Assessing the quality of products from cloned cattle: an integrative approach

Scientific expertise was developed during a 3-year study to evaluate a large number of bovine female clones (n=37; from 4 to 36 months of age) and their products through a multidisciplinary approach and compare them to non-cloned breed, age and sex-matched contemporary control animals (n=38) maintained under the same conditions at the same experimental farm of INRA. In clone and control groups, most parameters measured for health and development of the animals as well as evaluation of milk and meat products were within the normal range for the breed. The strict comparison between cloned animals and controls allowed us to detect slight significant differences between the two groups. Cloned heifers reached puberty significantly later (+62 days) and at higher body weight (+56kg) than controls. There were slight differences in antigen-specific induced proliferation of lymphocytes after vaccination with ovalbumin before 10 months of age, but responses were normal responses in older animals. There were differences in the fatty acid (FA) composition of milk and muscle arising from two families of clones, suggesting a possible deviation in lipid metabolism as assessed by higher Delta-9 desaturase activity indices in both milk and muscle from clones compared to controls. Nutritional evaluation of milk and meat using the rat model did not reveal any difference between products derived from clones versus controls.     

Effects of biostimulation and nutritional supplementation on pubertal age and pregnancy rates of Nelore heifers (Bos indicus) in a tropical environment

To determine effects of biostimulation (BIO) and dietary supplementation (BIO+S) on pubertal age and pregnancy rates, Nelore heifers (n=392) were randomly assigned to one of four treatment groups (n=98/group). All animals were in tropical environmental conditions, in the middle-west region of Brazil, grazing in pastures of Brachiaria brizantha, cv. Marandu; Panicum Maximum, cv. Tanzania and Brachiaria humidícula. The heifers of the BIO group were kept in the presence of bulls while being maintained on pasture; the animals in the BIO+S group were kept in the presence of bulls while being managed on pasture and were fed a diet with greater energy and protein content to produce 0.49 kg of BW gain/day; the animals in control group (the NBIO) were kept away from bulls and under pasture conditions; and the animals in the NBIO+S group were kept away from bulls, were maintained on pasture, and were fed the same diet as the BIO+S group. Heifers were bred at 22-23 months of age, and pregnancy diagnosis was made 45 days after the end of the breeding season. There were differences (P<0.05) between groups regarding pubertal heifers up to 19 months (NPH), final body weight (FBW) and pregnancy rates (P<0.01), with an advantage for the animals in the BIO and BIO+S groups. Although the effect of a diet with greater protein and energy content was not clear in this experiment, the exposure of heifers to a male during the prepubertal period decreased age at the first breeding season, resulting in a significant reduction in age of first pregnancy in Nelore heifers kept under extensive management systems in a tropical environment.     

Indirect immunohistochemistry on skin biopsy for the detection of persistently infected cattle with bovine viral diarrhoea virus in Italian dairy herds

Indirect immunohistochemistry (IHC) on skin biopsies for identification of persistently infected (PI) animals has been used as a parallel test to antigen and antibody ELISAs in a bovine viral diarrhoea (BVD) voluntary control program. The aim was to evaluate the reliability and feasibility of IHC on ear skin tissues to detect PI animals in field conditions, including both adult and calves under 6 months of age. In animals over 6 months of age skin biopsy and blood sample were collected at the same time, whereas in young calves blood sampling was performed when animals reached 6 months of age. One hundred and sixty-five animals were tested and immunohistochemical results were compared with those of antigen ELISA. In case of inconclusive results virus isolation and virus neutralization assays were performed. Agreement K value was 0.96. Immunohistochemical staining in positive animals was clearly detectable in the keratinocytes of the epidermis and adnexa.     

Effects of early weaning and housing conditions on the development of stereotypies in farmed mink

Female mink pups were weaned at 6, 8 or 10 weeks of age and subjected to two different housing conditions. They were either kept together with a single male sibling in traditional mink cages (30x45x90 cm) or housed socially with all litter-mates in an alternative system consisting of three adjoining traditional cages (90x45x90 cm). All cages were supplied with nest boxes. At 5 months of age, the siblings were removed leaving the females socially isolated in the two different cage systems. Females' stereotypies were quantified by repeated scanning observations under the social housing conditions immediately before removal of the siblings, and again at the age of 7 and 9 months, when the animals had stayed solitary in the two systems for 2 and 4 months. Solitary females showed significantly more stereotypies than females under social housing conditions in both cage systems. Stereotypies were more frequent in the smaller traditional cages. Stereotypies declined from 7 to 9 months of age among solitary animals in traditional cages but not in alternative cages. Early-weaned solitary females in traditional cages showed more stereotypies than later-weaned animals, but only when measured at the age of 7 months. It is suggested that early weaning, individual housing and small cages promote the development of stereotypies in farmed mink. The influence of early weaning on stereotypies seems to decline with age, while effects related to individual housing and small cages appear to be more persistent.     

Acetylcholine Synthesis and Release in the Extensor Digitorum Longus Muscle of Mature and Aged Rats

Uptake of labeled choline and its incorporation into acetylcholine (ACh) were assayed at the neuromuscular junction of the extensor digitorum longus (EDL) muscle of rats aged 11 (mature adult) and 27 (aged) months. Under resting conditions, there were no significant differences in muscle ACh or choline levels. Following a 1-h incubation in labeled choline, however, tissue from the younger rats contained significantly greater amounts of labeled choline and labeled ACh; the specific activities of ACh and choline were nearly 10-fold higher in the 11-month-old animals, indicating reduced uptake of labeled choline in the older animals. ACh and choline efflux rates under resting conditions did not change with age, indicating an uncoupling of exogenous choline uptake and ACh efflux in EDL during aging. During nerve stimulation (1 Hz), the amount of labeled choline incorporated into ACh was 150% greater in the aged animals. The specific activity of ACh released during stimulation was correspondingly greater in the 27-month-old animals, although total ACh released did not change appreciably with age. There were no age-related differences in choline acetyltransferase activity. Contrasting results were obtained from diaphragm in previous studies; the linkage between choline uptake and ACh efflux was maintained during rest and stimulation in the diaphragm. Hypothetically, these differences between EDL and diaphragm may be related to their diverse activation patterns: EDL is recruited much less frequently and less regularly than diaphragm, a continually active vital muscle.     

The hepatic cytochrome level in the cat (Felis catus): normal value and variations in relation to some biological parameters.

1. The average level of the hepatic hydroxylating cytochrome was determined in the cat (0.380 +/- 0.085 nmol.mg-1 proteins). 2. Great individual differences were noted. 3. The level of cytochrome increases with age in the immature (age less than or equal to 8.5 months) and young adults (age: 10-18 months). It is diminished in the older animals. 4. The measured average level is lower than that in other species, and approaches the human one. 5. The induction capacity of phenobarbital is lower in the cat than in other mammals, for example the rat or the mouse.     

Mechanisms for delayed density-dependent reproductive traits in field voles, Microtus agrestis: the importance of inherited environmental effects

Reproductive traits of voles vary with the phases of the population density fluctuations. We sought to determine whether the source of this variation resides in the individuals or in their environment. Overwintering field voles ( Microtus agrestis ) from two cyclic out-of-phase populations (increase and peak phases) were sampled in early spring and bred in the laboratory for two generations under standardised conditions with ambient light and temperature. Monitoring of the source populations by capture-mark-recapture showed large differences in reproductive performance. In the increase area, reproduction started six weeks earlier, the probability of maturation of young-of-the-year was more than ten times higher during mid-summer, and reproduction continued nearly two months later in the autumn than in the peak area. These differences were not found to be associated with a difference in age structure of overwintered animals between the two areas (assessed by the distribution of eye lens masses from autopsy samples). Although the population differences in reproductive traits were to some degree also present among the overwintered animals in the laboratory, we found no difference in reproductive traits in the laboratory-born generations. There was a strongly declining seasonal trend in probability of sexual maturation both in the field and in the laboratory under ambient light conditions. However, in the field there were large population differences in the steepness of the seasonal decline that were not seen under the standardised laboratory conditions. We conclude that seasonal decline in maturation rates is governed by change in photoperiod, but that the population level variation in the shape of the decline is caused by a direct response to the environment and not due to variation in any intrinsic state of the individuals reflecting the environment experienced by the previous generation(s).     

Influences of month of birth and age on patterns of luteinizing hormone secretion in prepubertal heifers

An experiment was done to determine if month of birth and age influenced patterns of luteinizing hormone (LH) secretion in prepubertal heifers. Mean concentrations of LH increased linearly (P < .05) in March-born heifers between one and seven months of age. This was partially due to an increase in number of LH pulses. The prepubertal pattern of LH concentrations was quadratic (P < .05) for heifers born in September because concentrations were slightly higher (P = .15) than those in March-born heifers at one month of age. There were no differences between groups during the remainder of the prepubertal period (3 to 7 months). There was a tendency (P = .18) for September-born animals to reach puberty at younger ages than those born in March. September-born heifers also had greater (P = .06) average daily gains, but body weights at puberty were similar for the two groups. These results show that season of birth influenced LH concentrations at one month of age, but did not significantly affect the increase between three and seven months of age.     

Influence of pre- and post-pubertal grazing regimes on adult testicular morphology in extensively reared corriedale rams

The aim of the present study was to determine whether pre- and post-pubertal young rams on different grazing regimes, resulting in differences in live weight (LW), would show corresponding differences in testicular growth or testicular morphometry that could influence the reproductive traits of these rams upon reaching adulthood. Forty-one spring-born Corriedale rams were reared on either native pasture (low feeding level, Group L, n=22) or improved pasture (higher feeding level, Group H, n=19) from 1 to 7 months of age. Thereafter, half the animals in the native-pasture group were placed on improved pasture and vice versa, thus creating an additional four differential-grazing treatment groups (Groups LL, n=11; LH, n=11; HL, n=10; and HH, n=9). Animals were managed in this way until 18 months of age. Half the animals from each group were then castrated and their testes were subjected to morphometric analysis. The remaining animals (Groups LL, n=6; LH, n=6; HL, n=5; and HH, n=4) were managed together until 30 months of age (from 18 to 27 months on native pastures and from 27 to 30 months of age on improved pastures, at a stocking rate of two to three rams per hectare), whereupon they were also castrated for testicular morphometry. LW and scrotal circumference (SC) were recorded every 60 days. The stereological analysis of testicular parenchyma included counts of elongated spermatids and Sertoli cells. Differences (P<0.001) in LW were observed between feeding levels, even at 30 months of age. Differences (P<0.001) in SC existing at the end of the differential treatment (18 months of age) disappeared (n.s.) soon after. Most differences (P<0.05) in testicular morphometry existing at the end of the differential treatments were no longer significant 1 year later. It is concluded that changes in grazing management during pre- and post-pubertal periods can induce short-lived differences in testicular post-natal growth in Corriedale rams but do not influence testicular morphology or function later in life.     

Sympathetic regulation of adrenal medullary function during aging

Our recent studies on changes in sympathoadrenal medullary function with age in anesthetized Wistar rats were reviewed. Although secretion rates of adrenaline and noradrenaline from the adrenal gland under resting conditions varied among animals, they gradually increased after 300 days and reached a level 2-4 times higher at 800-900 days compared with that of 100 days. Spontaneous activity of a single sympathetic nerve fiber under resting conditions also increased during aging in a manner similar to the catecholamine secretion rates. Reflex responses of mass activity of adrenal sympathetic nerve fibers to stimulation of baroreceptor and cutaneous mechanoreceptors were compared in young adult (4 months old) and aged (26 months old) Wistar rats under strictly controlled conditions for anesthesia, respiration and body temperature. Under these conditions the reflex depression in response to baroreceptor stimulation and cutaneous brushing as well as reflex excitation in response to cutaneous pinching were quite well maintained in the aged rats.     

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.     

Influences of stress, age and sex on serum growth hormone and free fatty acid levels in cattle.

The serum growth hormone (BGH) and free fatty acid (FFA) concentrations of large groups of calves (male and female ranging in age from 8 to 14 days and from 3 to 6 months old), bulls, oxen, heifers and cows under normal conditions were compared with the serum BGH and FFA values of corresponding large groups of animals under stress conditions. Stress was caused by transport, as it routinely occurs, and was further reinforced by a stay of several hours in unfamiliar surroundings. Stress provokes a significant increase in serum FFA in all groups and induced a significant decrease in serum BGH in all groups, with the exception of the non-pregnant heifers. The male and female calves which were 8 to 14 days old, had significantly higher BGH levels than the older animals. The female calves from 3 to 6 months of age, and the heifers had significantly lower BGH levels than the cows. The males had higher serum BGH values than the females, but a significant difference could be demonstrated only between the male and female calves of both age-groups. The oxen, which were 1 to 3 years old, had significantly higher BGH levels than the non-pregnant heifers of the same age. It appeared that gestation had no influence on the serum BGH and FFA levels of both heifers and cows.     

Weekend versus working day: differences in telemetric blood pressure in male Wistar rats

Blood pressure (BP) is a frequently monitored parameter in research. Various methods are used to obtain BP values in animal models, but telemetry is the method of choice because it allows for continuous monitoring in conscious and freely moving animals. However, factors due to the animal facility, like activities and sound, can still influence measurements. We, therefore, retrospectively compared BP values in adult male Wistar rats during working hours with values from non-working days. Telemetry devices were implanted according to standard protocol. Values were obtained at the age of 6 and 12 months during working hours (Friday 10:00-16:00 h, lights on 06:00-18:00 h) and compared with data from the average of Saturday 10:00-16:00 h and Sunday 10:00-16:00 h, representing non-working days. Data were available from 12 and 7 rats at 6 months and 12 months of age respectively. Relative differences in heart rate, spontaneous locomotor activity, systolic and diastolic BP were 2.2% (P<0.001), 32.9% (P<0.05), 3.2% (P<0.05) and 3.7% (P<0.05), respectively, with no differences between the age groups. We have shown a significant and important difference between BP values obtained during working hours and non-working days using telemetry in adult male Wistar rats. This phenomenon has implications for the interpretation of BP measurements in animals.     

Biologic data of Macaca mulatta, Macaca fascicularis, and Saimiri sciureus used for research at the Fiocruz primate center

Physiological parameters of laboratory animals used for biomedical research is crucial for following several experimental procedures. With the intent to establish baseline biologic parameters for non-human primates held in closed colonies, hematological and morphometric data of captive monkeys were determined. Data of clinically healthy rhesus macaques (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis), and squirrel monkeys (Saimiri sciureus) were collected over a period of five years. Animals were separated according to sex and divided into five age groups. Hematological data were compared with those in the literature by Student's t test. Discrepancies with significance levels of 0.1, 1 or 5% were found in the hematological studies. Growth curves showed that the sexual dimorphism of rhesus monkeys appeared at an age of four years. In earlier ages, the differences between sexes could not be distinguished (p < 0.05). Sexual dimorphism in both squirrel monkeys and cynomolgus monkeys occurred at an age of about 32 months. Data presented in this paper could be useful for comparative studies using primates under similar conditions.     

Effects of cholecystokinin octapeptide (CCK-8) on food intake in adult and aged rats under different feeding conditions

The effects of CCK on food intake were investigated under fixed feeding conditions in comparison to a test meal taken after 16 h of food deprivation. The experiments were performed on young adult rats (8 weeks old) as well on aged rats (23 months old). Intraperitoneal CCK-8 (8 and 40 μg/kg) significantly reduced the size of a test meal following 16-h food deprivation. This effect was independent of the age of the rats. However, under fixed feeding conditions neither of the doses used in this study reduced food intake in the young adult rats, whereas the highest dose of 40 μg/kg did so in the aged rats. These results suggest that the hypophagic effect of exogenous CCK-8 depends on experimental conditions, food intake being reduced after a period of food deprivation but not under a fixed feeding regimen in adult animals. Furthermore, the data suggest that age is a factor contributing to the complex behavioral actions of CCK, because only old animals were more susceptible to an anorectic action of CCK under the fixed feeding schedule. An explanation may lie in an interaction of other known behavioral effects of CCK (e.g., anxiogenic, mnemonic action) with its effects under the different feeding schedules.     

Effect of a 3-hydroxypyridine-class antioxidant on the functional activity of the central nervous system in mice of different age groups

Characteristics of emotional reactivity, orienting-investigating behaviour and of preservation of conditioned reflex of passive avoidance were used for estimation of the functional activity of animals CNS and also of psychotropic effect of antioxidant 2-ethyl-6-methyl-3-oxypyridine (3-OP) on mice of different age. Studies were carried out on white outbred young (3 months) and elderly (15 months) male mice. Different character was observed of the emotional reactions of different age mice to aversive stimuli and to the open field conditions. Differences were noted of psychotropic effects of 3-OP in 3 and 15-months animals.     

Undernutrition of ewe lambs in utero and in early post-natal life does not affect hypothalamic-pituitary function in adulthood

The effect of undernutrition in utero, during late gestation (from day 100), and early neonatal life on hypothalamic-pituitary function was investigated in female lambs born to ewes fed rations calculated to provide either 100% (high; H) or 70% (low; L) of the energy requirements to sustain a twin pregnancy. Following parturition in early spring, ewes and lambs were maintained on pasture with sward heights of 6 cm (H) or 4 cm (L) until week 8 of lactation and then sward heights of 5 cm (H) or 3 cm (L) until weaning at week 14. Mean lamb birth weights were 18% lower in L than H animals (P<0.05) and mean liveweights were 23% lower in the L animals (P<0.001) at weaning at 14 weeks of age. Liveweight differences were not significant at, or after, 26 weeks of age. There were no significant differences between pre-pubertal H and L animals, either before (26 weeks) or after ovariectomy (31 weeks), with respect to hypothalamic or pituitary activity, as measured by LH pulse frequency, pulse amplitude or mean plasma LH and FSH concentrations and the responses to GnRH injection as measured by LH peak amplitude, respectively. Similarly there were no differences in any of these variables in pubertal animals at 18 months of age. At 31 weeks of age, H animals had significantly lower pituitary GnRH receptor binding (P<0.01) and lower ERalpha mRNA content (P<0.05) than L lambs. There were no differences with treatment in the abundance of mRNA for LHbeta, FSHbeta or GnRH-receptor at 31 weeks of age or in pubertal animals aged 18 months, when there were no significant differences with treatment in GnRH receptor binding or ERalpha mRNA expression. It is concluded that effects on lifetime reproductive function of female sheep of undernutrition during late gestation and early neonatal life are unlikely to be expressed through permanent changes in hypothalamic-pituitary function and are therefore attributable to effects exerted directly on the ovary.     

Long-term, low-level microwave irradiation of rats.

Our goal was to investigate effects of long-term exposure to pulsed microwave radiation. The major emphasis was to expose a large sample of experimental animals throughout their lifetimes and to monitor them for effects on general health and longevity. An exposure facility was developed that enabled 200 rats to be maintained under specific-pathogen-free (SPF) conditions while housed individually in circularly-polarized waveguides. The exposure facility consisted of two rooms, each containing 50 active waveguides and 50 waveguides for sham (control) exposures. The experimental rats were exposed to 2,450-MHz pulsed microwaves at 800 pps with a 10-microseconds pulse width. The pulsed microwaves were square-wave modulated at 8-Hz. Whole body calorimetry, thermographic analysis, and power-meter analysis indicated that microwaves delivered at 0.144 W to each exposure waveguide resulted in an average specific absorption rate (SAR) that ranged from 0.4 W/kg for a 200-g rat to 0.15 W/kg for an 800-g rat. Two hundred male, Sprague-Dawley rats were assigned in equal numbers to radiation-exposure and sham-exposure conditions. Exposure began at 8 weeks of age and continued daily, 21.5 h/day, for 25 months. Animals were bled at regular intervals and blood samples were analyzed for serum chemistries, hematological values, protein electrophoretic patterns, thyroxine, and plasma corticosterone levels. In addition to daily measures of body mass, food and water consumption by all animals, O2 consumption and CO2 production were periodically measured in a sub-sample (N = 18) of each group. Activity was assessed in an open-field apparatus at regular intervals throughout the study. After 13 months, 10 rats from each group were euthanatized to test for immunological competence and to permit whole-body analysis, as well as gross and histopathological examinations. At the end of 25 months, the survivors (11 sham-exposed and 12 radiation-exposed rats) were euthanatized for similar analyses. The other 157 animals were examined histopathologically when they died spontaneously or were terminated in extremis.     

Podolian beef production on pasture and in confinement

From February to August 2007 the effect of rearing system (confined (C) v. free ranging (FR)) and season (spring v. summer) was evaluated on behaviour, immune and blood parameters and beef production (experiment 1). From February to August 2008, the effect of rearing system was only evaluated on beef production (experiment 2). A total of 12 Podolian young bulls were used each year. They were slaughtered at 18 and 15 months of age in 2007 and 2008, respectively. Herbage mass and pasture composition were monitored during the 2 years. Pasture availability, in terms of herbage mass and composition, as well as its seasonal development, was similar in the 2 years. In the first experiment, FR animals spent more time walking (P < 0.05), feeding (P < 0.001) and standing (P < 0.01) and showed a lower number of agonistic (P < 0.05) and non-agonistic social interaction than C animals (P < 0.01). Significant lower concentrations of serum urea nitrogen (P < 0.001) and creatinine (P < 0.10) in FR animals indicated a lower protein nutritional status due to inadequate protein availability at pasture. As a consequence, average daily gains (P < 0.05), slaughter weight (P < 0.05) and body condition score (P < 0.01) were lower in grazing animals as compared with C bulls. Cellular immune responsiveness was higher in FR animals (P < 0.05). Similarly, antibody titre to keyhole limpet hemocyanin was higher in FR bulls at the 2nd and 3rd month after antigen injection (P < 0.05), whereas it tended to be higher at the 4th month (P < 0.10). In both experiments, grazing negatively affected meat colour in terms of lightness. Eighteen-month-old bulls also showed lower final weight, weight gain and body conditions when kept outdoor: a possible consequence of nutrient deficits, as suggested by the metabolic status of FR animals. The same animals, however, benefited from FR in terms of natural behaviour expression and immune responsiveness. When the experiment was replicated the subsequent year, on animals slaughtered at 15 months of age, no differences between the performances of FR and C animals were detected. The earlier slaughter age system was also proportionally less dependent on external inputs as grazing was not extended to the dry season when herbage mass availability was lower.