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Effects of prenatal stress on the fetal calf
DOMESTIC ANIMAL E N D O C R I N O L O G Y
ELSEVIER
Vol. 14(2):73-80, 1997
EFFECTS OF PRENATAL STRESS ON THE FETAL CALF D.C. Lay, Jr., 1 R.D. Randel, 2,* T.H. Friend,** J.A. Carroll, 3 T.H. Welsh, Jr.,** O.C. Jenkins,*** D.A. Neuendorff,* D.M. Bushong,** and G.M. Kapp 4 *Texas A&M University Agricultural Research and Extension Center, Overton, Texas 75684-0038 USA **Department of Animal Science, Texas A&M University and Texas Agricultural Experiment Station, College Station, Texas 77843-2471 USA and ***Department of Statistics, Texas A&M University, College Station, Texas 77843 USA Received May 8, 1996
Twelve pregnant Brahman cows were randomly assigned to one of two treatment groups: 1) transported in a stock trailer for 24.2 km, unloaded at a second farm and penned for 1 hr, and then returned to the original farm (TRANS, n = 6); or 2) walked through the handling facilities (SHAM, n = 6). Treatments were repeated at 60, 80, 100, 120, and 140 d of gestation. Calves were delivered by cesarean section on d 266 of gestation. The male:female ratio was 4:2 and 5:1 for the TRANS and SHAM treatment groups, respectively. Before calf removal and severance of the umbilical blood flow, a blood sample was collected from the calf to determine plasma concentrations of adrenocorticotropin (ACTH) and cortisol. The calf was then sedated and exsanguinated, after which pituitary and adrenal glands were collected. The adrenals were immediately weighed, and a crosssection from the left adrenal was stored in 4% paraformaldehyde until being embedded in paraffin. Eight sections from each adrenal were sliced (5 t~m), fixed, and then stained with Harris' hematoxylin and eosin. Areas of the cortex and medulla were calculated with a computerized digitizing unit and tracing of the viewed section. The TRANS calves had heavier body weights (BW) (28.7 vs. 23.9 + 1.8 kg; P < 0.07), pituitary glands (12.63 vs. 8.24 + 1.I0 g/kg BW; P < 0.008), and heart weights (5.58 vs. 5,17 _+0.58 g/kg BW; P < 0.05) than did the SHAM calves. Plasma concentrations of ACTH and cortisol did not differ between SHAM and TRANS calves (57 vs. 82 _+ 14 pg/ml and 7.0 vs. 6.7 + 0.9 ng/ml, respectively; P > 0.2). Adrenal gland weight and medulla-to-cortex ratio did not differ between SHAM and TRANS calves (0.61 and 0.73 + 0.03 g and 0.97 and 0.99 + 0.12 g, respectively; P > 0.2). These results suggest that the altered response to stress in prenatally stressed calves is not associated with morphological changes in the adrenal gland but may be due to effects of prenatal stress on the fetal pituitary. O Elsevier Science Inc. 1997 INTRODUCTION Prenatal stress has been shown to alter the hypothalamic-pituitary-adrenal (HPA) response to stress in 3- to 90-d-old rats (1) and 10- and 150-d-old calves (2). Alteration in the HPA response was evidenced by the plasma concentration of cortisol being either greater and(or) maintained for a longer period in situations of stress. These observed effects of prenatal stress are analogous to the results of research using ~'handling" as a stressor of neonatal rodents (e.g., 3-5). The mechanism(s) by which prenatal stress affects plasma cortisol concentration is not known, but it is hypothesized that maternal cortisol crosses the placenta to moderate the HPA axis of the developing fetus (3). Research using radioactive corticosterone injected into pregnant rats has shown that corticosteroid does © Elsevier Science Inc. 1997 655 Avenue of the Americas, New York, NY 10010
0739-7240/97/$17.00 PII $0739-7240(96)00115-4
74
LAY ET AL.
cross the placenta and binds in the fetal hypothalamus (6). The stimulation of plasma glucocorticoids in neonates may be responsible for the greater weight of the ventral cortex and subcortical brain in rats that were "handled" in infancy (7). Although the effects of prenatal stress in rodents have been documented, the mechanism(s) that causes a prenatally stressed animal to respond to a stressor differently from a nonprenatally stressed animal is unknown. Sustained plasma glucocorticoid concentrations could be due to an increase in the amount of cortisol being secreted and(or) a decrease in the rate at which it is cleared from the plasma. Secretion rate relies on many factors, including: the negative feedback effects of plasma cortisol, the release of corticotropin-releasing hormone, and the release of adrenocorticotropic hormone (ACTH). Henry et al. (1) examined one aspect of negative feedback control and found that types I and II glucocorticoid receptors in the hippocampus for corticosterone were less dense in prenatally stressed rats. These receptors are, in part, responsible for the negative feedback control of cortisol secretion (8,9). Lay et al. (10) found that prenatally stressed calves had slower cortisol clearance rates than did controls. Slower clearance rates and an altered negative feedback system are two possible mechanisms to explain the greater plasma cortisol concentrations of prenatally stressed animals in response to stress. It is also important to examine other aspects of the HPA axis to determine their importance in the altered stress response of prenatally stressed animals. The objective of this experiment was to determine if physiological and(or) morphological differences exist in prenatally stressed calves that may he responsible for an alteration of the stress response. Plasma concentrations of cortisol and ACTH, body and organ weights, and adrenal morphology were examined to identify potential differences between control calves and prenatally stressed calves.
MATERIALS AND METHODS Twelve pregnant Brahman cows were randomly assigned to one of two treatment groups: transported in a stock trailer for 24.2 km, unloaded at a second ranch and penned for 1 hr, and then returned to the original ranch (TRANS, n = 6); or merely walked through the handling facilities at the home ranch (SHAM, n = 6). In order to precisely estimate the chronological age of the conceptus, the Brahman cows were artificially inseminated. Pregnancy status was determined by rectal palpation before being assigned to treatment. Treatments were repeated at 60, 80, 100, 120, and 140 d of gestation (+5 d maximum). All cows from each treatment were maintained in the same herd. When a cow reached one of the five designated stages of gestation, the entire herd was brought into a corral and sorted. Those cows not at the appropriate stage of gestation to receive their treatment were returned to pasture, while the cows who were to receive treatment were herded through the facility. After body weight was recorded, a blood sample was collected via tail venipuncture and immediately placed in an ice bath until centrifugation. The TRANS cows were loaded onto a trailer and transported for 24.2 km. One-and-one-half hours later, when the TRANS cows returned, all cows were again herded through the facility for the collection of another blood sample and the recording of body weight. Calves were delivered by cesarean section on d 266 of gestation. Cesarean sections were performed under the guidelines of the Animal Care and Use Committee of Texas A&M University. Increases in fetal cortisol stimulate the normal induction of parturition (1 l). This elevated concentration of circulating cortisol gradually decreases over a 2-d period after parturition (12). If the fetal HPA axis differs between treatments, then differences between plasma concentrations of cortisol necessary to induce parturition, or
PRENATAL STRESS ON THE FETAL CALF
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otherwise associated with parturition, may exist between treatments and could alter the morphology of the fetal adrenal. For this reason, cesarean sections were performed before the expected delivery date. In preparation for the cesarean sections, dams were given local iniections of 1% lidocaine at the point of incision, Cows were not given a general anesthetic because it could affect the data collected from the fetal calf, and previous experience found that cows exhibited only minor discomfort during this procedure. During the cesarean section and before the umbilical cord was severed, the calf's head was extended out of the abdominal cavity and a 5-ml jugular vein blood sample was collected with a solution of 1% NaEDTA as an anticoagulant. The calf was then removed from the uterus, given 1 ml of xylazine intravenously, weighed, and exsanguinated. Sedation with xylazine does not affect plasma cortisol concentration (13); in addition, the short duration between sedation and adrenal gland collection was assumed not to affect adrenal morphology. The blood samples from the calves provided endocrine data on whether baseline differences in the HPA axis existed in utero. The calf's adrenal glands and pituitary gland were immediately collected and weighed. The liver, kidney, and heart were also weighed. For histological examination, the left adrenal gland was removed and a 4- to 5-ram section was collected from the elongated lobe. This section was then stored in 4% paraformaldehyde until being embedded in paraffin. Tissue sections (n = 8) of 5-~m thickness were placed on slides to provide essentially concentric circles to represent the entire cross-section of the cortical and medullary components of the gland. Eight sections (5 ~m) from the embedded adrenal glands were fixed and then stained with Harris' hematoxylin and eosin. Adrenal gland sections were viewed with a microscope at 2.5x magnification. Areas of the cortex and medulla were calculated with a computerized digitizing unit and tracing of the viewed section. Plasma cortisol and ACTH concentrations were determined on duplicate samples with commercially available RIA kits (Pantex, Santa Monica, CA, and ICN Biomedicals Inc., Los Angeles, CA, respectively). These antibodies have been validated for cattle by the Texas Veterinary Medical Diagnostic Laboratory and have been used extensively in our laboratory (e.g., Refs. 14-19). Samples were reassayed if the duplicates differed by more than 5%. The intra-assay coefficient of variation (CV) for cortisol was 12.9%, and the interassay CV was 5.8%. The intra-assay CV for ACTH was 9.2%, and the inter-assay CV was 11%. The cross-reactivity values of the cortisol antiserum were as follows: cortisol, 100%; prednisolone, 40%; l l-desoxycortisol, 13.3%; corticosterone, 10.5%; and cortisone, prednisone, and dexamethasone <3.1% (analyses by Pantex). The cross-reactivity values of the ACTH antiserum were as follows: ACTH (1-24), 102%; and melenkephalin, leu-enkephalin, neurotensin, f3-endorphin, substance, P, somatostatin, and o~-melanocyte stimulating hormone <0.5% (analyses by ICN). Data for cortisol, body weight, organ weight as a proportion of body weight, and ratio of medulla to cortex were analyzed by use of the Wilcoxon-Mann-Whitney test (20). RESULTS Calves from the TRANS treatment had greater body weights on d 266 of gestation than did SHAM calves (P < 0.07; Table 1). Because body weight differed between the treatments, the remaining organ weight data were analyzed as organ weight-to-body weight ratios. These analyses found that TRANS calves had greater pituitary gland (P < 0.008: Table 1) and heart weights (P < 0.05). Although mean adrenal weight appeared to be greater for TRANS calves, there was no statistical difference after correcting for the larger birth weight.
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TABLE 1. DATA FOR CALVESDELIVEREDBY CESAREANSECTIONON D 266 OF GESTATION.a Parameter Body weight (BW) (kg) Right adrenal (mg/kg BW) Left adrenal (mg/kg BW) Adrenal cortex (mm 2) Adrenal medulla (ram 2) Pituitary (mg/kg BW) Anterior pituitary (mg/kg BW) Right kidney (g/kg BW) Left kidney (g/kg BW) Liver (g/kg BW) Heart (g/kg BW) Cortisol (ng/ml) ACTH (pg/ml)
Sham
Transportation
23.93 + 1.12u 20.43 + 4.26 20.98 + 4.33 8.10 5:0.75 8.07 5:1.47 8.24 5:1.70 a 6.38 5:1.35 1,76 5:0.34 1.76 5:0.34 14.26 5:2.70 5.17 5:1.01 t" 7.12 + 1.64 57.19 5:6.36
28.71 + 2.48 c 26.80 5:2.22 25.77 5:1.84 9.13 5:0.55 8.78 5:0.51 12,63 5:0.65 e 8.66 5:0,51 2.17 5:0.10 2.20 5:0.08 20.50 5:2.30 5.58 + 0.15 g 6.69 + 1.05 81.86 +_21.30
"Data are presented as the mean -t- SE. Organ weight data were adjusted according to body weight before statistical analysis was performed. b.c Numbers within rows with different superscripts differ (P < 0.07). d,e Numbers within rows with different superscripts differ (P < 0.008). f'g Numbers within rows with different superscripts differ (P < 0.05).
The fetal adrenal gland appeared to be as well organized as that of an adult. The zona glomerulosa, zona fasiculata, zona reticularis, and adrenal medulla were clearly separated and organized (Figures 1 and 2). However, some of the adrenals did appear to have distinct medullary cell areas within the cortisol area. The TRANS calves did have heavier heart weights (P < 0.05; Table 1); however, there were no treatment differences for kidney or liver weights (P < 0.10; Table 1). Neither plasma concentrations of cortisol nor ACTH differed between treatments (P < 0.20; Table 1), and values were comparable to previously reported baseline concentrations for cattle (14-16,21).
Figure 1. Cross-section of the left adrenal gland of a Brahman calf (266 d gestational age). The dam of the calf was subjected to transportation at 60, 80, 100, 120, and 140 d of gestation; therefore, the calf was considered to be exposed to "prenatal" stress. Section is viewed at 1.5x magnification. The zona glomerulosa (G), zona fasiculata (F), zona reticularis (R), and the medulla (M) are indicated on the figure. The size bar represents 1 ram.
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Figure 2. Cross-section of the left adrenal gland of a Brahman calf (266 d gestational age). The danl of the calf was not subjected to transportation during gestation; therefore, the calf was considered as a "control" for this study. Section is viewed at 1.5× magnification. Tile zona glomerulosa (G), zona fasiculata (F), zona reticularis (R), and the medulla (M) are indicated on the figure. The size bar represents 1 ram.
DISCUSSION The application of transportation to pregnant Brahman cattle appeared to universally increase both body weight and organ weight of the fetal calf. Because body weight proved to be greater for the T R A N S calves, organ weights were compared on a gram-to-kilogram of body weight basis and still appeared to be greater (Table I). However, not all of these differences proved to be significant. This study used only six calves for each treatment. Possibly, a larger sample size would help to clarify the effect of prenatal stress on fetal growth parameters. Transportation was used as a stressor in this study. The component(s) of the transportation treatment that may be responsible for the treatment differences is an important consideration. Not only were these cows transported, but they were also unloaded and held at another facility for 1 hr. There were no other animals at this facility; however, environmental factors other than transportation could still have affected the cows at this time. Additionally, these cows had calves that remained at the original farm. Clearly, the transportation stress imposed on these cows was a multifaceted stressor composed of transportation, relocation, and isolation from their offspring. Although comparisons between the cow and rat are tenuous, to date, the only species on which physiological prenatal stress studies have been conducted in depth are the rodent and the cow (this study). Obviously, these species are extremely different, and any mechanism acting via the placenta would likely differ. The placentation of the rodent is characterized as hemochorial, whereas that o f the cow is epitheliochorial. Therelore, the placenta of the cow offers a more significant barrier to maternal hormonal influences, With these differences in mind, we offer the following thoughts as to the mechanism responsible for the effects of prenatal stress. In the abundant literature on the effects of prenatal stress on rodents, none could be found that reported birth weight. Genetic potential cannot be changed by prenatal stress:
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therefore, possible causes for the weight difference could be due to either nutritional or hormonal influences. If the transportation treatment somehow influenced the behavior of the cow such that she increased her feed intake, or if it increased the blood flow to the fetus, then fetal birth weights could be increased. However, transportation resulted in larger pituitary glands in the calves, which may suggest a hormonal cause for the increased birth weight. Possibly, the larger pituitary gland may have been secreting more growth hormone, thereby causing the TRANS calves to be heavier. Generally, fetal growth is attributed to genetic potential as well as placental size, although insulin-like growth factor-II has been shown to have significant influence at least in the rodent (22). In support for the effects of stress increasing growth hormone, Nogami et al. (23) found that dexamethasone induced the differentiation of growth hormone-producing cells at an early age in the rat. In contrast to data for humans and rats, which found that glucocorticoids could stimulate growth hormone release, Sartin et al. (24) found that cortisol inhibited growth hormone release from the pituitary in sheep. Accordingly, any increase in fetal plasma cortisol due to maternal stress should have a negative effect on body weight. If the bovine pituitary can respond with a glucocorticoid-stimulated increase in growth hormone, similar to the rat, then this may be one possible explanation for the larger calves from TRANS cows found in this study. The effects of prenatal stress on fetal body weight warrant more detailed investigation. The heavier heart weight of the TRANS calves is an interesting finding that also deserves further attention. It is intriguing that the differences observed in this study are well after the application of transportation stress (120 d posttreatment). However, the effects observed in rodents have been similar. Henry et al. (I) subjected pregnant rats to restraint stress during the third week of gestation and noted the effects of prenatal stress 90 d later. The 90 d is less than our 120 d; however, the developmental stage of the rodent is much more advanced at this age than that of the cow. In our study, both control and treatment cows were housed together and treated identically, except for the application of transportation stress. The fact that treatment differences were noted well after the application of transportation illustrates the robustness of prenatal stress effects. If prenatal stress influences the function of the fetal HPA axis, then it is possible that plasma concentrations of cortisol and ACTH could differ at birth between calves from the TRANS and SHAM treatment groups. Shanks et al. (25) suggested that corticosteroid baseline differences between neonatally handled and unhandled rodents caused the differences in the corticosteroid response of these animals at maturity. Neither concentrations of plasma cortisol nor ACTH differed (P < 0.20) between treatments in fetal calves of this study. This suggests that if differences in the plasma cortisol response of calves in response to stress are affected by prenatal stress, then these differences are not due to altered baseline concentrations but to threshold differences in the activation of the HPA axis. The enlarged pituitary glands suggest that this component of the axis is responsible for the greater glucocorticoid response of prenatally stressed animals, perhaps in response to hypertrophy or hyperplasia consequences of increased corticotropin-releasing hormone and(or) vasopressin. Transportation and other common management procedures cause activation of the HPA axis. Our research has found that repeated activation of the HPA axis during pregnancy produced not only a larger fetal body weight at 266 d of gestation, but also heavier fetal pituitary glands. The pituitary gland is critical for many functions of the body including growth (growth hormone, thyroid-stimulating hormone [TSH], prolactin [PRL]), coping with stress (ACTH. TSH, PRL), and controlling reproduction (luteinizing hormone, fol-
PRENATAL STRESS ON THE FETAL CALF
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licle-stimulating hormone). Because such an important fetal endocrine gland is affected by maternal stress during gestation, the effects of prenatal stress may be profound and serve to precondition or imprint the postnatal experience on endocrine glands associated with growth, reproduction, and tolerance to biological stressors. Further research is warranted to determine the long-term effects of prenatal stress on reproduction, growth, and the stress response. ACKNOWLEDGMENTS/FOOTNOTES The authors thank Dr. Larry Johnson, Department of Veterinary Medicine, Texas A&M University, for help in quantifying adrenal gland areas and photography. This research was partially funded through the USDA Animal Health Formula Funds. The manuscript includes research supported and conducted by the Texas Agricultural Experiment Station, The Texas A&M University System. Furthermore, this study was a contribution to the Western Regional Research Project W-112, Reproductive Performance in Domestic Ruminants. Present Address: Dr. Donald C. Lay, Jr., Iowa State University, 337C Kildee Hall, Ames, IA 50011. 2 Correspondence: Dr. Ron D. Randel, Texas A&M University Agricultural Research and Extension Center, Overton, TX 75684-0038. 3Present Address: Dr. Jeff A. Carroll, Animal Physiology Research Unit, Rm S-107, ASRC, Univ. of Missouri, Columbia, MO 65221. 4 Present Address: Gabrielle M. Kapp, Laboratory Animal Resources and Research Facility, Texas A&M University, College Station, TX 77843-4473.
REFERENCES 1. Henry C, Kabbaj M, Simon H, LeMoal M, Maccari S. Prenatal stress increases the hypothalamo-pituitaryadrenal axis response in young and adult rats. J Neuroendocrinol 6:341-345, 1994. 2. Lay DC Jr, Friend TH, Randel RD, Jenkins OC, Neuendorff DA. Kapp GM, Bushong DM. ACTH dose response and some physiological effects of transportation on pregnant Brahman cattle. J Anim Sci 74:18061811, 1996. 3. Levine S, Mullins RF. Early experience and behavior: The psychobiology of development. In: Hormones in Infancy, Newton G and Levine S (eds). Charles C. Thomas, Springfield, IL, p. 168-197, 1968. 4. Levine S, Haltmeyer FF, Karas FC, Denenberg VH. Psychological and behavioral effects of infantile stimulation. Physiol Behav 2:55-59, 1967. 5. Denenberg VH. Effects of exposure to stressors in early life upon later behavioral and biological processes. In: Society, Stress and Disease, Levi L (ed), Oxford University Press, London, p. 269-281, 1975. 6. Zarrow MX, Philpott JE, Denenberg VH. Passage of C-4-corlicosterone from the rat mother to the fetus and neonate. Nature 226:1058-1059, 1970. 7. Tapp JT, Markowitz H. Infant handling: Effects on awfidance learning, brain weight, and cholinesterase activity. Science 140:486M-87, 1963. 8. McEwen BS, De Kloet ER, Rostene W. Adrenal steroid receptors and actions in the nervous system. Physiol Rev 66:1121-1188, 1986. 9. De Kloet ER, Reul JMHM. Feedback action and tonic influence of corticosteroids on brain function: A concept arising from the heterogeneity of brain receptor systems. Psychoneuroendocrinology 12:83-105, 1987. 10. Lay DC Jr, Friend TH, Randel RD, Neuendorff DA, Lanier EK, Bushong DM. Effects of prenatal stress on cortisol clearance rate and the behavioral and physiological response to branding in Brahman calves. J Anita Sci 73[Suppl. 1]:126, 1995. 11. Jainudeen MR, Hafez ESE. Gestation, prenatal physiology and parturition. In: Reproduction in Farm Animals, Hafez ESE (ed). Lea & Febiger, Philadelphia, p. 247-283, 1980. 12. Hoyer C, Grunert E, Jochle W. Plasma glucocorticoid concentrations in calves as an indicator of stress during parturition. Am J Vet Res 51:1882-1884, 1990. 13. Brearley JC, Dobson H, Jones RS. Investigations into the effect of two sedatives on the stress response in cattle. J Vet Pharmacol Ther 13:367-377, 1990. 14. Lay DC Jr, Friend TH, Grissom KK, Hale RL, Bowers CL. Novel breeding box has variable effects on heart rate and cortisol response of cattle. Appl Anim Behav Sci 35:1-10, 1992. 15. Lay DC Jr, Friend TH, Randel RD, Bowers CL, Neuendorff DA, Grissom KK. Does early maternal deprivation affect a calf's physiological and behavioral reactions to later stress'? J Anim Sci 70[Suppl. 1]:162. 1992. 16. Lay DC Jr, Friend TH, Grissom KK, Bowers CL, Mal ME. Effects of freeze or hot-iron branding of Angus calves on some physiological and behavioral indicators of steers. Appl Anita Behav Sci 33:137-147. 1992.
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17. Lay DC Jr, Friend TH, Randel RD, Bowers CL, Grissom KK, Jenkins OC. Behavioral and physiological effects of freeze or hot-iron branding on crossbred cattle. J Anim Sci 70:330-336, 1992. 18. Lay DC Jr, Friend TH, Bowers CL, Grissom KK, Jenkins OC. A comparative physiological and behavioral study of freeze and hot-iron branding using dairy cows. J Anita Sci 70:1121-1125, 1992. 19. Bowers CL, Friend TH, Grissom KK, Lay DC Jr. Confinement of lambs (Ovis aries) in metabolism stalls increased adrenal function, thyroxine and motivation for movement. Appl Anim Behav Sci 36:149-158, 1993. 20. Siegel S, Castellan NJ Jr. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, Inc., New York, 1988. 21. Sartin JL, Kemppainen RJ, Cummins KA, Williams JC. Plasma concentrations of metabolic hormones in high and low producing dairy cows. J Dairy Sci 71:650-657, 1988. 22. Owens JA. Endocrine and substrate control of fetal growth: placental and maternal influences and insulinlike growth factors. Reprod Fertil Dev 3:501-517, 1991. 23. Nogami H, Tachibana T, Katayama T, Ishikawa H. The free structure of dexamethasone-induced growth hormone cells in the anterior pituitary gland of the rat fetus. Arch Histol Cytol 58:581-589, 1995. 24. Sartin JL, Kemppainen RJ, Coleman ES, Steele B, Williams JC. Cortisol inhibition of growth hormonereleasing hormone-stimulated growth hormone release from cultured sheep pituitary cells. J Endocrinol 141:517-525, 1994. 25. Shanks N, Viau V, Plotsky PM, Meaney MJ. Early experience regulates the development of hypothalamic CRH gene expression and HPA responses to stress. Adaptive and Maladaptive Responses to Stress: Relationships Between Behavioral Responses and Neuroendocrine Systems. Proceedings of the International Society of Psychoneuroendocrinology XXIII Congress, Univ. of Wisconsin-Madison p. 190-192, 1992.
ELSEVIER
Vol. 14(2):73-80, 1997
EFFECTS OF PRENATAL STRESS ON THE FETAL CALF D.C. Lay, Jr., 1 R.D. Randel, 2,* T.H. Friend,** J.A. Carroll, 3 T.H. Welsh, Jr.,** O.C. Jenkins,*** D.A. Neuendorff,* D.M. Bushong,** and G.M. Kapp 4 *Texas A&M University Agricultural Research and Extension Center, Overton, Texas 75684-0038 USA **Department of Animal Science, Texas A&M University and Texas Agricultural Experiment Station, College Station, Texas 77843-2471 USA and ***Department of Statistics, Texas A&M University, College Station, Texas 77843 USA Received May 8, 1996
Twelve pregnant Brahman cows were randomly assigned to one of two treatment groups: 1) transported in a stock trailer for 24.2 km, unloaded at a second farm and penned for 1 hr, and then returned to the original farm (TRANS, n = 6); or 2) walked through the handling facilities (SHAM, n = 6). Treatments were repeated at 60, 80, 100, 120, and 140 d of gestation. Calves were delivered by cesarean section on d 266 of gestation. The male:female ratio was 4:2 and 5:1 for the TRANS and SHAM treatment groups, respectively. Before calf removal and severance of the umbilical blood flow, a blood sample was collected from the calf to determine plasma concentrations of adrenocorticotropin (ACTH) and cortisol. The calf was then sedated and exsanguinated, after which pituitary and adrenal glands were collected. The adrenals were immediately weighed, and a crosssection from the left adrenal was stored in 4% paraformaldehyde until being embedded in paraffin. Eight sections from each adrenal were sliced (5 t~m), fixed, and then stained with Harris' hematoxylin and eosin. Areas of the cortex and medulla were calculated with a computerized digitizing unit and tracing of the viewed section. The TRANS calves had heavier body weights (BW) (28.7 vs. 23.9 + 1.8 kg; P < 0.07), pituitary glands (12.63 vs. 8.24 + 1.I0 g/kg BW; P < 0.008), and heart weights (5.58 vs. 5,17 _+0.58 g/kg BW; P < 0.05) than did the SHAM calves. Plasma concentrations of ACTH and cortisol did not differ between SHAM and TRANS calves (57 vs. 82 _+ 14 pg/ml and 7.0 vs. 6.7 + 0.9 ng/ml, respectively; P > 0.2). Adrenal gland weight and medulla-to-cortex ratio did not differ between SHAM and TRANS calves (0.61 and 0.73 + 0.03 g and 0.97 and 0.99 + 0.12 g, respectively; P > 0.2). These results suggest that the altered response to stress in prenatally stressed calves is not associated with morphological changes in the adrenal gland but may be due to effects of prenatal stress on the fetal pituitary. O Elsevier Science Inc. 1997 INTRODUCTION Prenatal stress has been shown to alter the hypothalamic-pituitary-adrenal (HPA) response to stress in 3- to 90-d-old rats (1) and 10- and 150-d-old calves (2). Alteration in the HPA response was evidenced by the plasma concentration of cortisol being either greater and(or) maintained for a longer period in situations of stress. These observed effects of prenatal stress are analogous to the results of research using ~'handling" as a stressor of neonatal rodents (e.g., 3-5). The mechanism(s) by which prenatal stress affects plasma cortisol concentration is not known, but it is hypothesized that maternal cortisol crosses the placenta to moderate the HPA axis of the developing fetus (3). Research using radioactive corticosterone injected into pregnant rats has shown that corticosteroid does © Elsevier Science Inc. 1997 655 Avenue of the Americas, New York, NY 10010
0739-7240/97/$17.00 PII $0739-7240(96)00115-4
74
LAY ET AL.
cross the placenta and binds in the fetal hypothalamus (6). The stimulation of plasma glucocorticoids in neonates may be responsible for the greater weight of the ventral cortex and subcortical brain in rats that were "handled" in infancy (7). Although the effects of prenatal stress in rodents have been documented, the mechanism(s) that causes a prenatally stressed animal to respond to a stressor differently from a nonprenatally stressed animal is unknown. Sustained plasma glucocorticoid concentrations could be due to an increase in the amount of cortisol being secreted and(or) a decrease in the rate at which it is cleared from the plasma. Secretion rate relies on many factors, including: the negative feedback effects of plasma cortisol, the release of corticotropin-releasing hormone, and the release of adrenocorticotropic hormone (ACTH). Henry et al. (1) examined one aspect of negative feedback control and found that types I and II glucocorticoid receptors in the hippocampus for corticosterone were less dense in prenatally stressed rats. These receptors are, in part, responsible for the negative feedback control of cortisol secretion (8,9). Lay et al. (10) found that prenatally stressed calves had slower cortisol clearance rates than did controls. Slower clearance rates and an altered negative feedback system are two possible mechanisms to explain the greater plasma cortisol concentrations of prenatally stressed animals in response to stress. It is also important to examine other aspects of the HPA axis to determine their importance in the altered stress response of prenatally stressed animals. The objective of this experiment was to determine if physiological and(or) morphological differences exist in prenatally stressed calves that may he responsible for an alteration of the stress response. Plasma concentrations of cortisol and ACTH, body and organ weights, and adrenal morphology were examined to identify potential differences between control calves and prenatally stressed calves.
MATERIALS AND METHODS Twelve pregnant Brahman cows were randomly assigned to one of two treatment groups: transported in a stock trailer for 24.2 km, unloaded at a second ranch and penned for 1 hr, and then returned to the original ranch (TRANS, n = 6); or merely walked through the handling facilities at the home ranch (SHAM, n = 6). In order to precisely estimate the chronological age of the conceptus, the Brahman cows were artificially inseminated. Pregnancy status was determined by rectal palpation before being assigned to treatment. Treatments were repeated at 60, 80, 100, 120, and 140 d of gestation (+5 d maximum). All cows from each treatment were maintained in the same herd. When a cow reached one of the five designated stages of gestation, the entire herd was brought into a corral and sorted. Those cows not at the appropriate stage of gestation to receive their treatment were returned to pasture, while the cows who were to receive treatment were herded through the facility. After body weight was recorded, a blood sample was collected via tail venipuncture and immediately placed in an ice bath until centrifugation. The TRANS cows were loaded onto a trailer and transported for 24.2 km. One-and-one-half hours later, when the TRANS cows returned, all cows were again herded through the facility for the collection of another blood sample and the recording of body weight. Calves were delivered by cesarean section on d 266 of gestation. Cesarean sections were performed under the guidelines of the Animal Care and Use Committee of Texas A&M University. Increases in fetal cortisol stimulate the normal induction of parturition (1 l). This elevated concentration of circulating cortisol gradually decreases over a 2-d period after parturition (12). If the fetal HPA axis differs between treatments, then differences between plasma concentrations of cortisol necessary to induce parturition, or
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otherwise associated with parturition, may exist between treatments and could alter the morphology of the fetal adrenal. For this reason, cesarean sections were performed before the expected delivery date. In preparation for the cesarean sections, dams were given local iniections of 1% lidocaine at the point of incision, Cows were not given a general anesthetic because it could affect the data collected from the fetal calf, and previous experience found that cows exhibited only minor discomfort during this procedure. During the cesarean section and before the umbilical cord was severed, the calf's head was extended out of the abdominal cavity and a 5-ml jugular vein blood sample was collected with a solution of 1% NaEDTA as an anticoagulant. The calf was then removed from the uterus, given 1 ml of xylazine intravenously, weighed, and exsanguinated. Sedation with xylazine does not affect plasma cortisol concentration (13); in addition, the short duration between sedation and adrenal gland collection was assumed not to affect adrenal morphology. The blood samples from the calves provided endocrine data on whether baseline differences in the HPA axis existed in utero. The calf's adrenal glands and pituitary gland were immediately collected and weighed. The liver, kidney, and heart were also weighed. For histological examination, the left adrenal gland was removed and a 4- to 5-ram section was collected from the elongated lobe. This section was then stored in 4% paraformaldehyde until being embedded in paraffin. Tissue sections (n = 8) of 5-~m thickness were placed on slides to provide essentially concentric circles to represent the entire cross-section of the cortical and medullary components of the gland. Eight sections (5 ~m) from the embedded adrenal glands were fixed and then stained with Harris' hematoxylin and eosin. Adrenal gland sections were viewed with a microscope at 2.5x magnification. Areas of the cortex and medulla were calculated with a computerized digitizing unit and tracing of the viewed section. Plasma cortisol and ACTH concentrations were determined on duplicate samples with commercially available RIA kits (Pantex, Santa Monica, CA, and ICN Biomedicals Inc., Los Angeles, CA, respectively). These antibodies have been validated for cattle by the Texas Veterinary Medical Diagnostic Laboratory and have been used extensively in our laboratory (e.g., Refs. 14-19). Samples were reassayed if the duplicates differed by more than 5%. The intra-assay coefficient of variation (CV) for cortisol was 12.9%, and the interassay CV was 5.8%. The intra-assay CV for ACTH was 9.2%, and the inter-assay CV was 11%. The cross-reactivity values of the cortisol antiserum were as follows: cortisol, 100%; prednisolone, 40%; l l-desoxycortisol, 13.3%; corticosterone, 10.5%; and cortisone, prednisone, and dexamethasone <3.1% (analyses by Pantex). The cross-reactivity values of the ACTH antiserum were as follows: ACTH (1-24), 102%; and melenkephalin, leu-enkephalin, neurotensin, f3-endorphin, substance, P, somatostatin, and o~-melanocyte stimulating hormone <0.5% (analyses by ICN). Data for cortisol, body weight, organ weight as a proportion of body weight, and ratio of medulla to cortex were analyzed by use of the Wilcoxon-Mann-Whitney test (20). RESULTS Calves from the TRANS treatment had greater body weights on d 266 of gestation than did SHAM calves (P < 0.07; Table 1). Because body weight differed between the treatments, the remaining organ weight data were analyzed as organ weight-to-body weight ratios. These analyses found that TRANS calves had greater pituitary gland (P < 0.008: Table 1) and heart weights (P < 0.05). Although mean adrenal weight appeared to be greater for TRANS calves, there was no statistical difference after correcting for the larger birth weight.
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TABLE 1. DATA FOR CALVESDELIVEREDBY CESAREANSECTIONON D 266 OF GESTATION.a Parameter Body weight (BW) (kg) Right adrenal (mg/kg BW) Left adrenal (mg/kg BW) Adrenal cortex (mm 2) Adrenal medulla (ram 2) Pituitary (mg/kg BW) Anterior pituitary (mg/kg BW) Right kidney (g/kg BW) Left kidney (g/kg BW) Liver (g/kg BW) Heart (g/kg BW) Cortisol (ng/ml) ACTH (pg/ml)
Sham
Transportation
23.93 + 1.12u 20.43 + 4.26 20.98 + 4.33 8.10 5:0.75 8.07 5:1.47 8.24 5:1.70 a 6.38 5:1.35 1,76 5:0.34 1.76 5:0.34 14.26 5:2.70 5.17 5:1.01 t" 7.12 + 1.64 57.19 5:6.36
28.71 + 2.48 c 26.80 5:2.22 25.77 5:1.84 9.13 5:0.55 8.78 5:0.51 12,63 5:0.65 e 8.66 5:0,51 2.17 5:0.10 2.20 5:0.08 20.50 5:2.30 5.58 + 0.15 g 6.69 + 1.05 81.86 +_21.30
"Data are presented as the mean -t- SE. Organ weight data were adjusted according to body weight before statistical analysis was performed. b.c Numbers within rows with different superscripts differ (P < 0.07). d,e Numbers within rows with different superscripts differ (P < 0.008). f'g Numbers within rows with different superscripts differ (P < 0.05).
The fetal adrenal gland appeared to be as well organized as that of an adult. The zona glomerulosa, zona fasiculata, zona reticularis, and adrenal medulla were clearly separated and organized (Figures 1 and 2). However, some of the adrenals did appear to have distinct medullary cell areas within the cortisol area. The TRANS calves did have heavier heart weights (P < 0.05; Table 1); however, there were no treatment differences for kidney or liver weights (P < 0.10; Table 1). Neither plasma concentrations of cortisol nor ACTH differed between treatments (P < 0.20; Table 1), and values were comparable to previously reported baseline concentrations for cattle (14-16,21).
Figure 1. Cross-section of the left adrenal gland of a Brahman calf (266 d gestational age). The dam of the calf was subjected to transportation at 60, 80, 100, 120, and 140 d of gestation; therefore, the calf was considered to be exposed to "prenatal" stress. Section is viewed at 1.5x magnification. The zona glomerulosa (G), zona fasiculata (F), zona reticularis (R), and the medulla (M) are indicated on the figure. The size bar represents 1 ram.
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Figure 2. Cross-section of the left adrenal gland of a Brahman calf (266 d gestational age). The danl of the calf was not subjected to transportation during gestation; therefore, the calf was considered as a "control" for this study. Section is viewed at 1.5× magnification. Tile zona glomerulosa (G), zona fasiculata (F), zona reticularis (R), and the medulla (M) are indicated on the figure. The size bar represents 1 ram.
DISCUSSION The application of transportation to pregnant Brahman cattle appeared to universally increase both body weight and organ weight of the fetal calf. Because body weight proved to be greater for the T R A N S calves, organ weights were compared on a gram-to-kilogram of body weight basis and still appeared to be greater (Table I). However, not all of these differences proved to be significant. This study used only six calves for each treatment. Possibly, a larger sample size would help to clarify the effect of prenatal stress on fetal growth parameters. Transportation was used as a stressor in this study. The component(s) of the transportation treatment that may be responsible for the treatment differences is an important consideration. Not only were these cows transported, but they were also unloaded and held at another facility for 1 hr. There were no other animals at this facility; however, environmental factors other than transportation could still have affected the cows at this time. Additionally, these cows had calves that remained at the original farm. Clearly, the transportation stress imposed on these cows was a multifaceted stressor composed of transportation, relocation, and isolation from their offspring. Although comparisons between the cow and rat are tenuous, to date, the only species on which physiological prenatal stress studies have been conducted in depth are the rodent and the cow (this study). Obviously, these species are extremely different, and any mechanism acting via the placenta would likely differ. The placentation of the rodent is characterized as hemochorial, whereas that o f the cow is epitheliochorial. Therelore, the placenta of the cow offers a more significant barrier to maternal hormonal influences, With these differences in mind, we offer the following thoughts as to the mechanism responsible for the effects of prenatal stress. In the abundant literature on the effects of prenatal stress on rodents, none could be found that reported birth weight. Genetic potential cannot be changed by prenatal stress:
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therefore, possible causes for the weight difference could be due to either nutritional or hormonal influences. If the transportation treatment somehow influenced the behavior of the cow such that she increased her feed intake, or if it increased the blood flow to the fetus, then fetal birth weights could be increased. However, transportation resulted in larger pituitary glands in the calves, which may suggest a hormonal cause for the increased birth weight. Possibly, the larger pituitary gland may have been secreting more growth hormone, thereby causing the TRANS calves to be heavier. Generally, fetal growth is attributed to genetic potential as well as placental size, although insulin-like growth factor-II has been shown to have significant influence at least in the rodent (22). In support for the effects of stress increasing growth hormone, Nogami et al. (23) found that dexamethasone induced the differentiation of growth hormone-producing cells at an early age in the rat. In contrast to data for humans and rats, which found that glucocorticoids could stimulate growth hormone release, Sartin et al. (24) found that cortisol inhibited growth hormone release from the pituitary in sheep. Accordingly, any increase in fetal plasma cortisol due to maternal stress should have a negative effect on body weight. If the bovine pituitary can respond with a glucocorticoid-stimulated increase in growth hormone, similar to the rat, then this may be one possible explanation for the larger calves from TRANS cows found in this study. The effects of prenatal stress on fetal body weight warrant more detailed investigation. The heavier heart weight of the TRANS calves is an interesting finding that also deserves further attention. It is intriguing that the differences observed in this study are well after the application of transportation stress (120 d posttreatment). However, the effects observed in rodents have been similar. Henry et al. (I) subjected pregnant rats to restraint stress during the third week of gestation and noted the effects of prenatal stress 90 d later. The 90 d is less than our 120 d; however, the developmental stage of the rodent is much more advanced at this age than that of the cow. In our study, both control and treatment cows were housed together and treated identically, except for the application of transportation stress. The fact that treatment differences were noted well after the application of transportation illustrates the robustness of prenatal stress effects. If prenatal stress influences the function of the fetal HPA axis, then it is possible that plasma concentrations of cortisol and ACTH could differ at birth between calves from the TRANS and SHAM treatment groups. Shanks et al. (25) suggested that corticosteroid baseline differences between neonatally handled and unhandled rodents caused the differences in the corticosteroid response of these animals at maturity. Neither concentrations of plasma cortisol nor ACTH differed (P < 0.20) between treatments in fetal calves of this study. This suggests that if differences in the plasma cortisol response of calves in response to stress are affected by prenatal stress, then these differences are not due to altered baseline concentrations but to threshold differences in the activation of the HPA axis. The enlarged pituitary glands suggest that this component of the axis is responsible for the greater glucocorticoid response of prenatally stressed animals, perhaps in response to hypertrophy or hyperplasia consequences of increased corticotropin-releasing hormone and(or) vasopressin. Transportation and other common management procedures cause activation of the HPA axis. Our research has found that repeated activation of the HPA axis during pregnancy produced not only a larger fetal body weight at 266 d of gestation, but also heavier fetal pituitary glands. The pituitary gland is critical for many functions of the body including growth (growth hormone, thyroid-stimulating hormone [TSH], prolactin [PRL]), coping with stress (ACTH. TSH, PRL), and controlling reproduction (luteinizing hormone, fol-
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licle-stimulating hormone). Because such an important fetal endocrine gland is affected by maternal stress during gestation, the effects of prenatal stress may be profound and serve to precondition or imprint the postnatal experience on endocrine glands associated with growth, reproduction, and tolerance to biological stressors. Further research is warranted to determine the long-term effects of prenatal stress on reproduction, growth, and the stress response. ACKNOWLEDGMENTS/FOOTNOTES The authors thank Dr. Larry Johnson, Department of Veterinary Medicine, Texas A&M University, for help in quantifying adrenal gland areas and photography. This research was partially funded through the USDA Animal Health Formula Funds. The manuscript includes research supported and conducted by the Texas Agricultural Experiment Station, The Texas A&M University System. Furthermore, this study was a contribution to the Western Regional Research Project W-112, Reproductive Performance in Domestic Ruminants. Present Address: Dr. Donald C. Lay, Jr., Iowa State University, 337C Kildee Hall, Ames, IA 50011. 2 Correspondence: Dr. Ron D. Randel, Texas A&M University Agricultural Research and Extension Center, Overton, TX 75684-0038. 3Present Address: Dr. Jeff A. Carroll, Animal Physiology Research Unit, Rm S-107, ASRC, Univ. of Missouri, Columbia, MO 65221. 4 Present Address: Gabrielle M. Kapp, Laboratory Animal Resources and Research Facility, Texas A&M University, College Station, TX 77843-4473.
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17. Lay DC Jr, Friend TH, Randel RD, Bowers CL, Grissom KK, Jenkins OC. Behavioral and physiological effects of freeze or hot-iron branding on crossbred cattle. J Anim Sci 70:330-336, 1992. 18. Lay DC Jr, Friend TH, Bowers CL, Grissom KK, Jenkins OC. A comparative physiological and behavioral study of freeze and hot-iron branding using dairy cows. J Anita Sci 70:1121-1125, 1992. 19. Bowers CL, Friend TH, Grissom KK, Lay DC Jr. Confinement of lambs (Ovis aries) in metabolism stalls increased adrenal function, thyroxine and motivation for movement. Appl Anim Behav Sci 36:149-158, 1993. 20. Siegel S, Castellan NJ Jr. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, Inc., New York, 1988. 21. Sartin JL, Kemppainen RJ, Cummins KA, Williams JC. Plasma concentrations of metabolic hormones in high and low producing dairy cows. J Dairy Sci 71:650-657, 1988. 22. Owens JA. Endocrine and substrate control of fetal growth: placental and maternal influences and insulinlike growth factors. Reprod Fertil Dev 3:501-517, 1991. 23. Nogami H, Tachibana T, Katayama T, Ishikawa H. The free structure of dexamethasone-induced growth hormone cells in the anterior pituitary gland of the rat fetus. Arch Histol Cytol 58:581-589, 1995. 24. Sartin JL, Kemppainen RJ, Coleman ES, Steele B, Williams JC. Cortisol inhibition of growth hormonereleasing hormone-stimulated growth hormone release from cultured sheep pituitary cells. J Endocrinol 141:517-525, 1994. 25. Shanks N, Viau V, Plotsky PM, Meaney MJ. Early experience regulates the development of hypothalamic CRH gene expression and HPA responses to stress. Adaptive and Maladaptive Responses to Stress: Relationships Between Behavioral Responses and Neuroendocrine Systems. Proceedings of the International Society of Psychoneuroendocrinology XXIII Congress, Univ. of Wisconsin-Madison p. 190-192, 1992.