- Email: [email protected]
Hair cortisol determination in sows in two consecutive reproductive cycles
reproductive biology 14 (2014) 218–223
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/repbio
Original Research Article
Hair cortisol determination in sows in two consecutive reproductive cycles Maria Laura Bacci *, Eleonora Nannoni, Nadia Govoni, Fabrizio Scorrano, Augusta Zannoni, Monica Forni, Giovanna Martelli, Luca Sardi Department of Veterinary Medical Sciences, DIMEVET, University of Bologna, Via Tolara di Sopra 50, Ozzano dell'Emilia, 40064 Bologna, Italy
article info
abstract
Article history:
Hair analysis has been proposed as a minimally invasive technique capable of furnishing
Received 11 July 2013
information regarding the stress response during medium- and long-term periods. Bristle
Received in revised form
samples were collected from the rump region of sows at three key physiological phases (before
12 May 2014
delivery – BD; weaning time – WT; pregnancy diagnosis – PD) during consecutive reproductive
Accepted 7 June 2014
cycles in order to test swine hair as a reliable matrix of cortisol evaluation. Cortisol was extracted from the bristles and assayed using radioimmunoassay. The highest mean hair
Keywords:
cortisol concentrations were demonstrated (p < 0.001) at the PD time points (20.1 .95 and
Swine
16.29 2.15 pg/mg). Moreover, cortisol was significantly higher (p < 0.001) at BD2 (10.48
Hair
0.96 pg/mg) as compared to BD1 (5.17 0.51 pg/mg) and WT1 (6.01 0.47 pg/mg). The
Cortisol
various physiological phases had a significant effect on cortisol concentration (p < 0.00001)
Chronic stress
with a higher cortisol concentration found during late pregnancy and lactation than in early-
Reproduction
mid pregnancy. This could be due not only to the physiological hormonal status, but also to the different housing conditions (single crates vs. group housing). The season of the year was also observed to have an effect (p < 0.005), with the lowest cortisol concentration recorded during the hot season. # 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
1.
Introduction
Animal welfare becomes increasingly important in the context of intensive breeding. Although some objective parameters for animal welfare have already been identified [1], they are not
efficient when applied to chronic stress conditions. Animal stress responses are linked to many parameters, both subjective and those related to the environment; they depend on the nature, intensity and duration of the stressor. Much attention has been paid to acute stressors; however, in intensive breeding, the most adverse stressors are often
* Corresponding author. Tel.: +39 0512097912; fax: +39 051 2097899. E-mail addresses: [email protected], [email protected] (M.L. Bacci). http://dx.doi.org/10.1016/j.repbio.2014.06.001 1642-431X/# 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
reproductive biology 14 (2014) 218–223
chronic and induce negative effects, swine summer infertility being one such well-known example [2,3]. Although the problem has been extensively studied, and the major stressors, such as temperature and photoperiod, are known, the underlying hormonal mechanisms are not fully understood. Cortisol is often used as a stress biomarker due to the fact that adverse situations trigger an adrenal response, but it is also secreted under favorable conditions, and its circadian biorhythm may have physiological functions in mammals. This causes difficulties in differentiating between the physiological pattern of cortisol secretion and the response to stress. Blood, milk, saliva and urine (short-medium term), or feces and hair (medium-long term) were employed to assess changes in cortisol concentrations [4]. Blood/saliva sampling is stressful per se and confuses data interpretation [5–7]. On the other hand, urine, feces or hair sampling requires less intense handling, but the exact timing of the cortisol deposition is not known. The aims of the present study were to: (1) develop and validate a non–invasive technique for cortisol assessment in porcine bristle, and (2) examine the medium-long term changes of cortisol concentrations in the bristles of breeding sows under farm conditions.
2.
Material and methods
2.1.
Reagents and animals
The isopropanol and methanol were purchased from Carlo Erba (MI, Italy). The bovine serum albumin (BSA), sodium phosphate dibasic dihydrate (Na2HPO4), ethylendiaminetetraacetic acid disodium salt (EDTA Na2), sodium azide (NaN3), heat activated charcoal, dextran and cortisol were obtained from Sigma–Aldrich (St. Louis, MO, USA). The 1,2,6,7-3H cortisol was purchased from Perkin-Elmer (Shelton, CT, USA). Thirty, randomly selected, hybrid Goland sows (white skin, no black hair; mean parity: 4.68 3.1) from a breeding herd in Northern Italy were used in the study. Breeding herd management was based on a batch farrowing system every 3rd week, which led to natural estrus synchronization by
219
weaning. The animals were reared according to the current Italian legislation which implements Council Directive 2008/ 120 EC on pig protection. This study was conducted during two consecutive reproductive cycles occurring in different seasons.
2.2.
Experimental design
From the time of pregnancy diagnosis until 5 days before the expected delivery date (approximately 9 weeks), the sows were kept in collective pens with free access to an external area (gestation area) (Fig. 1). The sows were then moved to the farrowing house where they were individually housed in farrowing stalls until weaning. Lactation lasted approximately 4 weeks. At weaning, the sows entered the mating house where they were individually housed in single crates until pregnancy was diagnosed. They were then moved again to the gestation area and were re-grouped. The sows were monitored from February to November. Thirty and 25 (five sows were culled) sows were used during the first and the second reproductive cycles, respectively. During the entire trial, all the sows were in good health. In each cycle, hair samples were collected: (1) five days pre-partum (before delivery in the first and in the second reproductive cycles: BD1, BD2, respectively), (2) 25–28 days postpartum (weaning time in the first reproductive cycle: WT1) and (3) 70–75 days postpartum, during pregnancy in the first and the second reproductive cycles (PD1, PD2, respectively). The gestation house had a negative pressure ventilation system while the farrowing house was heated. Room temperature (RT) was recorded in both houses by means of data loggers (Model 175 H2, Testo, Lenzkirch, Germany). The monthly mean RT ranged from 29 8C (July) to 17 8C (November) (Fig. 1). Regarding the experimental period, the environmental temperature data were obtained from a nearby weather station (Fig. 2). The hair was shaved off manually, close to the skin on the rump region, from a maximum area of 49 cm2, alternately on the two sides, avoiding reddened or irritated areas. The samples were placed into small plastic bags, individually marked and stored at 4 8C. In addition, the hair re-growth (3 sows) was measured with a decimal Vernier caliper after one month. The mean rate of hair regrowth was 0.7 0.2 cm/month.
Fig. 1 – Schematic representation of the experimental design. AI: artificial insemination; BD1 and BD2: before delivery sampling times; PD1 and PD2: pregnancy diagnosis sampling times; WT1: sampling at weaning (BD1, PD1, WT1: 30 sows; BD2, PD2: 25 sows); RT: room temperature; Environ. T: environmental temperature; Av.: average.
220
reproductive biology 14 (2014) 218–223
2.4.
Statistical analysis
Statistical analysis was carried out using Statistica ver 10.0 (StatSoft, Inc., Tulsa, OK, USA). The cortisol data are presented as mean SEM. The data were analyzed using one-way ANOVA followed by the Duncan multiple range test. To evaluate the effects of the reproductive phase (PD vs. BD) and the season, two-way ANOVA was used.
3. Fig. 2 – Environmental temperature recorded during the experiment in the vicinity of the sow piggery. Average (av), minimal (min) and maximal (max) temperature for each day are provided.
2.3.
Measurement of hair cortisol concentration
The bristle sample (250 mg) was washed with water, air dried at RT, placed in a polypropylene tube and covered with isopropanol (5 mL) [8,9]. The tube was gently mixed (3 min at RT), centrifuged (1500 g for 1 min) and the isopropanol was discarded. The bristle sample was again washed with isopropanol, air dried and fully pulverized. The pulver (60 mg) was placed in a glass vial with 2 mL of methanol and incubated overnight at RT with continuous gentle agitation for steroid extraction. After centrifugation (1500 g for 5 min), 1.8 mL of methanol was collected, transferred into a glass tube and evaporated to dryness under an air-stream suction hood (37 8C). The dry extracts were stored at 20 8C. In order to obtain a sample pool for the radioimmunoassay (RIA) validation, 60 mg of pulverized hair from each sow were pooled together, extracted with methanol, dried and analyzed in triplicate during each RIA. Cortisol concentration was determined using a validated radioimmunoassay [10]. The dried extracts were dissolved in RIA buffer (0.3 mL; 74.26 mmol/L Na2HPO4, 12.49 mmol/L EDTA Na, 7.69 mmol/L NaN3) containing 0.1% BSA (pH 7.5) and were shaken for 10 min. The sample (0.1 mL), 1,2,6,7-3H cortisol (0.1 mL, 30 pg/tube) and rabbit anti-cortisol serum (0.1 mL; 1:20,000) were incubated overnight (4 8C); 1 mL of charcoal– dextran solution (0.25%, charcoal and 0.02% dextran in RIA buffer) was then added to the tubes. After 15 min (4 8C), the tubes were centrifuged (15 min, 3000 g), the supernatant was decanted and the radioactivity was immediately measured using a b scintillation counter (Packard C1600, Perkin-Elmer). The cross reactions of various steroids with the rabbit anticortisol serum were as follows: cortisol 100%, corticosterone 9.5%, cortisone 5.3%, 11a-deoxycortisol 5.0%, prednisolone 4.60%, 20a-dihydrocortisone 0.4%, progesterone <0.001% and testosterone <0.001%. Mean cortisol recovery was 96.6 2.2%. The intra- and inter-assay coefficients of variation (5 determinations in triplicate) were 2.76% and 8.5%, respectively. The assay sensitivity was 4.81 pg/tube and was defined as the dose of hormone at 90% binding (B/B0). A high degree of parallelism between the cortisol standards and the porcine hair cortisol was confirmed by a regression test (r2 = 0.99; p < 0.01).
Results
The highest mean hair cortisol concentrations were found at PD1 (20.1 .95 pg/mg) and PD2 (16.29 2.15 pg/mg). Although hair cortisol did not differ significantly between PD1 and PD2, a declining tendency (p < 0.058) was observed during the hot season (Fig. 3). Mean hair cortisol concentrations were significantly higher (p < 0.001) at PD1/PD2 as compared to the other phases. Moreover, the cortisol was significantly higher (p < 0.001) at BD2 (10.48 0.96 pg/mg) in comparison to BD1 (5.17 0.51 pg/mg) and WT1 (6.01 0.47 pg/mg). The twoway ANOVA revealed significant effects regarding the reproductive phase (p < 0.00001) and season (p < 0.005), but there was no significant interaction. On the basis of the mean cortisol concentration, the sows were divided into three categories: (1) 1–10, (2) 10–20 and (3) >20 pg/mg (Fig. 4). At WT1, all the sows fell into the lowest category. At BD1 and BD2, the majority of the sows (90.9% and 55.55%, respectively) were in the lowest category, and the remaining sows were in the middle category (9.1% and 44.45%, respectively). At PD1 (96%) and PD2 (72.23%), the majority of the sows were in either the middle or the high cortisol category.
4.
Discussion
The evaluation of the response to stress, based solely on plasma cortisol concentration, requires a detailed knowledge of the physiological and behavioral patterns of the species and
Fig. 3 – Cortisol concentration (mean W SEM) in sow hair. PD: diagnosis of pregnancy; BD: five days before delivery; WT: weaning time (28 days after delivery). PD1: April; BD1: June; WT1: July; PD2: September; BD2: November (BD1, PD1, WT1: 30 sows; BD2, PD2: 25 sows). Means without common superscripts are significantly different (p < 0.001).
reproductive biology 14 (2014) 218–223
221
Fig. 4 – Proportion of sows belonging to different hair cortisol concentration categories. PD: diagnosis of pregnancy; BD: five days before delivery; WT: weaning time (28 days after delivery). PD1: April; BD1: June; WT1: July; PD2: September; BD2: November (BD1, PD1, WT1: 30 sows; BD2, PD2: 25 sows). The cortisol concentration ‘‘>20 pg/mg’’ was present only at PD1 and PD2.
of the type of animal. The cortisol secretion, in addition to being determined by natural biorhythms, is highly regulated and is also affected by the invasive nature of blood sampling. Other biological fluids used for cortisol analysis which can be collected in a non-invasive manner, such as urine or saliva [11] require normalization [12] and provide information on acute stress responses only over a short-medium period of time. The special feature of hair is that it can provide information regarding exposure to endogenous or exogenous compounds over a medium-long period of time. However, additional studies are needed to determine how long the compounds remain in the hair [4,13]. Hair sampling is a simple noninvasive painless procedure; it can be carried out by a nonprofessional and the samples are easy to store and transport [4]. Hair cortisol, as a response endpoint to stressors, has been examined in rhesus macaques [9], wildlife [14,15], dogs and cats [16]. In the current study, a technique for cortisol determination in sow bristles using extraction and RIA was developed, and hair cortisol concentration in sows during the different phases of the reproductive cycle was measured. Cortisol is a steroid hormone considered to be secreted for the most part by the adrenal cortex. Although there is no evidence of skin cortisol production, its synthesis by keratinocytes has been suggested in one report [17]. On the other hand, circulating cortisol reaches the hair papilla and may be accumulated in cells during keratinization; in this study, very slow hair growth was observed. Mowafy and Cassens have reported that swine hair reached a skin depth of approximately 3–4 mm [18]. We therefore assumed that, in pigs, hair samples would provide information relating to a period varying from one to two months before the date of collection, with the exclusion of the last 15 days. Hence, the BD time points provided information regarding a period in which pregnant sows experienced positive conditions with some acute stress. Instead, the PD time points
provided information regarding a period of an increasingly more stressful situation during which the animals were placed in farrowing stalls in which they farrowed and nursed the piglets [19]. Since cortisol blood concentration rises sharply before and during delivery, and slowly returns to normal values at weaning [20], we analyzed hair samples collected at 28 days after farrowing (WT1). However, the WT1 mean cortisol concentration did not differ when compared to BD1, probably due to poor hair growth which prevented the measurement of the accumulation of cortisol in this short period of time; thus, WT sampling was not repeated for the second reproductive cycle. Our data showed that, at BD1 and BD2, the mean cortisol concentrations were significantly lower than at PD1and PD2, indicating a clear influence of the reproductive phase in agreement with the cortisol blood concentration reported by others [21,22]. Significant differences were not found between PD1 and PD2, although, at PD2, the cortisol concentration tended to be lower. However, the cortisol concentration at BD1 was significantly lower than that recorded at BD2. The reliability of the results found at BD1 was confirmed by the identical results obtained at WT1. These data confirmed an important seasonal effect, probably linked to the different conditions of temperature and daylight. The BD2 sampling took place at the end of November while the BD1 sampling took place in June. From this point of view, it was also possible to explain the different trends observed for the cortisol values between PD1 and PD2. At PD2 (September), the cortisol concentration tended to be lower than that obtained at PD1 (April), reflecting the mean blood cortisol concentration of the previous months. In fact, when the adverse situation is prolonged, the circulating cortisol concentrations not only diminish (during heat stress or crowding) [23,24], but the adrenocorticotropic hormone challenge also produces a more limited response [25].
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reproductive biology 14 (2014) 218–223
We can therefore affirm that, during warmer months, such as May, June, July and August, the mean cortisol blood concentration is strongly reduced in sows regardless of the reproductive phase, and vice versa in colder months (October, November, February and March), indicating a seasonal fluctuation of this hormone. To date, seasonal cortisol fluctuations have been described in unrestrained red deer [26], in humans [27,28], in goats [29], in sheep [30], in horses [31] and in squirrels [32]. In swine, seasonal infertility is well known [2,33] and is linked to a complex interaction of environmental factors which, in turn, are mediated by many endocrine signals. Fluctuations in the monthly farrowing rate are observed in the same period in the Northern and Southern hemispheres. Recently, in a survey involving 18,653 sows which was carried out in Northern Italy, the highest rates of return to estrus after artificial insemination were described in July, August and September (19.62%, 17.88%, 17.60% respectively) [34]. In light of our results, it could be hypothesized that this reduction in cortisol secretion during the hot season is one of several factors influencing swine fertility. Therefore, physiological status, rearing conditions and season could act as variables capable of influencing cortisol secretion and its accumulation in hair. When individual sows were evaluated, an elevated number having a cortisol concentration greater than 20 pg/mg was observed only at PD. On the contrary, at BD1, BD2 and WT1, the majority of subjects had a low concentration of cortisol. Since this study involved only a limited number of animals, an exhaustive comparative standard of reference for individual sows under specific physiological, rearing and environmental conditions cannot be provided; however, this will hopefully be possible in the future. It can therefore be concluded that cortisol measurement in swine hair is a reliable tool for obtaining information (which is otherwise very difficult to collect) regarding cortisol concentration measured over a medium-long period of time. It is noteworthy that this technique is not invasive or influenced by sampling, and that it can easily be carried out on many animals directly on the farm. Additional research is needed in order to test whether it is possible to identify the threshold values suitable for identifying problematic animals.
Conflict of interest statement No conflict of interest is declared by any of the authors.
Acknowledgement Work supported by the University of Bologna.
references
[1] EFSA. The welfare of intensively kept pigs. Report of the Veterinary Scientific Committee; 1997, http://ec.europa.eu/ food/fs/sc/oldcomm4/out17_en.pdf.
[2] Love RJ. Definition of seasonal infertility problem in pigs. Vet Rec 1978;103:443–6. [3] Love RJ, Evans G, Klupiec C. Seasonal effects on fertility in gilts and sows. J Reprod Fertil Suppl 1993;48:191–206. [4] Meyer JS, Novak MA. Minireview: hair cortisol: a novel biomarker of hypothalamic-pituitary-adrenocortical activity. Endocrinology 2012;153(9):4120–7. [5] Blackshaw JK, Blackshaw AW. Limitations of salivary and blood cortisol determinations in pigs. Vet Res Commun 1989;13:265–71. [6] Cook CJ, Mellor DJ, Harris PJ, Ingram JR, Matthews LR. Hands-on and hands-off measurement. In: Moberg GP, Mench JA, editors. The biology of animal stress. Wallingford, UK: CABI Publishing; 2000. p. 123–46. [7] Scipioni R, Martelli G, Volpelli LA. Assessment of welfare in pigs. Italian J Anim Sci 2009;8(Suppl. 1):117–37. [8] Hamel AF, Meyer JS, Henchey E, Dettmer AM, Suomi SJ, Novak MA. Effects of shampoo and water washing on hair cortisol concentrations. Clin Chim Acta 2001;412:382–5. [9] Davenport MD, Tiefenbacher S, Lutz CK, Novak MA, Meyer JS. Analysis of endogenous cortisol concentrations in the hair of rhesus macaques. Gen Comp Endocrinol 2006;147:255–61. [10] Tamanini C, Giordano N, Chiesa F, Seren E. Plasma cortisol variations induced in the stallion by mating. Acta Endocrinol 1983;02:447–50. [11] Pol F, Courboulay V, Cotte JP, Martrenchar A, Hay M, Mormède P. Urinary cortisol as an additional tool to assess the welfare of pregnant sows kept in two types of housing. Vet Res 2002;33:13–22. [12] Merlot E, Mounier AM, Prunier A. Endocrine response of gilts to various common stressors: a comparison of indicators and methods of analysis. Physiol Behav 2011;102:259–65. [13] Gow R, Thomson S, Rieder M, Van Uum S, Koren G. An assessment of cortisol analysis in hair and its clinical applications. Forensic Sci Int 2010;196:32–7. [14] Koren L, Mokady O, Karaskov T, Klein J, Koren G, Geffen E. A novel method using hair for determining hormonal levels in wildlife. Anim Behav 2002;63:403–6. [15] Bechshøft TØ, Sonne C, Dietz R, Born EW, Novak MA, Henchey E, et al. Cortisol levels in hair of East Greenland polar bears. Sci Total Environ 2011;409:831–4. [16] Accorsi PA, Carloni E, Valsecchi P, Viggiani R, Gamberoni M, Tamanini C, et al. Cortisol determination in hair and in faeces from domestic cats and dogs. Gen Comp Endocrinol 2008;155:398–402. [17] Cirillo N, Prime SS. Keratinocytes synthesize and activate cortisol. J Cell Biochem 2011;112:1499–505. [18] Mowafy M, Cassens RG. Hair growth in the domestic pigs: histological aspects. J Am Leather Chem Assoc 1976;71:64–70. [19] Kemp B, Soede NM. Reproductive issues in welfare-friendly housing systems in pig husbandry: a review. Reprod Domest Anim 2012;47(Suppl. 5):51–7. [20] Whitely JL, Wilcox DL, Newton JA, Bryant-Greenwood GD, Hartmann PE. Total and free plasma concentration of progesterone, cortisol and oestradiol-17beta during pregnancy, parturition and early lactation in sows. Aust J Biol Sci 1984;37:267–76. [21] Hay M, Meunier-Salaun MC, Brulaud F, Monnier M, Mormede P. Assessment of hypothalamic-pituitary-adrenal axis and sympathetic nervous system activity in pregnant sows through the measurement of glucocorticoids and catecholamines in urine. J Anim Sci 2000;78:420–8. [22] Osterlundh I, Holst H, Magnusson U. Hormonal and immunological changes in blood and mammary secretion in the sow at parturition. Theriogenology 1998;50:465–77. [23] Kattesh HG, Korneggay ET, Knight JW, Gwazdauskas FG, Thomas HR, Notter D. Glucocorticoid concentrations,
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corticosteroids binding protein characteristics and reproduction performance of sows and gilts subjected to applied stress during mid-gestation. J Anim Sci 1980;50: 897–905. Sutherland MA, Niekamp SR, Rodriguez-Zas SL, SalakJohnson JL. Impacts of chronic stress and social status on various physiological and performance measures in pigs of different breeds. J Anim Sci 2006;84:588–96. Peltoniemi OAT, Love RJ, Klupiec C, Evans G. Effect of feed restriction and season on LH and prolactin secretion, adrenal response, insulin and FFA in group housed pregnant gilts. Anim Reprod Sci 1997;49:179–90. Ingram JR, Crockford JN, Matthews LR. Ultradian, circadian and seasonal rhythms in cortisol secretion and adrenal responsiveness to ACTH and yarding in unrestrained red deer (Cervus elaphus) stags. J Endocrinol 1999;162:289–300. Matchock RL, Dorn LD, Susman EJ. Diurnal and seasonal cortisol, testosterone, and DHEA rhythms in boys and girls during puberty. Chronobiol Int 2007;24:969–90. Miller GE, Chen E, Zhou ES. If it goes up, must it come down? Chronic stress and the hypothalamic-pituitaryadrenocortical axis in human. Psychol Bull 2007;133:25–45.
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[29] Alila-Johansson A, Eriksson L, Soveri T, Laakso ML. Serum cortisol levels in goats exhibit seasonal but not daily rhythmicity. Chronobiol Int 2003;20(1):65–79. [30] Ssewannyana E, Lincoln GA, Linton EA, Lowry PJ. Regulation of the seasonal cycle of b-endorphin and ACTH secretion into the peripheral blood of rams. J Endocrinol 1990;124(3):443–54. [31] Cordero M, Brorsen BW, McFarlane D. Circadian and circannual rhythms of cortisol, ACTH, and a-melanocytestimulating hormone in healthy horses. Domest Anim Endocrinol 2012;43(4):317–24. [32] Boswell T, Woods SC, Kenagy GJ. Seasonal changes in body mass, insulin, and glucocorticoids of free-living goldenmantled ground squirrels. Gen Comp Endocrinol 1994;96:339–46. [33] Einarsson JS, Brandt Y, Lundeheim N, Madej A. Stress and its influence on reproduction in pigs: a review. Acta Vet Scand 2008;50:48. http://dx.doi.org/10.1186/1751-0147-50-48. [34] Mazzoni C, Tonon F, Borri E, Raffi V, Scollo A, Bonilauri P, et al. Regular and irregular return to heat distribution in a sample of sows in Northern Italy. Summa Animali da Reddito 2010;1:44–50.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/repbio
Original Research Article
Hair cortisol determination in sows in two consecutive reproductive cycles Maria Laura Bacci *, Eleonora Nannoni, Nadia Govoni, Fabrizio Scorrano, Augusta Zannoni, Monica Forni, Giovanna Martelli, Luca Sardi Department of Veterinary Medical Sciences, DIMEVET, University of Bologna, Via Tolara di Sopra 50, Ozzano dell'Emilia, 40064 Bologna, Italy
article info
abstract
Article history:
Hair analysis has been proposed as a minimally invasive technique capable of furnishing
Received 11 July 2013
information regarding the stress response during medium- and long-term periods. Bristle
Received in revised form
samples were collected from the rump region of sows at three key physiological phases (before
12 May 2014
delivery – BD; weaning time – WT; pregnancy diagnosis – PD) during consecutive reproductive
Accepted 7 June 2014
cycles in order to test swine hair as a reliable matrix of cortisol evaluation. Cortisol was extracted from the bristles and assayed using radioimmunoassay. The highest mean hair
Keywords:
cortisol concentrations were demonstrated (p < 0.001) at the PD time points (20.1 .95 and
Swine
16.29 2.15 pg/mg). Moreover, cortisol was significantly higher (p < 0.001) at BD2 (10.48
Hair
0.96 pg/mg) as compared to BD1 (5.17 0.51 pg/mg) and WT1 (6.01 0.47 pg/mg). The
Cortisol
various physiological phases had a significant effect on cortisol concentration (p < 0.00001)
Chronic stress
with a higher cortisol concentration found during late pregnancy and lactation than in early-
Reproduction
mid pregnancy. This could be due not only to the physiological hormonal status, but also to the different housing conditions (single crates vs. group housing). The season of the year was also observed to have an effect (p < 0.005), with the lowest cortisol concentration recorded during the hot season. # 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
1.
Introduction
Animal welfare becomes increasingly important in the context of intensive breeding. Although some objective parameters for animal welfare have already been identified [1], they are not
efficient when applied to chronic stress conditions. Animal stress responses are linked to many parameters, both subjective and those related to the environment; they depend on the nature, intensity and duration of the stressor. Much attention has been paid to acute stressors; however, in intensive breeding, the most adverse stressors are often
* Corresponding author. Tel.: +39 0512097912; fax: +39 051 2097899. E-mail addresses: [email protected], [email protected] (M.L. Bacci). http://dx.doi.org/10.1016/j.repbio.2014.06.001 1642-431X/# 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
reproductive biology 14 (2014) 218–223
chronic and induce negative effects, swine summer infertility being one such well-known example [2,3]. Although the problem has been extensively studied, and the major stressors, such as temperature and photoperiod, are known, the underlying hormonal mechanisms are not fully understood. Cortisol is often used as a stress biomarker due to the fact that adverse situations trigger an adrenal response, but it is also secreted under favorable conditions, and its circadian biorhythm may have physiological functions in mammals. This causes difficulties in differentiating between the physiological pattern of cortisol secretion and the response to stress. Blood, milk, saliva and urine (short-medium term), or feces and hair (medium-long term) were employed to assess changes in cortisol concentrations [4]. Blood/saliva sampling is stressful per se and confuses data interpretation [5–7]. On the other hand, urine, feces or hair sampling requires less intense handling, but the exact timing of the cortisol deposition is not known. The aims of the present study were to: (1) develop and validate a non–invasive technique for cortisol assessment in porcine bristle, and (2) examine the medium-long term changes of cortisol concentrations in the bristles of breeding sows under farm conditions.
2.
Material and methods
2.1.
Reagents and animals
The isopropanol and methanol were purchased from Carlo Erba (MI, Italy). The bovine serum albumin (BSA), sodium phosphate dibasic dihydrate (Na2HPO4), ethylendiaminetetraacetic acid disodium salt (EDTA Na2), sodium azide (NaN3), heat activated charcoal, dextran and cortisol were obtained from Sigma–Aldrich (St. Louis, MO, USA). The 1,2,6,7-3H cortisol was purchased from Perkin-Elmer (Shelton, CT, USA). Thirty, randomly selected, hybrid Goland sows (white skin, no black hair; mean parity: 4.68 3.1) from a breeding herd in Northern Italy were used in the study. Breeding herd management was based on a batch farrowing system every 3rd week, which led to natural estrus synchronization by
219
weaning. The animals were reared according to the current Italian legislation which implements Council Directive 2008/ 120 EC on pig protection. This study was conducted during two consecutive reproductive cycles occurring in different seasons.
2.2.
Experimental design
From the time of pregnancy diagnosis until 5 days before the expected delivery date (approximately 9 weeks), the sows were kept in collective pens with free access to an external area (gestation area) (Fig. 1). The sows were then moved to the farrowing house where they were individually housed in farrowing stalls until weaning. Lactation lasted approximately 4 weeks. At weaning, the sows entered the mating house where they were individually housed in single crates until pregnancy was diagnosed. They were then moved again to the gestation area and were re-grouped. The sows were monitored from February to November. Thirty and 25 (five sows were culled) sows were used during the first and the second reproductive cycles, respectively. During the entire trial, all the sows were in good health. In each cycle, hair samples were collected: (1) five days pre-partum (before delivery in the first and in the second reproductive cycles: BD1, BD2, respectively), (2) 25–28 days postpartum (weaning time in the first reproductive cycle: WT1) and (3) 70–75 days postpartum, during pregnancy in the first and the second reproductive cycles (PD1, PD2, respectively). The gestation house had a negative pressure ventilation system while the farrowing house was heated. Room temperature (RT) was recorded in both houses by means of data loggers (Model 175 H2, Testo, Lenzkirch, Germany). The monthly mean RT ranged from 29 8C (July) to 17 8C (November) (Fig. 1). Regarding the experimental period, the environmental temperature data were obtained from a nearby weather station (Fig. 2). The hair was shaved off manually, close to the skin on the rump region, from a maximum area of 49 cm2, alternately on the two sides, avoiding reddened or irritated areas. The samples were placed into small plastic bags, individually marked and stored at 4 8C. In addition, the hair re-growth (3 sows) was measured with a decimal Vernier caliper after one month. The mean rate of hair regrowth was 0.7 0.2 cm/month.
Fig. 1 – Schematic representation of the experimental design. AI: artificial insemination; BD1 and BD2: before delivery sampling times; PD1 and PD2: pregnancy diagnosis sampling times; WT1: sampling at weaning (BD1, PD1, WT1: 30 sows; BD2, PD2: 25 sows); RT: room temperature; Environ. T: environmental temperature; Av.: average.
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2.4.
Statistical analysis
Statistical analysis was carried out using Statistica ver 10.0 (StatSoft, Inc., Tulsa, OK, USA). The cortisol data are presented as mean SEM. The data were analyzed using one-way ANOVA followed by the Duncan multiple range test. To evaluate the effects of the reproductive phase (PD vs. BD) and the season, two-way ANOVA was used.
3. Fig. 2 – Environmental temperature recorded during the experiment in the vicinity of the sow piggery. Average (av), minimal (min) and maximal (max) temperature for each day are provided.
2.3.
Measurement of hair cortisol concentration
The bristle sample (250 mg) was washed with water, air dried at RT, placed in a polypropylene tube and covered with isopropanol (5 mL) [8,9]. The tube was gently mixed (3 min at RT), centrifuged (1500 g for 1 min) and the isopropanol was discarded. The bristle sample was again washed with isopropanol, air dried and fully pulverized. The pulver (60 mg) was placed in a glass vial with 2 mL of methanol and incubated overnight at RT with continuous gentle agitation for steroid extraction. After centrifugation (1500 g for 5 min), 1.8 mL of methanol was collected, transferred into a glass tube and evaporated to dryness under an air-stream suction hood (37 8C). The dry extracts were stored at 20 8C. In order to obtain a sample pool for the radioimmunoassay (RIA) validation, 60 mg of pulverized hair from each sow were pooled together, extracted with methanol, dried and analyzed in triplicate during each RIA. Cortisol concentration was determined using a validated radioimmunoassay [10]. The dried extracts were dissolved in RIA buffer (0.3 mL; 74.26 mmol/L Na2HPO4, 12.49 mmol/L EDTA Na, 7.69 mmol/L NaN3) containing 0.1% BSA (pH 7.5) and were shaken for 10 min. The sample (0.1 mL), 1,2,6,7-3H cortisol (0.1 mL, 30 pg/tube) and rabbit anti-cortisol serum (0.1 mL; 1:20,000) were incubated overnight (4 8C); 1 mL of charcoal– dextran solution (0.25%, charcoal and 0.02% dextran in RIA buffer) was then added to the tubes. After 15 min (4 8C), the tubes were centrifuged (15 min, 3000 g), the supernatant was decanted and the radioactivity was immediately measured using a b scintillation counter (Packard C1600, Perkin-Elmer). The cross reactions of various steroids with the rabbit anticortisol serum were as follows: cortisol 100%, corticosterone 9.5%, cortisone 5.3%, 11a-deoxycortisol 5.0%, prednisolone 4.60%, 20a-dihydrocortisone 0.4%, progesterone <0.001% and testosterone <0.001%. Mean cortisol recovery was 96.6 2.2%. The intra- and inter-assay coefficients of variation (5 determinations in triplicate) were 2.76% and 8.5%, respectively. The assay sensitivity was 4.81 pg/tube and was defined as the dose of hormone at 90% binding (B/B0). A high degree of parallelism between the cortisol standards and the porcine hair cortisol was confirmed by a regression test (r2 = 0.99; p < 0.01).
Results
The highest mean hair cortisol concentrations were found at PD1 (20.1 .95 pg/mg) and PD2 (16.29 2.15 pg/mg). Although hair cortisol did not differ significantly between PD1 and PD2, a declining tendency (p < 0.058) was observed during the hot season (Fig. 3). Mean hair cortisol concentrations were significantly higher (p < 0.001) at PD1/PD2 as compared to the other phases. Moreover, the cortisol was significantly higher (p < 0.001) at BD2 (10.48 0.96 pg/mg) in comparison to BD1 (5.17 0.51 pg/mg) and WT1 (6.01 0.47 pg/mg). The twoway ANOVA revealed significant effects regarding the reproductive phase (p < 0.00001) and season (p < 0.005), but there was no significant interaction. On the basis of the mean cortisol concentration, the sows were divided into three categories: (1) 1–10, (2) 10–20 and (3) >20 pg/mg (Fig. 4). At WT1, all the sows fell into the lowest category. At BD1 and BD2, the majority of the sows (90.9% and 55.55%, respectively) were in the lowest category, and the remaining sows were in the middle category (9.1% and 44.45%, respectively). At PD1 (96%) and PD2 (72.23%), the majority of the sows were in either the middle or the high cortisol category.
4.
Discussion
The evaluation of the response to stress, based solely on plasma cortisol concentration, requires a detailed knowledge of the physiological and behavioral patterns of the species and
Fig. 3 – Cortisol concentration (mean W SEM) in sow hair. PD: diagnosis of pregnancy; BD: five days before delivery; WT: weaning time (28 days after delivery). PD1: April; BD1: June; WT1: July; PD2: September; BD2: November (BD1, PD1, WT1: 30 sows; BD2, PD2: 25 sows). Means without common superscripts are significantly different (p < 0.001).
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Fig. 4 – Proportion of sows belonging to different hair cortisol concentration categories. PD: diagnosis of pregnancy; BD: five days before delivery; WT: weaning time (28 days after delivery). PD1: April; BD1: June; WT1: July; PD2: September; BD2: November (BD1, PD1, WT1: 30 sows; BD2, PD2: 25 sows). The cortisol concentration ‘‘>20 pg/mg’’ was present only at PD1 and PD2.
of the type of animal. The cortisol secretion, in addition to being determined by natural biorhythms, is highly regulated and is also affected by the invasive nature of blood sampling. Other biological fluids used for cortisol analysis which can be collected in a non-invasive manner, such as urine or saliva [11] require normalization [12] and provide information on acute stress responses only over a short-medium period of time. The special feature of hair is that it can provide information regarding exposure to endogenous or exogenous compounds over a medium-long period of time. However, additional studies are needed to determine how long the compounds remain in the hair [4,13]. Hair sampling is a simple noninvasive painless procedure; it can be carried out by a nonprofessional and the samples are easy to store and transport [4]. Hair cortisol, as a response endpoint to stressors, has been examined in rhesus macaques [9], wildlife [14,15], dogs and cats [16]. In the current study, a technique for cortisol determination in sow bristles using extraction and RIA was developed, and hair cortisol concentration in sows during the different phases of the reproductive cycle was measured. Cortisol is a steroid hormone considered to be secreted for the most part by the adrenal cortex. Although there is no evidence of skin cortisol production, its synthesis by keratinocytes has been suggested in one report [17]. On the other hand, circulating cortisol reaches the hair papilla and may be accumulated in cells during keratinization; in this study, very slow hair growth was observed. Mowafy and Cassens have reported that swine hair reached a skin depth of approximately 3–4 mm [18]. We therefore assumed that, in pigs, hair samples would provide information relating to a period varying from one to two months before the date of collection, with the exclusion of the last 15 days. Hence, the BD time points provided information regarding a period in which pregnant sows experienced positive conditions with some acute stress. Instead, the PD time points
provided information regarding a period of an increasingly more stressful situation during which the animals were placed in farrowing stalls in which they farrowed and nursed the piglets [19]. Since cortisol blood concentration rises sharply before and during delivery, and slowly returns to normal values at weaning [20], we analyzed hair samples collected at 28 days after farrowing (WT1). However, the WT1 mean cortisol concentration did not differ when compared to BD1, probably due to poor hair growth which prevented the measurement of the accumulation of cortisol in this short period of time; thus, WT sampling was not repeated for the second reproductive cycle. Our data showed that, at BD1 and BD2, the mean cortisol concentrations were significantly lower than at PD1and PD2, indicating a clear influence of the reproductive phase in agreement with the cortisol blood concentration reported by others [21,22]. Significant differences were not found between PD1 and PD2, although, at PD2, the cortisol concentration tended to be lower. However, the cortisol concentration at BD1 was significantly lower than that recorded at BD2. The reliability of the results found at BD1 was confirmed by the identical results obtained at WT1. These data confirmed an important seasonal effect, probably linked to the different conditions of temperature and daylight. The BD2 sampling took place at the end of November while the BD1 sampling took place in June. From this point of view, it was also possible to explain the different trends observed for the cortisol values between PD1 and PD2. At PD2 (September), the cortisol concentration tended to be lower than that obtained at PD1 (April), reflecting the mean blood cortisol concentration of the previous months. In fact, when the adverse situation is prolonged, the circulating cortisol concentrations not only diminish (during heat stress or crowding) [23,24], but the adrenocorticotropic hormone challenge also produces a more limited response [25].
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We can therefore affirm that, during warmer months, such as May, June, July and August, the mean cortisol blood concentration is strongly reduced in sows regardless of the reproductive phase, and vice versa in colder months (October, November, February and March), indicating a seasonal fluctuation of this hormone. To date, seasonal cortisol fluctuations have been described in unrestrained red deer [26], in humans [27,28], in goats [29], in sheep [30], in horses [31] and in squirrels [32]. In swine, seasonal infertility is well known [2,33] and is linked to a complex interaction of environmental factors which, in turn, are mediated by many endocrine signals. Fluctuations in the monthly farrowing rate are observed in the same period in the Northern and Southern hemispheres. Recently, in a survey involving 18,653 sows which was carried out in Northern Italy, the highest rates of return to estrus after artificial insemination were described in July, August and September (19.62%, 17.88%, 17.60% respectively) [34]. In light of our results, it could be hypothesized that this reduction in cortisol secretion during the hot season is one of several factors influencing swine fertility. Therefore, physiological status, rearing conditions and season could act as variables capable of influencing cortisol secretion and its accumulation in hair. When individual sows were evaluated, an elevated number having a cortisol concentration greater than 20 pg/mg was observed only at PD. On the contrary, at BD1, BD2 and WT1, the majority of subjects had a low concentration of cortisol. Since this study involved only a limited number of animals, an exhaustive comparative standard of reference for individual sows under specific physiological, rearing and environmental conditions cannot be provided; however, this will hopefully be possible in the future. It can therefore be concluded that cortisol measurement in swine hair is a reliable tool for obtaining information (which is otherwise very difficult to collect) regarding cortisol concentration measured over a medium-long period of time. It is noteworthy that this technique is not invasive or influenced by sampling, and that it can easily be carried out on many animals directly on the farm. Additional research is needed in order to test whether it is possible to identify the threshold values suitable for identifying problematic animals.
Conflict of interest statement No conflict of interest is declared by any of the authors.
Acknowledgement Work supported by the University of Bologna.
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