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Sows exposed to octylphenol in early gestation: No estrogenic effects in male piglets, but increased rate of stillbirth
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Theriogenology 78 (2012) 1494 –1499 www.theriojournal.com
Sows exposed to octylphenol in early gestation: No estrogenic effects in male piglets, but increased rate of stillbirth Birgitta Graléna, Weethima Visalvethayab, Kalle Ljungvalla, Wichai Tantasuparukb, Leif Norrgrenc, Ulf Magnussona,* b
a Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand c Section of Pathology, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden
Received 24 January 2012; received in revised form 20 June 2012; accepted 21 June 2012
Abstract Octylphenol is an industrial chemical with estrogenic effects both in vitro and in vivo. In this study the effects of short-term intramuscular exposure to 0.1 mg/kg of body weight and 1.0 mg/kg of body weight in early gestation were evaluated in pregnant sows with respect to reproductive parameters in the newborn male piglets, as compared with male piglets from unexposed control sows. The male piglets were examined immediately after birth with respect to the macroscopic appearance of the reproductive organs and testosterone concentration in serum. It was not possible to identify any estrogenic effects in the newborn male piglets. However, in the sows exposed at the highest level of octylphenol, there was an increased number of stillborn piglets and an increased proportion of sows with stillborn piglets in the litter (P ⬍ 0.05). This was an unexpected finding which has not been reported previously. © 2012 Elsevier Inc. All rights reserved. Keywords: Endocrine disruption; Pig; Octylphenol; Stillbirth; Male
1. Introduction It is generally recognized that a growing number of chemicals present in the environment possess estrogenic, antiandrogenic, or other hormonal activities, and are therefore referred to as endocrine disruptors (EDs) [1]. The increasing and ubiquitous presence of EDs in the environment which coincide with possible emerging adverse trends in human reproductive health as discussed by Vidaeff and Sever [2], translates into a
* Corresponding author. Tel.: ⫹46 (0) 18 671000; fax: ⫹46 (0)18 673545. E-mail address: [email protected] (U. Magnusson). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2012.06.028
public health concern regarding the EDs [2]. Notably, the knowledge of the exposure of humans to xenobiotic compounds is scattered and the vast majority of the data of in vivo effects these compounds may have are limited to laboratory animals [3]. Here, we wanted to expand this knowledge base by investigating possible effects of one ED in a large mammal, the pig. Octylphenol, which is a nonionic surfactant widely used in a variety of industrial applications, has been detected in the aquatic environment worldwide [4,5] as well as in human urine [6]. This compound is known from both in vitro and in vivo studies to mainly have estrogenic effects in rats which can be linked to endocrine disruption and deranged development [7–9]. Such
B. Gralén et al. / Theriogenology 78 (2012) 1494 –1499
effects are for example reduced size of the sexual organs and disrupted spermatogenesis [8,9]. There is also a report on transgenerational effects in pigs [10]. In rats, it has been described that the size of all male reproductive organs is programmed by androgen exposure during a short period termed the masculinization programming window (MPW), which precedes the morphologic sexual differentiation [11]. Exposure to endocrine-disrupting chemicals during the MPW of fetal male rats significantly reduced penis, ventral prostate, and seminal vesicle size, along with anogenital distance [11]. The pig, in contrast to laboratory rodents, has longer gestation and prepubertal periods like, for example, humans. This has previously been suggested as an advantage of the pig as a model animal in research regarding EDs [12,13]. Additionally, polytocous species like the pig allow the recording of important reproductive traits, such as litter size [12]. In occidental pig breeds, gonads begin to form on the ventromedial surfaces of the mesonephroi approximately 20 to 21 days postcoitum and male sexual differentiation takes place approximately 26 days postcoitum [14]. Thus, analogous to the situation in the rat, it can be expected that there is a sensitive window for exposure to xenobiotic compounds preceding the morphologic differentiation also in the pig. In this study we test the hypothesis that exposure to the endocrine-disrupting compound octylphenol during the anticipated sensitive window of the development of the reproductive system in the male piglet causes disturbances in the gross anatomy of the reproductive tract of the newborn male piglet as well as the neonatal male sex hormone metabolism. Additionally, gestational parameters in the exposed sows were examined. 2. Materials and methods 2.1. Animals and herd management The animals in this study were kept at a pig breeding farm in Nakorn-Pathom province, 60 km west of Bangkok, Thailand. The sows were bred with boars of Duroc, Yorkshire, or Landrace breed. During gestation the sows were kept in individual stalls in open buildings without walls (N ⫽ 7, referred to as Unit 1 to 7 in this study). They were moved into individual farrowing pens 2 to 4 days before estimated farrowing. The sows were fed 2.0 kg/day of commercial pig feed containing 17% crude protein and 2900 kcal/kg (Thai Food Co., Ltd., Bangkok, Thailand), and had free access to tap water. The weight of the sows was
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estimated to 160 kg and they were in second to fourth gestation. The sows were visually monitored for clinical disease and no clinical signs were observed through the study period. All sows were vaccinated against Aujeszky’s disease, Parvo virus, and atrophic rhinitis. Sow reproduction data, including gestational length, litter size, number of stillborn or abnormal offspring, and gestational length of previous pregnancies, was collected at the farm. All animals in this study were treated according to the ethical standards of Chulalongkorn University. 2.2. Experimental design Pregnancy diagnosis was performed by transabdominal ultrasonography on mated sows on Day 18 or 19 after last AI. Forty-two pregnant sows were randomly selected and divided into three groups; a low exposure group (N ⫽ 14), a high exposure group (N ⫽ 14), and a control group (N ⫽ 14). During Day 20 to 24 in gestation the sows in the low exposure group were given a daily 0.1 mg/kg body weight (bw) im injection of octylphenol (97% pure, Sigma-Aldrich Chemicals, Stockholm, Sweden) diluted in 2 mL corn oil. During the same period the sows in the high exposure group were given a daily 1.0 mg/kg bw im injection of octylphenol diluted in 2 mL corn oil. The sows in the control group were given a daily intramuscular injection of 2 mL corn oil during the same period. The sows were kept on the farm together with other sows in Unit 1 to 7 during the entire gestation. Within 24 h after farrowing total litter weight was recorded and up to five male piglets were randomly selected from each litter. The weight of each male piglet was recorded on the farm before transportation to the Livestock hospital and Veterinary Student Training Center, Chulalongkorn University, 10 km from the farm. At the university blood was collected before the piglets were euthanized and subjected to postmortem examination. Seven sows were excluded from the study because of farrowing after the collection of piglets was finished. Piglets were collected from 13 sows in the low exposure group, from 10 sows in the high exposure group. and from 12 sows in the control group. 2.3. Blood sampling Blood samples were collected by jugular venipuncture in nonheparinized blood tubes. All samples were centrifuged for 10 min at 400 X g within an hour after collection and serum was stored at ⫺20 °C. The serum was transferred to Chulalongkorn University, Bangkok where the blood samples were heat-treated and refrozen before transportation by air to Swedish University of
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Agricultural Sciences, Uppsala, where they were stored at ⫺20 °C. 2.4. Gross postmortem examination and tissue sampling After blood sampling the male piglets were euthanized by intravenous injection of thiopental sodium and subjected to postmortem examination as previously described [15]. Briefly, the testes, epididymides, bulbourethral glands, and seminal vesicles were carefully dissected and weighed. Also, the length of the bulbourethral glands, the penis, the anogenital distance, and the crown to rump distance were recorded, as was chryptorchidism and other abnormalities. The penis was dissected free from the prepuce and the length was measured from the tip to the sigmoid flexure. The testes, epididymides, bulbourethral glands, seminal vesicles, and the prepuce were immediately fixed for histologic examination. The sows and the female piglets were not further examined. 2.5. Analysis of testosterone in plasma Testosterone in serum was determined as a means to assess effects on the male sex hormone metabolism. A solid-phase radioimmunoassay (Coat-A-Count Total Testosterone, Siemens Healthcare Diagnostics Inc., Los Angeles, CA, USA) previously described for analysis of porcine serum was used [16]. The intra-assay CVs were 3.84% at 4.77 nmol/L, 2.6% at 15.97 nmol/L, and 2.02% at 28.9 nmol/L. The interassay CVs were 6.07% at 4.77 nmol/L, 2.16% at 15.97 nmol/L, and 6.07 at 15.97 nmol/L. The average analytical detection limit of the assay was 0.114 nmol/L. 2.6. Histologic analysis relative to tubular cross sectional area After the postmortem examination and weighing of the testes, the whole testes were immersion-fixed in Bouin’s solution for 24 h, washed in water, dehydrated with ethanol, and embedded in paraffin. Subsequently, 5-m sections were cut, mounted on glass slides, and stained with hematoxylin and eosin. Three images from each of the hematoxylin and eosin-stained sections of the testes were captured at magnification X 4 on a Leica DMRB microscope equipped with an Invenio 3S digital camera supplied by DeltaPix Aps (Maalov, Denmark). The resulting image covered an area of 2.7 by 2.0 mm. The images were opened in the ImageJ software (http:// rsbweb.nih.gov/ij/) and from each image a representative area without artifacts was selected, yielding a re-
gion of interest in each image. In the cropped image, a mask was created where the tubuli were manually outlined and the relative cross sectional area of the tubuli was automatically calculated and expressed as a percentage of the designated region of interest. All manual operations were performed by the same person who was unaware of the identity of the sections. 2.7. Statistical evaluations A general linear model was developed, including the effects of treatment, unit in the farm where the sows were kept, and parity number on difference in gestational length compared with the previous gestation, gestational length, difference in litter size compared with the previous gestation, litter size, number of male and female fetuses, and the number of stillborn fetuses, and least squares were analyzed using the general linear model procedure of the SAS-software (Version 9.1, Cary, NC, USA). A mixed linear model was developed for the effect of treatment on piglet body weight, crown to rump distance, anogenital distance, left and right testicular weight, weight of the epididymides, weight of the seminal vesicles, weight of the bulbourethral glands, length of the bulbourethral glands, penis length, testosterone level in serum, and relative tubular area of the testes, and the least square means for the different treatment groups were compared using the MIXED procedure of the SAS software. In all MIXED analyses the litter was used as the experimental unit. The proportion of litters with stillborn piglets was analyzed with chi-square test. In all statistical evaluations an ␣ value of 5% was considered significant. 3. Results 3.1. Sows The number of stillborn piglets was higher (P ⬍ 0.05) in the high exposure group compared with the control, and the number of male piglets was higher (P ⬍ 0.05) in the high exposure group versus the low exposure group (Table 1). There were no differences in gestational length or litter size between treatments. The proportion of litters with stillborn piglets was higher (P ⬍ 0.05) in the high exposure group compared with the control group. 3.2. Gross postmortem examination of piglets There were no differences between treatment groups in any of the parameters, piglet body weight, crown to
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Table 1 Summary of gestational data from control sows and sows exposed to octylphenol (OP) at two different levels.
Gestational length Litter size Number of males/females per litter Proportion of litters with stillborn piglets Number of stillborn piglets Number of stillborn males/females per litter Mummified fetuses
Control (SD) N ⫽ 12
0.1 mg OP/kg bw per day (SD) N ⫽ 13
1 mg OP/kg bw per day (SD) N ⫽ 10
115 days (1.8) 11.1 (2.5) 5.5 (2.0)/5.1 (1.7) 2/12 0.4 (0.7) 0.3 (0.6)/0.07 (0.3) 0.07 (0.3)
115 days (1.8) 11.6 (3.1) 5.4 (2.4)/5.6 (1.8) 4/13 0.5 (0.7) 0.3 (0.5)/0.2 (0.4) 0.2 (0.4)
115 days (2.8) 11.8 (2.0) 5.9 (1.2)/4.4 (1.5) 7/10* 1.1 (1)* 0.7 (0.7)/0.4 (0.7) 0.4 (1)
bw, body weight. * Significant difference (P ⬍ 0.05) from control group.
rump distance, anogenital distance, left and right testicular weight, weight of the epididymides, weight of the seminal vesicles, weight of the bulbourethral glands, length of the bulbourethral glands, or penis length (Table 2). 3.3. Relative testes tubular cross-sectional area The relative area of the tubuli in the cross section of the testes ranged from 10.7% to 31.9%. There were no differences between the treatment groups in the crosssectional area of the tubuli of the testes. 3.4. Testosterone levels in serum The concentrations of serum testosterone ranged from 2.0 to 29 nmol/L in the newborn piglets. There were no differences between the treatment groups in the levels of testosterone in serum (Table 2).
4. Discussion In the experiment described in this report a low dose of octylphenol during early gestation in pregnant sows did not cause any measurable effects on reproductive traits in newborn male piglets. In contrast, there was a significant increase in the number of litters with stillborn piglets in the highest exposure group. The male piglets were the focus of interest in this study. Extensive data were therefore gathered and the data are provided in detail in Table 2 as reference values for some parameters in the newborn male piglet, which are sparse in the literature. It was hypothesized that exposure to the estrogenic compound octylphenol during a critical window of development would alter the morphology of the male reproductive tract and the male sex hormone metabolism in the newborn piglet, as
Table 2 Summary of the morphologic parameters from newborn male control piglets and piglets exposed to octylphenol (OP).
Bw (kg) Crown to rump length (mm) Anogenital distance (mm) Left testis weight (g) Right testis weight (g) Paired epididymides weight (g) Paired seminal vesicles weight (g) Left bulbourethral weight (g) Right bulbourethral weight (g) Left bulbourethral length (mm) Right bulbourethral length (mm) Penis length (mm) Testosterone in serum (nmol/L) Relative tubular area left testis (%) Relative tubular area right testis (%) Data are mean (SD). bw, body weight.
Control (SD) N ⫽ 53
0.1 mg OP/kg bw per day (SD) N ⫽ 55
1 mg OP/kg bw per day (SD) N ⫽ 48
1.53 (0.30) 316 (20.3) 122 (12.1) 0.34 (0.12) 0.35 (0.13) 0.46 (0.11) 0.25 (0.08) 0.30 (0.08) 0.31 (0.08) 18.7 (2.0) 19.0 (2.5) 84.5 (8.7) 7.21 (2.08) 22.0 (3.6) 23.8 (3.3)
1.48 (0.33) 314 (22.3) 124 (13.3) 0.39 (0.21) 0.38 (0.21) 0.47 (0.14) 0.26 (0.09) 0.29 (0.08) 0.29 (0.09) 18.5 (2.0) 18.6 (2.3) 83.9 (12.4) 7.6 (3.1) 22.3 (3.4) 24.1 (4.1)
1.44 (0.32) 308 (23.4) 122 (13.6) 0.36 (0.17) 0.35 (0.17) 0.45 (0.32) 0.24 (0.09) 0.26 (0.08) 0.27 (0.08) 18.3 (1.7) 18.3 (1.7) 80.7 (8.34) 8.0 (4.2) 21.5 (3.7) 23.1 (3.3)
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previously described in prepubertal boars exposed to di(2-ethylhexyl) phthalate [13]. McCoard et al. [14] described the timing of morphologic events in the sexual differentiation in male piglets. However, in rats the morphologic changes in sexual differentiation is preceded by the so-called MPW by a short period of time [11]. Although, the onset of the morphologic sexual differentiation in the pigs has been determined as occurring approximately Day 26 of pregnancy; there are no records of the exact timing of the period analogous to the MPW in the rat. In studies in rats, where adverse effects have been seen on male reproductive organs and steroidogenesis after maternal exposure to octylphenol, doses of 50 to 100 mg/kg bw have been used in different dosage regimens and the animals were examined at several developmental stages [8,9,17]. In our study the intention was to use dosages as low as possible, but still within a range that have caused reproductive disturbances in previous studies in pigs [10]. In order to ascertain the exposure a parenteral route of administration was chosen. Thus, in the present study, dosages were 0.1 and 1.0 mg/kg bw im. Furthermore, the piglets were examined immediately after birth, before the onset of pubertal changes. Both the comparably low dosages and the early examination may explain the lack of effects on the piglets in this study. However, doses of octylphenol of 1 mg/L of drinking water can affect reproductive parameters mildly in rats when administered postnatally, but these rats were examined later in life [18]. Although a dose of 1 mg/kg bw octylphenol can adversely affect fertility in pigs [10], it can be concluded that the dosage regimen used in the present study did not cause any detectable adverse effects in the reproductive system of newborn male piglets. In summary this may be due to one of the following reasons: the dosages used do not affect the sexual development in pigs; the changes may not have been evident until after sexual maturity; or the exposure did not take place during the sensitive window of the development of the male reproductive system. In contrast, this report demonstrates an increased rate of stillborn piglets in sows exposed to octylphenol for only a short period during the first trimester of gestation. It has previously been demonstrated that stillbirth in pigs can increase after exposure of the pregnant sows to estrogenic mycotoxins [19]. In other species, Tyl et al. [20] described the effects of long-term estradiol treatment during gestation in mice, which included an increase in pups that were born dead. Sharpe et al. [18] reported that diethylstilbestrol reduced the litter
size and increased the proportion of male pups in rat litters after the dams had been exposed to the substance from before the onset of gestation to birth. There were no conclusions regarding the mechanism responsible for the loss of pups in either of these two reports. Likewise, it is difficult to explain this finding in the present study, as the study was designed to investigate the effects on male offspring rather than on the sows themselves. Even so, this finding warrants that, in future studies of developmental effects of EDs on offspring examinations of the dam should not be overlooked. Furthermore, the numbers of male piglets per sow in the high exposure group was higher than it was in the low exposure group. It has previously been hypothesized that high levels of estrogen in African wild dogs can skew the sex ratio toward more male offspring per litter [21]. However, the exact mechanism behind that effect is unknown. Similarly, the increased proportion of male pups in rats exposed to diethylstilbestrol during pregnancy remains unexplained [18]. 4.1. Conclusions In this study on the effects of octylphenol during early gestation in a large mammal model, the hypothesized effects on male reproductive parameters were not evident in the offspring. However, there was a surprising, but unexplained, increased rate of stillbirth that warrants further investigation. Acknowledgments Financial support was provided by the Swedish International Development Cooperation Agency. References [1] Toppari J, Skakkebaek NE. Sexual differentiation and environmental endocrine disrupters. Baillieres Clin Endocrinol Metab 1998;12:143–56. [2] Vidaeff AC, Sever LE. In utero exposure to environmental estrogens and male reproductive health: a systematic review of biological and epidemiologic evidence. Reprod Toxicol 2005; 20:5–20. [3] Martin OV, Lester JN, Voulvoulis N, Boobis AR. Human health and endocrine disruption: a simple multicriteria framework for the qualitative assessment of end point specific risks in a context of scientific uncertainty. Toxicol Sci 2007;98:332– 47. [4] Tsuda T, Takino A, Kojima M, Harada H, Muraki K, Tsuji M. 4-Nonylphenols and 4-tert-octylphenol in water and fish from rivers flowing into Lake Biwa. Chemosphere 2000;41:757– 62. [5] Blackburn MA, Kirby SJ, Waldock MJ. Concentrations of alkylphenol polyethoxylates entering UK estuaries. Mar Pollut Bull 1999;38:109 –18. [6] Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL. Exposure of the U.S. population to bisphenol A and 4-tertiary-
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octylphenol: 2003-2004. Environ Health Perspect 2008;116: 39 – 44. Pocar P, Augustin R, Gandolfi F, Fischer B. Toxic effects of in vitro exposure to p-tert-octylphenol on bovine oocyte maturation and developmental competence. Biol Reprod 2003;69: 462– 8. Aydog˘an M, Barlas N. Effects of maternal 4-tert-octylphenol exposure on the reproductive tract of male rats at adulthood. Reprod Toxicol 2006;22:455– 60. Boockfor FR, Blake CA. Chronic administration of 4-tert-octylphenol to adult male rats causes shrinkage of the testes and male accessory sex organs, disrupts spermatogenesis, and increases the incidence of sperm deformities. Biol Reprod 1997; 57:267–77. Bøgh IB, Christensen P, Dantzer V, Groot M, Thøfner IC, Rasmussen RK, et al. Endocrine disrupting compounds: effect of octylphenol on reproduction over three generations. Theriogenology 2001;55:131–50. MacLeod DJ, Sharpe RM, Welsh M, Fisken M, Scott HM, Hutchison GR, et al. Androgen action in the masculinization programming window and development of male reproductive organs. Int J Androl 2010;33:279 – 87. Magnusson U. Can farm animals help to study endocrine disruption? Domest Anim Endocrinol 2005;29:430 –5. Ljungvall K, Karlsson P, Hultén F, Madej A, Norrgren L, Einarsson S, et al. Delayed effects on plasma concentration of testosterone and testicular morphology by intramuscular lowdose di(2-ethylhexyl)phthalate or oestradiol benzoate in the prepubertal boar. Theriogenology 2005;64:1170 – 84. McCoard SA, Wise TH, Ford JJ. Expression levels of Mullerian-inhibiting substance, GATA4 and 17␣-hydroxylase/17,20-
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lyase cytochrome P450 during embryonic gonadal development in two diverse breeds of swine. J Endocrinol 2002;175:365–74. Ljungvall K, Veeramachaneni DN, Hou M, Hultén F, Magnusson U. Morphology and morphometry of the reproductive organs in prepubertal and postpubertal male pigs exposed to di(2-ethylhexyl) phthalate before puberty: precocious development of bulbourethral glands. Theriogenology 2008;70:984 –91. Andersson H, Wallgren M, Rydhmer L, Lundström K, Andersson K, Forsberg M. Photoperiodic effects on pubertal maturation of spermatogenesis, pituitary responsiveness to exogenous GnRH, and expression of boar taint in crossbred boars. Anim Reprod Sci 1998;54:121–37. Sainath SB, Meena R, Kumar CH, Kalapana P, Swetha KN, Devi NS, et al. Embryonic exposure to octylphenol induces changes in testosterone levels and disrupts reproductive efficiency in rats at their adulthood. Food Chem Toxicol 2011;49: 983–90. Sharpe RM, Fisher JS, Millar MM, Jobling S, Sumpter JP. Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production. Environ Health Perspect 1995;103:1136 – 43. Ványi A, Bata A, Glávits R, Kovács F. Perinatal oestrogen syndrome in swine. Acta Vet Hung 1994;42:433– 46. Tyl RW, Myers CB, Marr MC, Sloan CS, Castillo NP, Veselica MM, et al. Two-generation reproductive toxicity study of dietary bisphenol A in CD-1 (Swiss) mice. Toxicol Sci 2008;104: 362– 84. Creel S, Creel NM, Monfort SL. Birth order, estrogens and sex-ratio adaptation in African wild dogs (Lycaon pictus). Anim Reprod Sci 1998;53:315–20.
Theriogenology 78 (2012) 1494 –1499 www.theriojournal.com
Sows exposed to octylphenol in early gestation: No estrogenic effects in male piglets, but increased rate of stillbirth Birgitta Graléna, Weethima Visalvethayab, Kalle Ljungvalla, Wichai Tantasuparukb, Leif Norrgrenc, Ulf Magnussona,* b
a Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand c Section of Pathology, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden
Received 24 January 2012; received in revised form 20 June 2012; accepted 21 June 2012
Abstract Octylphenol is an industrial chemical with estrogenic effects both in vitro and in vivo. In this study the effects of short-term intramuscular exposure to 0.1 mg/kg of body weight and 1.0 mg/kg of body weight in early gestation were evaluated in pregnant sows with respect to reproductive parameters in the newborn male piglets, as compared with male piglets from unexposed control sows. The male piglets were examined immediately after birth with respect to the macroscopic appearance of the reproductive organs and testosterone concentration in serum. It was not possible to identify any estrogenic effects in the newborn male piglets. However, in the sows exposed at the highest level of octylphenol, there was an increased number of stillborn piglets and an increased proportion of sows with stillborn piglets in the litter (P ⬍ 0.05). This was an unexpected finding which has not been reported previously. © 2012 Elsevier Inc. All rights reserved. Keywords: Endocrine disruption; Pig; Octylphenol; Stillbirth; Male
1. Introduction It is generally recognized that a growing number of chemicals present in the environment possess estrogenic, antiandrogenic, or other hormonal activities, and are therefore referred to as endocrine disruptors (EDs) [1]. The increasing and ubiquitous presence of EDs in the environment which coincide with possible emerging adverse trends in human reproductive health as discussed by Vidaeff and Sever [2], translates into a
* Corresponding author. Tel.: ⫹46 (0) 18 671000; fax: ⫹46 (0)18 673545. E-mail address: [email protected] (U. Magnusson). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2012.06.028
public health concern regarding the EDs [2]. Notably, the knowledge of the exposure of humans to xenobiotic compounds is scattered and the vast majority of the data of in vivo effects these compounds may have are limited to laboratory animals [3]. Here, we wanted to expand this knowledge base by investigating possible effects of one ED in a large mammal, the pig. Octylphenol, which is a nonionic surfactant widely used in a variety of industrial applications, has been detected in the aquatic environment worldwide [4,5] as well as in human urine [6]. This compound is known from both in vitro and in vivo studies to mainly have estrogenic effects in rats which can be linked to endocrine disruption and deranged development [7–9]. Such
B. Gralén et al. / Theriogenology 78 (2012) 1494 –1499
effects are for example reduced size of the sexual organs and disrupted spermatogenesis [8,9]. There is also a report on transgenerational effects in pigs [10]. In rats, it has been described that the size of all male reproductive organs is programmed by androgen exposure during a short period termed the masculinization programming window (MPW), which precedes the morphologic sexual differentiation [11]. Exposure to endocrine-disrupting chemicals during the MPW of fetal male rats significantly reduced penis, ventral prostate, and seminal vesicle size, along with anogenital distance [11]. The pig, in contrast to laboratory rodents, has longer gestation and prepubertal periods like, for example, humans. This has previously been suggested as an advantage of the pig as a model animal in research regarding EDs [12,13]. Additionally, polytocous species like the pig allow the recording of important reproductive traits, such as litter size [12]. In occidental pig breeds, gonads begin to form on the ventromedial surfaces of the mesonephroi approximately 20 to 21 days postcoitum and male sexual differentiation takes place approximately 26 days postcoitum [14]. Thus, analogous to the situation in the rat, it can be expected that there is a sensitive window for exposure to xenobiotic compounds preceding the morphologic differentiation also in the pig. In this study we test the hypothesis that exposure to the endocrine-disrupting compound octylphenol during the anticipated sensitive window of the development of the reproductive system in the male piglet causes disturbances in the gross anatomy of the reproductive tract of the newborn male piglet as well as the neonatal male sex hormone metabolism. Additionally, gestational parameters in the exposed sows were examined. 2. Materials and methods 2.1. Animals and herd management The animals in this study were kept at a pig breeding farm in Nakorn-Pathom province, 60 km west of Bangkok, Thailand. The sows were bred with boars of Duroc, Yorkshire, or Landrace breed. During gestation the sows were kept in individual stalls in open buildings without walls (N ⫽ 7, referred to as Unit 1 to 7 in this study). They were moved into individual farrowing pens 2 to 4 days before estimated farrowing. The sows were fed 2.0 kg/day of commercial pig feed containing 17% crude protein and 2900 kcal/kg (Thai Food Co., Ltd., Bangkok, Thailand), and had free access to tap water. The weight of the sows was
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estimated to 160 kg and they were in second to fourth gestation. The sows were visually monitored for clinical disease and no clinical signs were observed through the study period. All sows were vaccinated against Aujeszky’s disease, Parvo virus, and atrophic rhinitis. Sow reproduction data, including gestational length, litter size, number of stillborn or abnormal offspring, and gestational length of previous pregnancies, was collected at the farm. All animals in this study were treated according to the ethical standards of Chulalongkorn University. 2.2. Experimental design Pregnancy diagnosis was performed by transabdominal ultrasonography on mated sows on Day 18 or 19 after last AI. Forty-two pregnant sows were randomly selected and divided into three groups; a low exposure group (N ⫽ 14), a high exposure group (N ⫽ 14), and a control group (N ⫽ 14). During Day 20 to 24 in gestation the sows in the low exposure group were given a daily 0.1 mg/kg body weight (bw) im injection of octylphenol (97% pure, Sigma-Aldrich Chemicals, Stockholm, Sweden) diluted in 2 mL corn oil. During the same period the sows in the high exposure group were given a daily 1.0 mg/kg bw im injection of octylphenol diluted in 2 mL corn oil. The sows in the control group were given a daily intramuscular injection of 2 mL corn oil during the same period. The sows were kept on the farm together with other sows in Unit 1 to 7 during the entire gestation. Within 24 h after farrowing total litter weight was recorded and up to five male piglets were randomly selected from each litter. The weight of each male piglet was recorded on the farm before transportation to the Livestock hospital and Veterinary Student Training Center, Chulalongkorn University, 10 km from the farm. At the university blood was collected before the piglets were euthanized and subjected to postmortem examination. Seven sows were excluded from the study because of farrowing after the collection of piglets was finished. Piglets were collected from 13 sows in the low exposure group, from 10 sows in the high exposure group. and from 12 sows in the control group. 2.3. Blood sampling Blood samples were collected by jugular venipuncture in nonheparinized blood tubes. All samples were centrifuged for 10 min at 400 X g within an hour after collection and serum was stored at ⫺20 °C. The serum was transferred to Chulalongkorn University, Bangkok where the blood samples were heat-treated and refrozen before transportation by air to Swedish University of
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Agricultural Sciences, Uppsala, where they were stored at ⫺20 °C. 2.4. Gross postmortem examination and tissue sampling After blood sampling the male piglets were euthanized by intravenous injection of thiopental sodium and subjected to postmortem examination as previously described [15]. Briefly, the testes, epididymides, bulbourethral glands, and seminal vesicles were carefully dissected and weighed. Also, the length of the bulbourethral glands, the penis, the anogenital distance, and the crown to rump distance were recorded, as was chryptorchidism and other abnormalities. The penis was dissected free from the prepuce and the length was measured from the tip to the sigmoid flexure. The testes, epididymides, bulbourethral glands, seminal vesicles, and the prepuce were immediately fixed for histologic examination. The sows and the female piglets were not further examined. 2.5. Analysis of testosterone in plasma Testosterone in serum was determined as a means to assess effects on the male sex hormone metabolism. A solid-phase radioimmunoassay (Coat-A-Count Total Testosterone, Siemens Healthcare Diagnostics Inc., Los Angeles, CA, USA) previously described for analysis of porcine serum was used [16]. The intra-assay CVs were 3.84% at 4.77 nmol/L, 2.6% at 15.97 nmol/L, and 2.02% at 28.9 nmol/L. The interassay CVs were 6.07% at 4.77 nmol/L, 2.16% at 15.97 nmol/L, and 6.07 at 15.97 nmol/L. The average analytical detection limit of the assay was 0.114 nmol/L. 2.6. Histologic analysis relative to tubular cross sectional area After the postmortem examination and weighing of the testes, the whole testes were immersion-fixed in Bouin’s solution for 24 h, washed in water, dehydrated with ethanol, and embedded in paraffin. Subsequently, 5-m sections were cut, mounted on glass slides, and stained with hematoxylin and eosin. Three images from each of the hematoxylin and eosin-stained sections of the testes were captured at magnification X 4 on a Leica DMRB microscope equipped with an Invenio 3S digital camera supplied by DeltaPix Aps (Maalov, Denmark). The resulting image covered an area of 2.7 by 2.0 mm. The images were opened in the ImageJ software (http:// rsbweb.nih.gov/ij/) and from each image a representative area without artifacts was selected, yielding a re-
gion of interest in each image. In the cropped image, a mask was created where the tubuli were manually outlined and the relative cross sectional area of the tubuli was automatically calculated and expressed as a percentage of the designated region of interest. All manual operations were performed by the same person who was unaware of the identity of the sections. 2.7. Statistical evaluations A general linear model was developed, including the effects of treatment, unit in the farm where the sows were kept, and parity number on difference in gestational length compared with the previous gestation, gestational length, difference in litter size compared with the previous gestation, litter size, number of male and female fetuses, and the number of stillborn fetuses, and least squares were analyzed using the general linear model procedure of the SAS-software (Version 9.1, Cary, NC, USA). A mixed linear model was developed for the effect of treatment on piglet body weight, crown to rump distance, anogenital distance, left and right testicular weight, weight of the epididymides, weight of the seminal vesicles, weight of the bulbourethral glands, length of the bulbourethral glands, penis length, testosterone level in serum, and relative tubular area of the testes, and the least square means for the different treatment groups were compared using the MIXED procedure of the SAS software. In all MIXED analyses the litter was used as the experimental unit. The proportion of litters with stillborn piglets was analyzed with chi-square test. In all statistical evaluations an ␣ value of 5% was considered significant. 3. Results 3.1. Sows The number of stillborn piglets was higher (P ⬍ 0.05) in the high exposure group compared with the control, and the number of male piglets was higher (P ⬍ 0.05) in the high exposure group versus the low exposure group (Table 1). There were no differences in gestational length or litter size between treatments. The proportion of litters with stillborn piglets was higher (P ⬍ 0.05) in the high exposure group compared with the control group. 3.2. Gross postmortem examination of piglets There were no differences between treatment groups in any of the parameters, piglet body weight, crown to
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Table 1 Summary of gestational data from control sows and sows exposed to octylphenol (OP) at two different levels.
Gestational length Litter size Number of males/females per litter Proportion of litters with stillborn piglets Number of stillborn piglets Number of stillborn males/females per litter Mummified fetuses
Control (SD) N ⫽ 12
0.1 mg OP/kg bw per day (SD) N ⫽ 13
1 mg OP/kg bw per day (SD) N ⫽ 10
115 days (1.8) 11.1 (2.5) 5.5 (2.0)/5.1 (1.7) 2/12 0.4 (0.7) 0.3 (0.6)/0.07 (0.3) 0.07 (0.3)
115 days (1.8) 11.6 (3.1) 5.4 (2.4)/5.6 (1.8) 4/13 0.5 (0.7) 0.3 (0.5)/0.2 (0.4) 0.2 (0.4)
115 days (2.8) 11.8 (2.0) 5.9 (1.2)/4.4 (1.5) 7/10* 1.1 (1)* 0.7 (0.7)/0.4 (0.7) 0.4 (1)
bw, body weight. * Significant difference (P ⬍ 0.05) from control group.
rump distance, anogenital distance, left and right testicular weight, weight of the epididymides, weight of the seminal vesicles, weight of the bulbourethral glands, length of the bulbourethral glands, or penis length (Table 2). 3.3. Relative testes tubular cross-sectional area The relative area of the tubuli in the cross section of the testes ranged from 10.7% to 31.9%. There were no differences between the treatment groups in the crosssectional area of the tubuli of the testes. 3.4. Testosterone levels in serum The concentrations of serum testosterone ranged from 2.0 to 29 nmol/L in the newborn piglets. There were no differences between the treatment groups in the levels of testosterone in serum (Table 2).
4. Discussion In the experiment described in this report a low dose of octylphenol during early gestation in pregnant sows did not cause any measurable effects on reproductive traits in newborn male piglets. In contrast, there was a significant increase in the number of litters with stillborn piglets in the highest exposure group. The male piglets were the focus of interest in this study. Extensive data were therefore gathered and the data are provided in detail in Table 2 as reference values for some parameters in the newborn male piglet, which are sparse in the literature. It was hypothesized that exposure to the estrogenic compound octylphenol during a critical window of development would alter the morphology of the male reproductive tract and the male sex hormone metabolism in the newborn piglet, as
Table 2 Summary of the morphologic parameters from newborn male control piglets and piglets exposed to octylphenol (OP).
Bw (kg) Crown to rump length (mm) Anogenital distance (mm) Left testis weight (g) Right testis weight (g) Paired epididymides weight (g) Paired seminal vesicles weight (g) Left bulbourethral weight (g) Right bulbourethral weight (g) Left bulbourethral length (mm) Right bulbourethral length (mm) Penis length (mm) Testosterone in serum (nmol/L) Relative tubular area left testis (%) Relative tubular area right testis (%) Data are mean (SD). bw, body weight.
Control (SD) N ⫽ 53
0.1 mg OP/kg bw per day (SD) N ⫽ 55
1 mg OP/kg bw per day (SD) N ⫽ 48
1.53 (0.30) 316 (20.3) 122 (12.1) 0.34 (0.12) 0.35 (0.13) 0.46 (0.11) 0.25 (0.08) 0.30 (0.08) 0.31 (0.08) 18.7 (2.0) 19.0 (2.5) 84.5 (8.7) 7.21 (2.08) 22.0 (3.6) 23.8 (3.3)
1.48 (0.33) 314 (22.3) 124 (13.3) 0.39 (0.21) 0.38 (0.21) 0.47 (0.14) 0.26 (0.09) 0.29 (0.08) 0.29 (0.09) 18.5 (2.0) 18.6 (2.3) 83.9 (12.4) 7.6 (3.1) 22.3 (3.4) 24.1 (4.1)
1.44 (0.32) 308 (23.4) 122 (13.6) 0.36 (0.17) 0.35 (0.17) 0.45 (0.32) 0.24 (0.09) 0.26 (0.08) 0.27 (0.08) 18.3 (1.7) 18.3 (1.7) 80.7 (8.34) 8.0 (4.2) 21.5 (3.7) 23.1 (3.3)
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previously described in prepubertal boars exposed to di(2-ethylhexyl) phthalate [13]. McCoard et al. [14] described the timing of morphologic events in the sexual differentiation in male piglets. However, in rats the morphologic changes in sexual differentiation is preceded by the so-called MPW by a short period of time [11]. Although, the onset of the morphologic sexual differentiation in the pigs has been determined as occurring approximately Day 26 of pregnancy; there are no records of the exact timing of the period analogous to the MPW in the rat. In studies in rats, where adverse effects have been seen on male reproductive organs and steroidogenesis after maternal exposure to octylphenol, doses of 50 to 100 mg/kg bw have been used in different dosage regimens and the animals were examined at several developmental stages [8,9,17]. In our study the intention was to use dosages as low as possible, but still within a range that have caused reproductive disturbances in previous studies in pigs [10]. In order to ascertain the exposure a parenteral route of administration was chosen. Thus, in the present study, dosages were 0.1 and 1.0 mg/kg bw im. Furthermore, the piglets were examined immediately after birth, before the onset of pubertal changes. Both the comparably low dosages and the early examination may explain the lack of effects on the piglets in this study. However, doses of octylphenol of 1 mg/L of drinking water can affect reproductive parameters mildly in rats when administered postnatally, but these rats were examined later in life [18]. Although a dose of 1 mg/kg bw octylphenol can adversely affect fertility in pigs [10], it can be concluded that the dosage regimen used in the present study did not cause any detectable adverse effects in the reproductive system of newborn male piglets. In summary this may be due to one of the following reasons: the dosages used do not affect the sexual development in pigs; the changes may not have been evident until after sexual maturity; or the exposure did not take place during the sensitive window of the development of the male reproductive system. In contrast, this report demonstrates an increased rate of stillborn piglets in sows exposed to octylphenol for only a short period during the first trimester of gestation. It has previously been demonstrated that stillbirth in pigs can increase after exposure of the pregnant sows to estrogenic mycotoxins [19]. In other species, Tyl et al. [20] described the effects of long-term estradiol treatment during gestation in mice, which included an increase in pups that were born dead. Sharpe et al. [18] reported that diethylstilbestrol reduced the litter
size and increased the proportion of male pups in rat litters after the dams had been exposed to the substance from before the onset of gestation to birth. There were no conclusions regarding the mechanism responsible for the loss of pups in either of these two reports. Likewise, it is difficult to explain this finding in the present study, as the study was designed to investigate the effects on male offspring rather than on the sows themselves. Even so, this finding warrants that, in future studies of developmental effects of EDs on offspring examinations of the dam should not be overlooked. Furthermore, the numbers of male piglets per sow in the high exposure group was higher than it was in the low exposure group. It has previously been hypothesized that high levels of estrogen in African wild dogs can skew the sex ratio toward more male offspring per litter [21]. However, the exact mechanism behind that effect is unknown. Similarly, the increased proportion of male pups in rats exposed to diethylstilbestrol during pregnancy remains unexplained [18]. 4.1. Conclusions In this study on the effects of octylphenol during early gestation in a large mammal model, the hypothesized effects on male reproductive parameters were not evident in the offspring. However, there was a surprising, but unexplained, increased rate of stillbirth that warrants further investigation. Acknowledgments Financial support was provided by the Swedish International Development Cooperation Agency. References [1] Toppari J, Skakkebaek NE. Sexual differentiation and environmental endocrine disrupters. Baillieres Clin Endocrinol Metab 1998;12:143–56. [2] Vidaeff AC, Sever LE. In utero exposure to environmental estrogens and male reproductive health: a systematic review of biological and epidemiologic evidence. Reprod Toxicol 2005; 20:5–20. [3] Martin OV, Lester JN, Voulvoulis N, Boobis AR. Human health and endocrine disruption: a simple multicriteria framework for the qualitative assessment of end point specific risks in a context of scientific uncertainty. Toxicol Sci 2007;98:332– 47. [4] Tsuda T, Takino A, Kojima M, Harada H, Muraki K, Tsuji M. 4-Nonylphenols and 4-tert-octylphenol in water and fish from rivers flowing into Lake Biwa. Chemosphere 2000;41:757– 62. [5] Blackburn MA, Kirby SJ, Waldock MJ. Concentrations of alkylphenol polyethoxylates entering UK estuaries. Mar Pollut Bull 1999;38:109 –18. [6] Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL. Exposure of the U.S. population to bisphenol A and 4-tertiary-
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