Physiology & Behavior, Voi. 15, pp. 487--489. Pergamon Press and Brain Research Publ., 1975. Printed in the U.S.A BRIEF COMMUNICATION Effects of Early Handling Upon Puberty: Correlations with Adrenal Stress Responsiveness ] GARY SIECK 2 AND JUDITH A. RAMALEY Department of Physiology and Biophysics, University of Nebraska Medical Center Omaha, NE 68105 (Received 13 January 1975) SIECK, G. AND J. A. KAMALEY. Effects of early handlingupon puberty: correlations with adrenal stress responsiveness. PHYSIOL. BEHAV. 15(4) 487-489, 1975. - Handling of rat pups for 5 rain once a week prior to weaning to permit weighing of the litter significantly delays vaginal opening and ovulation and is associated with a reduction in the response to an ether stress administered at 60 days of age. Handling combined with replacement of the bedding material results in a further delay in puberty and a further reduction in the stress response. It is therefore necessary to follow uniform handling procedures in any study of a developmental process. Puberty Handling Maturation Adrenal Stress EARLY handling of immature rats has been shown either to advance, delay or have no effect on the onset of sexual maturity, depending upon how often the rats are handled, the intensity of handling and the time at which rats are handled [2, 5, 6]. Early handling also will affect adult behavior and adrenocortical responsiveness [1,4]. In examining the literature it appeared that a common handling pattern employed in growth experiments, namely weekly handling for obtaining growth rates, has not been studied in terms of its long-range developmental effects. It also appeared that the conditions of animal care during development might be a varinble since some investigators routinely change the nesting materials at weekly or more frequent intervals while others do not disturb the animals until weaning age. The following experiments were per- formed to determine whether or not standard animal handling procedures in our laboratory would alter the onset of puberty and one measurement of adrenocortical function, the response to ether stress. METHOD Animals and Procedure Sprague-Dawley derived rats from Sascho Laboratories, Omaha, Nebraska were bred in our animal colony in a 14:10 light cycle (lights on at 0500 colony time, counting the midpoint of the dark period as colony midnight). Temperature was held at 23-24°C and food and water were available ad lib. The pregnant females were placed in translucent plastic nesting caps containing corncob litter. At I day of age, the litters were sexed and reduced to 6 females and 2 males. The litters were assigned at random to one of three treatment groups: (I) No further handling until weaning at 24 days of age; (2) Weekly removal of both mother and pups for 5 min to permit weighing of the pups (H group); (3) Weekly removal of mother and pups for 5 min for weighing and for changing of the nesting material NM group). In the latter 2 groups, the pups were kept on a warming pad while they were out of the cage to avoid undue maternal reaction to tlieir temperature (7) and were replaced in the cage in a small group to keep the degree of maternal retrieval behavior relatively uniform. At weaning the females were placed in groups of 3 in stainless steel hanging cages and checked daily for vaginal opening. The males were not studied further. The vagina was considered patent when a Pasteur pipette could be introduced easily to obtain a vaginal smear. The female rats were anesthetized with ether and laparotomized on the day of vaginal opening and examined for signs of ovulation (hemorrhagic ovarian follicles and dilated tubal ampullae). Rats that had not ovulated, were examined again when the vaginal smear pattern changed to estrous. The majority of rats had tubal ova on the morning of vaginal opening. In our colony, vaginal opening takes place in the early evening and ovulation between 2400 and 0400 on the light cycle used, here. At 60 days of age, the rats were exposed to a 2 rain ether stress at 0900. An initial blood sample was collected by cardiac puncture without anesthesia and a second blood Supported by NSF Grant GB 35730. 2 Supported by the Frank Morsmen Fellowship in Endocrinology, University of Nebraska Medical Center. 487