Postnatal alveolar development of the rabbit.

Previous studies of alveolarization have used rats or lambs; however, neither closely reflects human alveolar development. We characterized alveolar development in rabbits (n = 3-7 /group) at 28 days gestation (dg) to 9 mo to determine whether they followed the human pattern more closely. The right lung was made up of 30% alveolar and 50% duct space at 28 dg to 3 days and of 50 and 30%, respectively, at 14 days to 9 mo. Tissue fraction and alveolar wall thickness decreased by 40% 28 dg to birth. At birth, approximately 4.5% of the number of alveoli seen at 9 mo were present, with alveolar number increasing progressively well into adulthood. The rate of alveolar formation was high around birth, decreasing progressively with age. Alveolar volume increased more than twofold (28 dg to birth) and continued to increase postnatally to 16 wk. Surface fraction decreased by 17% (28 dg to 3 days), after which it remained uniform. Our findings suggest that the timing of onset of alveolarization in humans and rabbits is similar and that rabbits may be used to model postnatal influences on alveolar development.     

Distribution of pores within alveoli in the human lung

Usually the wall opposite the orifice of alveoli has been used to study interalveolar pores by scanning electron microscopy. To ascertain whether biased results may be obtained from this, the distribution of pores within alveoli was studied in human lungs. By the use of scanning electron photomicrographs, the number, major axes of pores, and proportional area of pores to the alveolar wall were estimated. The alveolar wall seen opposite the orifice was defined as the bottom wall. Average number of pores per alveolus was 13-21, and one-half of them was located in the bottom walls. The average length of major axes was 7-19 micron, and average area fractions were 0.8-5%. The distribution of the numerical density, area fraction, and size of pores was uniform regardless of their location within the alveolus and the size of alveoli. Thus pores can be compared using the bottom walls of alveoli. This will facilitate the study of the effects of age, smoking, and topography on pore size and frequency in humans.     

Development of alveolar septa and formation of alveolar pores during the early postnatal period in the rat lung

In order to investigate the formation of alveolar pores, lungs of rats, after intratracheal perfusion of glutaraldehyde, are processed at postnatal days 1, 7, 14, 16 and 21 for light and transmission electron microscopy and at days 7 and 16 for scanning electron microscopy. The initial low secondary crests of day 1 rapidly elongate to pleats subdividing the primary saccules. The ledges of some pleats partly grow toward each other as ring like diaphragms, leaving openings whose boundary is composed of alveolar epithelium separated by a basal lamina from a connective tissue sheath with capillaries. At day 7, in scanning electron microscopy the lumina of some rudimentary alveoli communicate by apertures of different sizes, as a result of the outgrowth of curved alveolar pleats which narrow to a ring-like aperture. The interalveolar openings observed in scanning electron microscopy resemble those investigated by light and transmission electron microscopy. The number of interalveolar pores increases from day 7 on; they become more and more frequent at days 14, 16 and 21, respectively. It appears that alveolar multiplication in newborn rats proceeds not only by segmentation of terminal respiratory units but also by compoundment of septa. The difference between genuine pores and transsections of folds in transmission electron microscopy will be given closer attention in this study. Also, the incidence and location of type II pneumocytes during rapid enlargement of the alveolar surface area is discussed.     

Morphometric analyses of the effects of thyrotrophin releasing hormone and cortisol on the lungs of fetal sheep

Morphometric analyses of ovine fetal lung parenchyma were undertaken in order to elucidate the roles of pituitary, thyroid and adrenocortical hormones in promoting the structural changes underlying the increased distensibility and stability present in mature fetal lungs. Twenty-six Romney fetuses were treated with either cortisol for 84 h from 125 days (4), pulsatile TRH for 6.5 days from 122 days (4), cortisol and TRH (12), or 0.9% NaCl solution (6). The left lungs were used for physiological studies (distensibility, V40) and the right lungs were prepared for electron microscopy. Using 32 regions of lung parenchyma per fetus, volume density, surface density and arithmetic mean thickness of the alveolar walls were calculated using point and intersection counts. Of the three regimens, treatment with TRH + cortisol (exposure to raised concentrations of cortisol, T3 and prolactin) induced significantly greater lung distensibility, the largest potential alveolar air space (62% of the parenchyma), the greatest alveolar surface area (113.7 mm2/mm3 x 10(-3)) and the thinnest alveolar walls (6.7 microns). We conclude that cortisol, T3 and prolactin act synergistically to promote maturational changes in the alveolar wall. While cortisol plays the major role, T3 and prolactin enhance the ability of the immature lung to respond to the cortisol.     

IL-1beta stimulates alveolar fluid absorption in fetal guinea pig lungs via the hypothalamus-pituitary-adrenal gland axis

We tested the hypothesis that interleukin (IL)-1beta-induced cortisol synthesis stimulates alveolar fluid clearance in preterm fetuses. IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. Fetuses were obtained by abdominal hysterotomy at 61 and 68 days gestation and instilled with isosmolar 5% albumin in the lungs, and alveolar fluid movement was measured over 1 h from the change in alveolar protein concentration. Alveolar fluid clearance was induced at 61 days gestation and stimulated at 68 days gestation by IL-1beta, which both were attenuated by cortisol synthesis inhibition. Plasma ACTH and cortisol concentrations were increased by IL-1beta at both gestational ages, and metyrapone reduced cortisol concentrations. IL-1beta was mostly low or undetectable in both fetal and maternal blood. Prenatal alveolar fluid clearance, when present as well as IL-1beta induced, was always propranolol and amiloride sensitive, suggesting that beta-adrenoceptor stimulation and amiloride-sensitive Na+ channels were critical for fluid absorption. IL-1beta increased lung beta-adrenoceptor density at gestation day 61, and cortisol synthesis inhibition attenuated the increased beta-adrenoceptor density. Epithelial Na+ channel and Na+-K+-ATPase subunit expressions were both increased by IL-1beta and attenuated by cortisol synthesis inhibition. These results may explain why babies delivered preterm after intrauterine inflammation have a reduced risk of developing severe respiratory distress.     

Activation of alveolar macrophages after plutonium oxide inhalation in rats: involvement in the early inflammatory response

Alveolar macrophages play an important role in the distribution, clearance and inflammatory reactions after particle inhalation, which may influence long-term events such as fibrosis and tumorigenesis. The objectives of the present study were to investigate the early inflammatory events after plutonium oxide inhalation in rats and involvement of alveolar macrophages. Lung changes were studied from 3 days to 3 months after inhalation of PuO2 of different isotopic compositions (70% or 97% 239Pu) and initial lung deposits (range 2.1 to 43.4 kBq/rat). Analyses of bronchoalveolar lavages showed early increases in the numbers of granulocytes, lymphocytes and multinucleated macrophages. The activation of macrophages was evaluated ex vivo by measurement of inflammatory mediator levels in culture supernatants. TNF-alpha and chemokine MCP-1, MIP-2 and CINC-1 production was elevated from 7 days after inhalation and remained so up to 3 months. In contrast, IL-1beta, IL-6 and IL-10 production was unchanged. At 6 weeks, pulmonary macrophage numbers and activation state were increased as observed from an immunohistochemistry study of lung sections with anti-ED1. Similarly, histological analyses of lung sections also showed evidence of inflammatory responses. In conclusion, our results indicate early inflammatory changes in the lungs of PuO2-contaminated animals and the involvement of macrophages in this process. A dose-effect relationship was observed between the amount of radionuclide inhaled or retained at the time of analysis and inflammatory mediator production by alveolar macrophages 14 days after exposure. For similar initial lung deposits, the inflammatory manifestation appears higher for 97% 239Pu than for 70% 239Pu.     

Age‐related changes in biological characteristics of human alveolar osteoblasts

Objectives: Age‐related changes are common in many tissues and organs. However, cell‐related causes in human alveolar bone remain unclear. This study has been carried out to explore the possibility that advancing age might change the biological characteristics of alveolar osteoblasts (AOBs) in women. Materials and methods: Alveolar osteoblasts from women donors (five women aged 33–38 years and five women aged 62–68 years) were cultured in vitro. The cells were serially passaged and maximal lifespan evaluated. Cell viability, ultramicrostructure and osteogenic differentiation ability were determined respectively, using MTT assay, transmission electron microscopy, alkaline phosphatase (ALP) activity assay and von Kossa staining assay. These parameters of the two groups of AOBs were evaluated. Results: When compared with cells from young adult donors, AOBs from elderly women exhibited lower maximal lifespan (P < 0.05). Mean rate of population doubling was lower in elderly donor cells compared to those from young adult cells (P < 0.05). Organelles from AOBs of elderly donors were much fewer than those from young donors. MTT value of elderly donor cells was significantly lower than those of young adult donors from day 2 (P < 0.05). Relative ratio of ALP activity in elderly donor cells was significantly lower than those of the young womens’ cells at 8, 12, 16 and 20 days (P < 0.05). Calcium nodules of young adult donors’ specimens were significantly more numerous and larger than those from elderly donors. Conclusions: Comparison of biological characteristics of AOBs from young adult women with those from elderly women in vitro revealed differences in proliferative capacity and bone formation functions, which decreased with aging. These data indicate that aging may play an important role in pathogenesis of human AOBs loss.     

Stereological analysis of hepatic fine structure in the Fischer 344 rat. Influence of sublobular location and animal age

Stereological analysis of hepatic fine structure in Fischer 344 male rats at 1, 6, 10, 16, 20, 25, and 30 mo of age revealed differences in the amounts and distributions of hepatocellular organelles as a function of sublobular location or animal age. Between 1 and 16 mo of age, both the centrolobular and periportal hepatocytes increased in volume by 65 and 35%, respectively. Subsequently, the cell volumes declined until the hepatocytes of 30-mo-old rats approached the size of those found in the youngest animals. Regardless of animal age, the centrolobular cells were consistently larger than the corresponding periportal hepatocytes. The cytoplasmic and ground substance compartments reflected similar changes in their volumes, although there was no significant alteration in the nuclear volume. The volumes of the mitochondrial and microbody compartments increased and decreased concomitant with the changes in average hepatocyte size. Both lobular zones in the 30-mo-old rats contained significantly smaller relative volumes of mitochondria than similar parenchyma in 16-mo-old animals. The volume density of the dense bodies (lysosomes) increased markedly in both lobular zones between 1 and 30 mo of age, confirming reports of an age-dependent increase in this organelle. The surface area of the endoplasmic reticulum in the centrolobular and periportal hepatocytes reached its maximum level in the 10-mo-old rats and subsequently declined to amounts which approximated those measured in the 1-mo-old animals. This age-related loss of intracellular membrane is attributable to a significant reduction in the surface area of the smooth-surfaced endoplasmic reticulum (SER) in animals beyond 16 mo of age. The amount of rough-surfaced endoplasmic reticulum (RER) in the periportal parenchymal cells was unaffected by aging, but the centrolobular hepatocytes of 30-mo-old animals contained 90% more RER than similar cells in the youngest rats. The centrolobular parenchyma contained more SER and the portal zones more RER throughout the age span studied. These quantitative data suggest that (a) certain hepatic fine structural parameters undergo marked changes as a function of animal age, (b) there exists a gradient in hepatocellular fine structure across the entire liver lobule, and (c) there are remarkable similarities in hepatocyte ultrastructure between very young and senescent animals, including cell size and the amount of SER.     

Activation of alveolar macrophages after lower respiratory tract infection.

Alveolar macrophage function has been studied in relation to bacterial infection of the lower respiratory tract. First, LRT macrophages were examined after exposure of rabbits to Listeria monocytogenes aerosols. Macrophages obtained from the LRT of animals 10 to 48 days after infection were activated, as evidenced by greater adherence to culture dishes and increased ability to ingest and kill both the original infecting organism and unrelated organisms, when compared to normal alveolar macrophages. Next, the in vitro effects on normal alveolar macrophages of incubation supernatants of control and antigen-stimulated lymphocytes (LRT and lymph node) from animals infected with L. monocytogenes or Streptococcus pneumoniae were evaluated. As manifested by increased adherence and phagocytosis, and an enhanced nonspecific bactericidal activity, alveolar macrophages were activated by the antigen-stimulated supernatants. These stimulated lymphocyte supernatants contain lymphokines (MIF), but the exact nature of the alveolar macrophage activating factor(s) remains to be determined. These observations, together with recent evidence that alveolar macrophages respond to lymphokines (MIF), suggest that the effector mechanism for cell-mediated immunity in the LRT is intact.     

Injury,inflammation, and remodeling in fetal sheep lung after intra-amniotic endotoxin

Chorioamnionitis is frequent in preterm labor and increases the risk of bronchopulmonary dysplasia. We hypothesized that intra-amniotic endotoxin injures the lung in utero, causing a sequence of inflammation and tissue injury similar to that which occurs in the injured adult lung. Preterm lamb lungs at 125 days gestational age were evaluated for indicators of inflammation, injury, and repair 5 h, 24 h, 72 h, and 7 days after 4 mg of intra-amniotic endotoxin injection. At 5 h, the epithelial cells in large airways expressed heat shock protein 70, and alveolar interleukin-8 was increased. Surfactant protein B (SP-B) decreased in alveolar type II cells at 5 h, and SP-B in lung tissue and alveolar lavage fluid increased by 72 h. By 24 h, neutrophils were recruited into the large airways, and cell death was the highest. Alveolar type II cells decreased by 25% at 24 h, and proliferation was highest at 72 h, consistent with tissue remodeling. Intra-amniotic endotoxin caused surfactant secretion, inflammation, cell death, and remodeling as indications of lung injury. The recovery phase was accompanied by maturational changes in the fetal lung.     

Quantitative analysis of development of mitochondrial ultrastructure in differentiating mouse hepatocytes during postnatal period

Between birth and 10 days of age, the volume density (volume/unit cytoplasmic volume) of the matrix, and the surface density (area/unit cytoplasmic volume) of the inner membrane and cristae increased in both periportal and perihepatic hepatocytes, and did not differ significantly between the cells of the two zones. After 10 days of age, however, the volume density of the matrix decreased in perihepatic cells and remained unchanged in periportal cells, and, therefore, it became greater in periportal cells than in perihepatic cells in 20-day-old and adult animals. The surface density of the inner membrane and cristae decreased in the cells of both zones. Further, the hepatocyte volume increased markedly, especially in perihepatic zones between 20 days of age and the adult. The results show that, in postnatally differentiating hepatocytes, mitochondria are likely to develop during early postnatal period, then the structural heterogeneity of mitochondria arises, and hepatocyte volume increases markedly during late postnatal period after weaning. Thus, the process of postnatal hepatocyte differentiation includes such several phases of development.     

Age-related changes in body weight,scrotal size and plasma testosterone levels in buffalo bulls (Bubalus bubalis)

Body weight, testicle size and peripheral testosterone concentrations were measured in 35 water buffalo bulls at 5, 15, 17, 21, 25 and 38 months of age. These parameters were studied in all animals during the same month (October), so the changes due to age were independent of changes in photoperiod and temperature. Body weight increased linearly with age. The testicular size measured in terms of scrotal circumference as related to age, showed a curvilinear increase; the average rate of testicular growth was maximum between 15 and 25 months. Plasma testosterone levels were low between 5 and 21 months. A significant rise in plasma concentration of testosterone was observed at 25 months reaching peak levels at 38 months. The mean age of sexually mature bulls at the time of first ejaculation of semen with motile sperm, was 24.9±0.9 months (n=9). It has been concluded that in the Nili-Ravi buffalo bulls the sexually quiescent period (prepubertal) extends up to 15 months of age and sexual maturation as indicated by the presence of motile sperm in the ejaculate is attained at about 25 months.     

Diabetes influences cardiac extracellular matrix remodelling after myocardial infarction and subsequent development of cardiac dysfunction

This study was conducted to examine the influence of acute streptozotocin‐induced diabetes on cardiac remodelling and function in mice subjected to myocardial infarction (MI) by coronary artery ligation. Echocardiography analysis indicated that diabetes induced deleterious cardiac functional changes as demonstrated by the negative differences of ejection fraction, fractional shortening, stroke volume, cardiac output and left ventricular volume 24 hrs after MI. Temporal analysis for up to 2 weeks after MI showed higher mortality in diabetic animals because of cardiac wall rupture. To examine extracellular matrix remodelling, we used fluorescent molecular tomography to conduct temporal studies and observed that total matrix metalloproteinase (MMP) activity in hearts was higher in diabetic animals at 7 and 14 days after MI, which correlated well with the degree of collagen deposition in the infarct area visualized by scanning electron microscopy. Gene arrays indicated temporal changes in expression of distinct MMP isoforms after 1 or 2 weeks after MI, particularly in diabetic mice. Temporal changes in cardiac performance were observed, with a trend of exaggerated dysfunction in diabetic mice up to 14 days after MI. Decreased radial and longitudinal systolic and diastolic strain rates were observed over 14 days after MI, and there was a trend towards altered strain rates in diabetic mouse hearts with dyssynchronous wall motion clearly evident. This correlated with increased collagen deposition in remote areas of these infarcted hearts indicated by Masson's trichrome staining. In summary, temporal changes in extracellular matrix remodelling correlated with exaggerated cardiac dysfunction in diabetic mice after MI.     

Alveolar type II-like cell colonies: effect of alveolar macrophages and macrophage-conditioned media

Alveolar type II-like colonies were obtained after a low density plating (5 X 10(3)/60 mm tissue culture dish) of primary type II cells. These colonies were formed only when type II cells were either cocultured with alveolar macrophages or with conditioned media generated by alveolar macrophages. Cells in the colonies appeared homogeneous and kept their lamellar bodies over a period of 8 weeks and more, as observed by electron microscopy. These cells reacted immunocytochemically with antibodies directed against the 32-38 kDa protein fractions of rat surfactant.     

Long-term consequences of compensatory lung growth

Left pneumonectomy or left nephrectomy was performed on 10-wk-old littermate male New Zealand White rabbits, and they were killed at 30 wk of age. Thirty-week-old male littermates served as controls. Nephrectomy was done to produce major tissue loss and trauma and to assess blood somatomedin C. At the end of the experiment, the right lungs of the pneumonectomy animals had a greater lung volume, weight, gas-exchanging surface area, and alveolar number than the nephrectomy animals and the controls, and their air spaces were the same size. When compared with both lungs of the nephrectomy group and the controls, lung weight was the same; lung volume, alveolar number, and protein were not significantly less in the pneumonectomy group, but gas-exchanging area (compared with controls only), DNA, and RNA were. After left nephrectomy, the right kidney increased in weight; nephrectomy had no effect on lung size or structure. We conclude that pneumonectomy at age 10 wk in male rabbits results in significant compensatory lung growth, including alveolar multiplication, and this persists to age 30 wk. Compensatory lung growth, however, was incomplete; that is, it did not reconstitute (equal) in all respects that of both lungs of the nephrectomy animals or the controls.     

Alveolar epithelial changes in rabbits after a 21-day exposure to 60% O2

This study characterizes the biochemical and physiological effects of prolonged exposure of rabbits to sublethal (60%) O2 concentrations. After 3 wk in 60% O2, rabbits had arterial PO2 values of 69 +/- 2 vs. 79 +/- 3 Torr for control animals (means +/- SE; P less than 0.05) and a small but significant rise in pulmonary wet weight-to-dry weight ratios (5.6 +/- 0.3 vs. 4.1 +/- 0.3; P less than 0.05). Alveolar permeability to solute, lung compliance, total lung capacity, and alveolar protein levels were unchanged from control, but the amount of lavagable alveolar phospholipid was 90% higher in the O2-exposed rabbits. The lipid biosynthetic ability of isolated alveolar type II pneumocytes, measured by radiolabeled precursor [3H]choline incorporation, indicated that type II cells isolated from hyperoxic animals synthesized phosphatidylcholine at a rate 110% higher than those from control animals. Laser flow cytometric analyses of isolated type II cells showed a significant increase in type II cell diameter, based on time-of-flight measurements, and an average 60% increase in lipid content per cell, based on phosphine-3R fluorescence intensity. These findings indicate that exposure to 60% O2 for 21 days results in a decrease in arterial PO2 and induces several important biochemical and morphological changes in alveolar type II pneumocytes.     

Quantitative analysis of Sertoli cells in neonatally oestrogen-treated rats

On Day 1 of age rats were treated with 500 micrograms oestradiol benzoate. Oestrogen-treated rats had increased numbers of Sertoli cells per reference area or volume, whereas the total number of cells per testis was unchanged. The mean nuclear size was significantly smaller in oestrogen-treated rats than in control rats, at 22 and 45 days of age. The volume density of the heterochromatin clumps decreased from 22 to 45 days of age in control rats (68% fall), the decrease being slower in oestrogenized animals (30% fall) during the same period. The differences were significant at 45 days of age only. The relative volume occupied by the nuclear membrane infoldings was significantly less in oestrogenized rats than in control ones at the two ages considered. Nucleolar development was delayed in oestrogen-treated rats, which had lower numbers of nuclear sections showing nucleoli, as well as a decrease in the nucleolar diameter. We suggest that these Sertoli cell alterations are due to the altered gonadotrophin and testosterone concentrations induced by the steroid treatment rather than to a direct effect of oestrogen.     

The modifications produced in allergic alveolitis and in Goodpasture's syndrome due to exposure to cigarette smoke.

Two groups of rats with experimental alveolitis were exposed to cigarette smoke. After comparing the results, the possible muffling effect of the cigarette smoke related to interstitial lung disease was discussed. 180 rats were divided into 6 groups of 30 animals each: Group 1: untreated controls; Group 2: exposed to cigarette smoke for 2 months; Group 3: sensitized with bovine albumin (BA) and exposed to an atmosphere with this antigen for two months, to reproduce a type of extrinsic allergic alveolitis (EAA); Group 4: treated with a single daily dose of anti-lung serum for three days followed by two days without treatment, to reproduce a type of Goodpasture's syndrome; Group 5: exposed to cigarette smoke and to BA; Group 6: exposed to cigarette smoke and treated with anti-lung serum. The animals were sacrificed and their lungs were treated for: Bronchoalveolar lavage (BAL), percentage lymphocyte count, polymorphonuclear (PMN) and alveolar macrophages (AM); semiquantitative and morphometric histological study. The semiquantitative study determined the area of the studied lung incision, affected by granulomae, increased alveolar aerial spaces, thickened alveolar walls and haemosiderine lung area. The morphometric study, based on the linear integration method, evaluated: the distance between two alveolar walls, the amount of interstice per field; and the number of AM with haemosiderine per field was counted. From the results we point out that the treated animals had significantly higher lymphocyte and BAL PMN counts than the untreated ones; no significant differences were found between the singly and doubly treated animals. The animals exposed to cigarette smoke and treated with anti-lung serum were those that showed the least number of lymphocytes and PMN of all the treated animals. The semiquantitative variables studied were all increased in comparison to the control group, most of the increases being significant. The morphometric variables revealed significant differences with respect to the untreated group, except for the animals which were treated with anti-lung serum and cigarette smoke, which showed a minimum decrease in the alveolar size and a slight increase of the interstitial tissue. Only one morphometric variable showed a significant difference between the group treated with anti-lung serum and the one treated with anti-lung serum and cigarette smoke: the number of AM with haemosiderine in the lung.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solute conductance of blood-gas barrier in hamsters exposed to hyperoxia

Hamsters were exposed to greater than 95% O2 continuously for up to 5 days to determine longitudinal changes in the diffusive conductance of the alveolar epithelium and capillary endothelium as a result of hyperoxia. Permeability X surface area (PS, cm3/s X 10(-4)) was measured by isolated, perfused lung techniques. Alveolar epithelium PS for [14C]sucrose and 125I-bovine serum albumin (BSA) were determined at seven exposure times. Control PS (sucrose) and PS(BSA) averaged 1.00 and 0.022, respectively. Values were unchanged until 4.5 days, when significant increases in both, but especially PS(BSA), occurred. After 5 days, PS values were 4.69 and 0.691, respectively. Capillary endothelium PS for 125I-BSA and fluoresceinisothiocyanate dextran-150 (D-150) were measured at four exposure times. Control endothelium PS(BSA) and PS(D-150) averaged 0.232 and 0.048, respectively. These values were also unchanged after 4 days but increased to 0.440 and 0.131 after 5 days. Wet lung weight significantly increased after only 4 days. Hyperoxia thus increased both endothelium and epithelium PS, but epithelium changes were much greater. These functional changes do not occur for several days, occur simultaneously, and follow increases in lung wet weight.     

Effects of early handling on heifers' subsequent reactivity to humans and to unfamiliar situations

The influence of early handling on the behavioural and physiological responses of Friesian heifers to unfamiliar situations and human manipulation was studied. Forty abimals were used. The animals were reared in 4 groups of 10, under standard artificial husbandry conditions. Animals of one group (control animals) were handled only in respect of the demands of normal husbandry. Experimental animals were subjected to additional handling, which consisted of brushing twice a day and leading with a halter. All handled animals received the same amount of handling (30 days) but at different periods in life: 3 days/week from 0 to 3 months of age (Group 0–3); 3 days/week from 6 to 9 months of age (Group 6–9); or 3 days/month from 0 to 9 months of age (Group –9).

When the animals were 15 months old, each heifer, from each treatment group, was tested individually and once only in a set of behavioural tests designed to investigate fearfulness (in the presence or absence of human contact) and the ease with which the animal could be handled.

Animals from Group 0–9, and to a lesser extent those from Group 6–9, were less reactive than controls in tests involving the presence of a human. However, in most cases Group 0–3 animals did not differ significantly from controls. Thus, it would appear that only prolonged handling during early life substantially influences man-animal relationships.

In the fear-eliciting tests, which did not involve human contact, only the responses of Group 0–9 animals differed significantly from those of the controls. It is suggested that temporary prolonged handling influences the expression of fear responses in heifers.