36 ± 0.080 and and tended to be higher in the oral meloxicam group than day +20: 0.27 ± 0.018 p < 0.001 in all pair comparisons), and those in the control group on day +9 (p = 0.078 and p = there was an interaction between treatment and sampling day 0.056, respectively). IL-2 and IL-4 levels in piglet serum were not (p = 0.020). IgA levels in piglet serum from sows treated with significantly affected by sex of piglets (p = 0.596 and p = 0.868, oral meloxicam were significantly higher than in piglets from respectively) or by piglet position at the udder (p = 0.888 and p the control group on day +1 (p = 0.019) and day +9 (p = = 0.715, respectively) irrespective of the day studied. 0.011). However, on day +20, IgA level in piglet serum was not IFN-g levels in piglet serum (ng/mL) were affected by day significantly affected by treatment (p = 0.943). IgA levels in piglet after farrowing, and were lower on day +1 (6.00 ± 0.334) than serum were not significantly affected by sex (7.79 ± 0.944 in those on day +9 (10.74 ± 0.654) and day +20 (12.07 ± 0.758) (p females vs. 7.89 ± 1.001 in males; p = 0.633) or by piglet position < 0.001 in all cases). at the udder (anterior teats: 7.67 ± 1.048, middle teats: 7.17 ± IFN-g levels in piglet serum were not significantly affected by 1.328 and posterior teats: 9.07 ± 1.448, p = 0.725) irrespective of treatment (p = 0.409), sex of piglets (p = 0.858), or piglet position the day studied. at the udder (p = 0.320), irrespective of the day studied. Concentration of Cytokines (IL-2, IL-4 and DISCUSSION IFN-g) in Colostrum or Milk of Sows and in In the present study, both treatment groups (oral meloxicam Piglet Serum and control) were well-matched in terms of performance Concentration of interleukins (IL-2 and IL-4) and interferon variables recorded during farrowing. Early administration of gamma (IFN-g) in colostrum or milk of sows and in piglet serum oral meloxicam treatment did not negatively affect total piglets by treatment effect on days +1, +9, and +20 after farrowing are born alive per litter, total duration of farrowing, treatments shown in Figure 2. administered during farrowing (such as oxytocin) or the number Frontiers in Veterinary Science | www.frontiersin.org 6 February 2021 | Volume 8 | Article 574250 Navarro et al. Oral Meloxicam Administration in Sows FIGURE 2 | Concentration of IL-4, IL-2, and IFN-g at days +1, +9, and +20 after farrowing regarding treatment received by sows (control vs. oral meloxicam) in sow colostrum and milk (a, c, e) and piglet serum (b, d, f). Tendency differences were established at p < 0.1(#). Boxes represent the interquartile range (IQ = Q3 – Q1), horizontal lines inside the boxes represent the median and the cross (+) represents the mean values of the data. Whisker bars were calculated from the IQ (Upper: Q3 + 1.5 × IQ; lower: Q1 - 1.5 × IQ) and reflect the variability of the data outside Q1 and Q3. Points outside the box-and-whiskers plot represent extreme values of the population. of manual interventions during farrowing. Hence, it appears that to observe differences in piglet mortality (piglet mortality in our the use of oral meloxicam during parturition (more specifically study was 6.8%). between the first and the third piglet born) did not interfere with Piglet weights at birth were similar (16, 17) or slightly the progression of the birth process. higher (3, 22, 23) than values reported in other studies. The administration of oral meloxicam at the beginning of farrowing Piglet Mortality and Growth tended to enhance the ADG of piglets from day +9 to weaning, In agreement with other authors that studied the effect of and particularly for the lightest piglets. A similar effect was NSAIDs around farrowing (17, 18, 20, 21), oral meloxicam described by Mainau et al., in two studies (18, 19), treating administered to healthy sows did not show an effect on pre- the sows around farrowing with injectable and with oral weaning piglet mortality. On the contrary, Homedes et al. (14), meloxicam. Tenbergen et al. (17), injected meloxicam intra- in a large-scale study on commercial farms with a high incidence muscularly within 12 h of farrowing and found that piglet ADG of pre-weaning mortality (±10%), showed lower pre-weaning tended to be higher for piglets from the meloxicam group piglet mortality after ketoprofen administration to sows within sows than for control piglets in medium-sized litters (11–13 12 h after farrowing. Homedes et al. (14) explained such an piglets). Ketoprofen is another AINE used in pig production, effect due to higher milk production by the sow ketoprofen- but Viitasaari et al. (21) and Ison et al. (15), who both injected treatment group. We assume that a larger sample size enrolling sows with ketoprofen during farrowing, did not find that it different farms with high pre-weaning mortality would be needed had any effect on piglet average daily gain to weaning. These Frontiers in Veterinary Science | www.frontiersin.org 7 February 2021 | Volume 8 | Article 574250 Navarro et al. Oral Meloxicam Administration in Sows discrepancies in the effects of NSAIDs administered to healthy on the intestinal mucosa (1). Mainau et al. (18) found that sows around farrowing on piglet growth could be due to different the administration of meloxicam orally at the beginning of factors such as the active principle administered. Meloxicam is a farrowing (on average, when 2.6 piglets had already been born) selective COX-2 inhibitor and may be a more specific treatment increased the concentration of IgG in the serum of piglets. In for inflammation caused by farrowing than a non-selective COX the present study, sows were treated early at the beginning inhibitor, like ketoprofen (24). The time of administration is of farrowing, when early colostrum (with the highest IgG another important factor to take into consideration. Studies levels) has already been produced and thus the influence of administering meloxicam at the beginning of farrowing (17–19) treatment on the IgG serum levels of piglets fed with this show the effect on weaning weights of piglets and ADG. Thus, colostrum was lower. Nevertheless, weaker piglets and those born the active ingredient administered (preferably a selective COX-2 later during parturition are known to suffer from delayed and inhibitor) and the administration time (as soon as possible after reduced colostrum intake (31). These animals have lower survival farrowing starts) are presumably important factors to improve and growth rates, which may be improved by treatment with piglet growth and weight at weaning. meloxicam, as shown by our results with piglets born with a very light weight at birth, as well as by other studies (31). These Transfer of Passive and Active Immunity differences could be explained by a higher IgG and IgA immunity Colostrum intake is crucial for development of piglet immunity. transfer in the treatment group in these weaker animals, which In this study, and in accordance with normal colostrum and are likely to consume a smaller quantity of early-colostrum and a milk immunoglobulin kinetics (5), sow colostrum and milk larger proportion of mid- and late-colostrum. Unfortunately, in IgG and IgA showed an abrupt and steady decrease respectively this study a low percentage of piglets with low BW at birth were (Figures 1C,D). Interestingly, colostrum immunoglobulin blood sampled, thus hampering a proper analysis of their serum content on day +1 was higher in the oral meloxicam group then IgG and IgA levels. in the control group. The difference between groups was more Regarding colostrum and milk cytokines, higher levels of IL- pronounced in IgA than in IgG, which could be explained by 2, IL-4, and IFN-Y were found on day +1, which is likely to the switch between the IgG/IgA ratio after farrowing (5). One- be related to pain and to contamination of the reproductive hundred percent of colostrum IgG and 40% of colostrum IgA tract induced by farrowing. Milk cytokine levels moderately come from sow blood via an Ig receptor, whereas up to 60% of decreased between day +1 and +9 and increased again between IgA is directly synthesized in the mammary gland (1). Our data day +9 and +20, likely in response to the vaccination given support the local role of oral meloxicam in the mammary gland, to sows on day +16. Cytokines and lymphoid cells have been which likely decreases local inflammation, thus favoring both demonstrated to cross the intestinal barrier of newborn piglets immunoglobulin recruitment from plasma and local production (32–34). In piglet serum, cytokine levels measured on day +1 of IgA in plasma cells (1). Indeed, in vitro studies developed in are expected to be the result of both colostrum-derived cytokines cattle have shown the anti-inflammatory effect of meloxicam in and cytokines produced by the piglets. In contrast, taking into mammary epithelial cells (25). Furthermore, mastitis in cows has consideration the short half-life of these cytokines (minutes been associated with reduced pre-weaning immunity, growth, for IL-2 and IL-4 and a few hours for IFN-g) and the loss and health of the offspring (26, 27), so the anti-inflammatory of piglet intestinal permeability, cytokine levels on days +9 effect elicited by meloxicam treatment is presumed to have the and +20 reflect only the activity of the piglet immune system. opposite effect. Higher concentrations of all cytokines were found on day +9, In comparison with blood sampling, saliva sampling is likely due to the immune challenge elicited by tail docking (in generally considered to be a non-invasive and stress-free both sexes) and castration (in males), which were performed methodology (28). IgG levels in sow saliva are directly on their second day of life. Interestingly, higher IL-4 and IL- proportional to the levels in sow serum, whereas IgA in saliva 2 levels were measured in piglets from the meloxicam treated is mostly produced locally, so IgA levels are highly variable group on days day +9 (Figures 2B,D). Increased secretion of in response to environmental factors such as stress and oral IL-4 and IL-2 in piglets has been related to better Th2 and infections (29). In our study, saliva IgG levels, a marker of Th1 immune responses, respectively (8). Moreover, IL-4 induces plasma IgG levels, showed no differences between groups, antibody production and tissue repair, whereas IL-2 plays a which probably rules out a systemic effect of oral meloxicam major role in the activation of NK-cells and the generation administration on the Ig increase in piglets from treated sows. of effector and memory cells (9, 10). This positive influence IgA and IgG concentration in piglet serum during lactation of colostrum on the immune system development could be is the result of intake of immunoglobulins from colostrum. related to the transfer of colostrum-associated immune cells, The quick drop of IgA and the slow drop of IgG in piglet which are absorbed selectively in the newborn gut, although the serum is likely explained by the different half-lives of these precise mechanisms remain unclear (1). Therefore, it could be immunoglobulins in serum, being 6 days for IgA and 21 days hypothesized that meloxicam treatment around farrowing had an for IgG (30). IgA concentration was higher in piglet serum in impact on the concentration of immune cells in colostrum, but the oral meloxicam group on days +1 and +9. Interestingly, further studies are needed to investigate this hypothesis. diarrhea of newborn piglets is one of the biggest health issues This study was developed on a commercial farm with in pig production, and increased IgA levels could play a major really good sanitary and husbandry conditions. Further role in preventing these problems by their protective effect research is required to determine if these positive results on Frontiers in Veterinary Science | www.frontiersin.org 8 February 2021 | Volume 8 | Article 574250 Navarro et al. Oral Meloxicam Administration in Sows piglet welfare can be even more pronounced by studing a of the Universitat Autònoma de Barcelona (CEEAH-1591) and larger set of commercial farms with higher mortality rates the Generalitat de Catalunya (DMAH-6720). Written informed and lower growth rates during lactation. In summary, the consent was obtained from the owners for the participation of results of this study show that early administration of oral their animals in this study. meloxicam improves some aspects of piglet performance and welfare. Further research is needed to study whether AUTHOR CONTRIBUTIONS these effects are also observed in primiparous sows or could be improved by administering meloxicam before the onset EN: data acquisition, structuralization, and interpretation, of farrowing. drafting of the manuscript and final approval of the version to be published. EM: study concept and design, data acquisition, CONCLUSIONS data analysis and interpretation, drafting of the manuscript and final approval of the version to be published. RM: immunology The administration of meloxicam orally at the beginning of data analysis and interpretation, preparation of figures, and farrowing in multiparous sows increased the concentration of drafting of the manuscript and final approval of the version to immunoglobins and cytokines in sow colostrum and improved be published. DT and MS: data acquisition and final approval both humoral and cellular immune response in piglets. Pre- of the version to be published. XM: study concept and design, weaning growth of piglets, especially in piglets born with low BW, drafting of the manuscript and final approval of the version to be tended to be higher in the meloxicam-treated group than that in published. All authors contributed to the article and approved the the control group. submitted version. DATA AVAILABILITY STATEMENT ACKNOWLEDGMENTS The original contributions presented in the study are included The authors wish to thank the staff of the commercial farm Heura in the article/Supplementary Material, further inquiries can be S.L., Santa Perpètua de Mogoda, Barcelona, Spain. directed to the corresponding author/s. SUPPLEMENTARY MATERIAL ETHICS STATEMENT The Supplementary Material for this article can be found The experimental protocol described in this experiment was online at: https://www.frontiersin.org/articles/10.3389/fvets. approved by the Institutional Animal Care and Use Committee 2021.574250/full#supplementary-material REFERENCES 9. Abbas AK, Trotta E, Simeonov D, Marson A, Bluestone JA. Revisiting IL-2: biology and therapeutic prospects. Sci Immunol. (2018) 25:3;6. 1. Salmon H, Berri M, Gerdts V, Meurens F. Humoral and cellular factors doi: 10.1126/sciimmunolaat1482 of maternal immunity in swine. Dev Comp Immunol. (2009) 33:384–93. 10. Kumar V, Abbas AK, Aster JC. 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This 26. Heravi M.oussavi A, Danesh Mesgaran M, Gilbert RO. Effect of is an open-access article distributed under the terms of the Creative Commons mastitis during the first lactation on production and reproduction Attribution License (CC BY). The use, distribution or reproduction in other forums performance of Holstein cows. Trop Anim Health Prod. (2012) 44:1567–73. is permitted, provided the original author(s) and the copyright owner(s) are credited doi: 10.1007/s11250-012-0107-3 and that the original publication in this journal is cited, in accordance with accepted 27. Ferdowsi Nia E, Nikkhah A, Rahmani HR, Alikhani M, Mohammad Alipour academic practice. No use, distribution or reproduction is permitted which does not M, Ghorbani GR. Increased colostral somatic cell counts reduce pre-weaning comply with these terms. Frontiers in Veterinary Science | www.frontiersin.org 10 February 2021 | Volume 8 | Article 574250