A Novel Approach for Non-Invasive Continuous In-Line Control of Perfusion Cell Cultivations by Raman Spectroscopy.

Continuous manufacturing is becoming more important in the biopharmaceutical industry. This processing strategy is favorable, as it is more efficient, flexible, and has the potential to produce higher and more consistent product quality. At the same time, it faces some challenges, especially in cell culture. As a steady state has to be maintained over a prolonged time, it is unavoidable to implement advanced process analytical technologies to control the relevant process parameters in a fast and precise manner. One such analytical technology is Raman spectroscopy, which has proven its advantages for process monitoring and control mostly in (fed-) batch cultivations. In this study, an in-line flow cell for Raman spectroscopy is included in the cell-free harvest stream of a perfusion process. Quantitative models for glucose and lactate were generated based on five cultivations originating from varying bioreactor scales. After successfully validating the glucose model (Root Mean Square Error of Prediction (RMSEP) of ∼0.2 g/L), it was employed for control of an external glucose feed in cultivation with a glucose-free perfusion medium. The generated model was successfully applied to perform process control at 4 g/L and 1.5 g/L glucose over several days, respectively, with variability of ±0.4 g/L. The results demonstrate the high potential of Raman spectroscopy for advanced process monitoring and control of a perfusion process with a bioreactor and scale-independent measurement method.

Subchronic administration of fluoxetine impairs estrous behavior in intact female rats.

Treatment with serotonin reuptake inhibitors (SRIs) has been shown to cause reduced libido and anorgasmia in women. A large body of evidence suggests that serotonin may influence sexual behavior in estradiol + progesterone primed, gonadectomized female rats; however, the influence of selective SRIs on the estrous behavior of intact female rats has not been described previously. In the present study, the effect of 1 to 3 weeks of fluoxetine administration (10 mg/kg daily) on vaginal and behavioral estrus in intact female rats was studied; in addition, the effect of fluoxetine (same dose, 1-8 weeks) on copulatory behavior and on sexual motivation in hormone-primed gonadectomized rats was investigated. Subchronic administration of fluoxetine did not influence cyclicity as judged by the examination of vaginal smears but significantly reduced the percentage of rats displaying receptive behavior in the estrous phase. In addition, fluoxetine significantly reduced receptive behavior, including lordosis, in ovariectomized female rats primed with estradiol (6.25 micrograms/rat; -48 hr) plus progesterone (1.0 mg/rat, -4 hr); in contrast, sexual motivation--as reflected by the amount of time these rats elected to spend in the vicinity of a male rather than in the vicinity of a female or elsewhere--was little affected by the treatment.

Postnatal testicular secretions partially restore the disturbances in reproductive activity caused by prenatal hormonal manipulation.

Male rats that prenatally had been exposed to an antiestrogen, nitromifene citrate (CI 628), showed evidence of impaired defeminization and masculinization in adulthood, suggesting a role of endogenous estrogen for the sexual differentiation of the male. The present study was undertaken to investigate a possible role of postnatal testicular secretions for the above behavioral effects. Male rats were exposed prenatally to CI 628 (1 mg/rat) or saline, and castrated on Day 0, Day 10, or Day 90 after birth. After treatment with gonadal hormones in adulthood, the males were tested for feminine and masculine sexual behavior and for sexual orientation, both when sexually naive and after they had acquired sexual experience. The following conclusions were drawn: 1. Permanent deficits of lordotic behavior were observed in all experimental groups, suggesting the importance of prenatal estrogen for the defeminization process. 2. Hop/darting and ear wiggling behaviors were enhanced in Day-0 and Day-10 castrates, and blocked in the Day-90 castrates. The restitution of these behaviors to normal levels in Day-90 castrates suggests that, in addition to prenatal estrogen, postnatal testicular secretions also are involved in the behavioral defeminization process. 3. Prenatal estrogen contributes to masculinization as evidenced by the impaired ejaculatory behavior observed in all experimental groups. 4. Male-typical sexual orientation toward the female seems to be facilitated by prenatal estrogen. Both the masculinization and the defeminization of male-typical sexual orientation toward a female were impaired by castration at birth and at Day 10 in the experimental animals, but a full restoration of the sexual orientation toward females was seen in Day-90 castrates, suggesting the restorative role of postnatal testicular secretions for these processes.

The effects of lesions in the mesencephalic raphe systems on male rat sexual behavior and locomotor activity.

Lesions were produced in the median and the dorsal raphe nuclei of the rat, and the effects of these injuries on various behaviors were studied. The median raphe lesions had facilitatory effects on the male rat sexual behavior, as evidenced by a decrease in the number of intromissions prior to ejaculation, as well as shortening of the ejaculation latency and the postejaculatory interval. No effects were seen on the masculine sexual behavior after lesions in the dorsal raphe nucleus. The median raphe lesions drastically increased the locomotory activity whereas the dorsal raphe lesions did not. Treatment with FG5893, a 5-HT1A agonist/5-HT2A receptor antagonist, increased the locomotor activity of the dorsal-raphe-lesioned rats but did not affect the median-raphe-lesioned animals. These results suggest that the dorsal and the median raphe nuclei play different roles in the neural regulation of the sexual behavior and locomotor activity of the male rat. The median raphe nucleus appears to exert a modulatory influence upon these behaviors, which probably is mediated by serotonergic neural pathways.

Sexual preference and feminine and masculine sexual behavior of male rats prenatally exposed to antiandrogen or antiestrogen.

Male rats were prenatally (Day 10-19 of pregnancy) exposed to an antiestrogen, nitromifene citrate (CI628, 1 mg/rat), or an antiandrogen, cyproterone acetate (CA, 10 mg/rat), and in adulthood were examined for their exhibition of male-typical and female-typical behavior pattern. Treatment with CI628 abolished the capacity of the adult intact male to ejaculate, enhanced his potential to exhibit feminine sexual behavior, and decreased the intensity of the level of female-oriented behavior in a two-choice stimulus situation (estrous female vs active male). The administration of testosterone (T) did not alter these behaviors. Males exposed to CA showed low levels of lordosis behavior and normal levels of female-oriented preference. Further, they showed increased frequency of mounts and decreased number of intromissions, and only a few males ever ejaculated. Macroscopic inspection of the genital organs of the CI628-treated males revealed complete absence of the prostate. The dissections of the CA-treated males revealed a poorly developed penis and a blind-ending vagina. It was concluded that prenatal estrogen (E) is involved (1) in determining the development of mechanisms destined to mediate the display of male-typical behaviors in adulthood, (2) in suppressing the development of mechanisms of female-typical behaviors, and (3) seems to stimulate neural mechanisms influencing sexual preference behavior in the adult.

Adrenalectomy does not prevent the ability of 8-OH-DPAT to decrease the ejaculatory threshold in male rats.

The present experiments demonstrate that 8-OH-DPAT (0.25 mg/kg SC, - 15 min) produced a decrease in the ejaculatory threshold to the same extent in adrenalectomized male rats as in sham operated controls. Both groups of animals displayed a marked and statistically significant decrease in number of mounts and penile intromissions preceding ejaculation and in the ejaculation latency, as a result of treatment with 8-OH-DPAT. Adrenalectomy per se did not affect any aspect of the male rat's sexual behavior (latency to first intromission, number of mounts or penile intromissions, ejaculation latency or the postejaculatory interval). The surgical removal of the adrenals was verified by measurements of plasma corticosterone levels. It is concluded that well documented effects of 8-OH-DPAT on adrenal secretions do not contribute to its ability to decrease the ejaculatory threshold in male rats.

Age-dependent variations in the sexual preference of male rats.

Male rats were tested for their sexual preference behavior at either 37, 70, 90, or 150 days of age on three different occasions; while still sexually naive, after sexual experience with a receptive female, and while sexually aroused by having initiated copulation. These tests resulted in the following findings: a) 37-day-old sexually naive males showed a preference for other males and failed to show a preference for either sex after exposure to females; b) 70- and 90-day-old males showed a statistically significant preference for the female after acquiring sexual experience; c) 150-day-old animals showed a female-oriented preference only after being sexually aroused with two intromissions preceding the preference test, and d) none of the age groups tested showed a female-oriented preference without previous exposure to females. It was then concluded that a) female-oriented behavior requires sexual experience and b) the effects of experience varies with age.

Experience modulates the influence of gonadal hormones on sexual orientation of male rats.

The aim of this study was to investigate whether heterosexual experience influences the male's partner preference and the role of gonadal hormones, including several androgens and estradiol, on female-oriented behavior. In the first experiment, the animals were exposed to a four-stimuli test situation including a sexually active male, a castrated male, an estrous female, and an ovariectomized (ovx) female. Castrated naive and experienced male rats were implanted with either testosterone (T), estradiol (E2), or an empty tubing (blank) and compared to intact naive and experienced male rats. None of the naive animals, whether castrated or intact, showed a consistent preference for any of the four stimuli. Whereas when sexually experienced, only intact and T-treated males showed a female-oriented preference. In the second experiment, the animals were allowed to choose between an active male and an estrous female. Castrated naive male rats were implanted with either T, E2, or DHT, or injected daily SC with the synthetic nonaromatizable androgen, methyltrienelone (R 1881). Two groups of intact males, one consisting of experienced and the other of naive animals, were also included in this experiment. The experienced intact males showed a significant preference for the estrous female, while the intact naive males showed no preference for either of the two stimuli. After the animals had gained heterosexual experience, intact and androgen-treated males showed a significant preference for the female. Neither the administration of R 1881 nor E2 promoted a female-oriented behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of exposure to an estrous female on forebrain monoaminergic neurotransmission in the non-copulating male rat.

Regional changes in the rate of brain monoamine synthesis were monitored in male rats exposed to, but prevented from physical contact with, an estrous or an ovariectomized female. The in vivo rate of tyrosine and tryptophan hydroxylase activities were estimated by measuring the accumulation of DOPA and 5-HTP following inhibition of cerebral aromatic L-amino acid decarboxylase by means of 3-hydroxybenzylhydrazine (NSD-1015) treatment (100 mg/kg i.p.). 5 min upon NSD-1015 treatment, the males were exposed to an intact estrous female or an ovariectomized female for 20 min before decapitation and brain dissections. Exposure to an estrous female produced an increased rate of tyrosine and tryptophan hydroxylase activity in the medial prefrontal cortex, the dorso-lateral neostriatum and in the ventral neostriatum, in comparison with home-cage controls. By the same comparison, exposure to an ovariectomized female resulted in an increased rate of tyrosine hydroxylase activity in the medial prefrontal cortex, but not in the neostriatal areas, whereas tryptophan hydroxylase activity was unaffected. Finally, exposure to the empty test cage, with no stimulus females present, did not produce any statistically significant changes in the rate of tyrosine or tryptophan hydroxylase activity in any of the brain areas sampled. Taken together with recent findings from this laboratory, the present results demonstrate that the level of sexual motivation brought about by the olfactory, auditory and/or visual stimulation of a receptive female is associated with an increased demand on catecholamine and 5-hydroxytryptamine synthesis in the limbic forebrain of the male rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Sexual orientation and sexual motivation of the adult male rat.

Intact sexually experienced male rats were allowed to choose between staying in the vicinity of an estrous female or a sexually active male under different conditions of sexual arousal. In one group, the animals were allowed to perform two intromissions before the start of the partner preference test. In a second group, the subjects were allowed to reach a moderate level of sexual satiation by repeated ejaculations 24 h before testing. A third group was not given any pretreatment. Independently of the conditions of the pretreatment, all groups showed a clear female-oriented behavior. However, animals performing two intromissions before the test spent significantly more time with the female and showed a higher number of visits to her compared to the untreated controls. Males being sexually exhausted after repeated ejaculations showed a significantly lower level of female-oriented behavior than did the controls. It was concluded that the male's readiness to orient towards the female was affected by his motivational state.

Synergistic action of estradiol, progesterone and testosterone on rat proceptive behavior.

The role of estradiol (E), progesterone (P) and testosterone (T) in the control of rat feminine sexual behavior (receptivity, proceptivity and sexual orientation) was analyzed. The action of estradiol benzoate (EB, 1.25 micrograms/rat x 3 days) and P (0.5 mg/rat) was compared in ovariectomized (OVX) and ovariectomized-adrenalectomized (OVX + ADX) subjects. Administration of EB alone was followed by maximum levels of lordosis behavior and a male-directed orientation in both OVX and OVX + ADX females. A reduction in the level of proceptivity was observed in EB-treated OVX + ADX animals as compared with EB-treated OVX rats. The administration of P to OVX + ADX females resulted in an increase in proceptive behavior similar to that observed in OVX EB-treated animals. A further study analyzed the effect of combined administration of EB, P and various doses of T (30, 90 and 270 micrograms/rat) in OVX + ADX rats. A synergistic action of all three hormones on proceptivity was observed. Neither a further increase in lordosis nor in male-directed orientation was observed in EB-treated females after additional treatment with P or T independently or together. Finally, we studied the effect of adrenalectomy on the spontaneous onset of estrous behavior. Adrenalectomy did not modify any aspect of normal feminine sexual behavior, suggesting that the adrenals, in the presence of the ovaries, are of no critical importance for this behavior. The possible contribution of adrenal steroids to the expression of proceptivity is discussed.

Role of androgen, estrogen and sexual experience on the female rat's partner preference.

Virgin ovariectomized rats were implanted with Silastic tubings containing estradiol (E2, 5 mm), testosterone (T, 30 mm), dihydrotestosterone (DHT, 30 mm), or injected daily SC with the synthetic nonaromatizable androgen, methyltrienelone (R 1881, 5 mg.kg-1) daily for three weeks. Animals were tested for partner preference behavior (staying in the vicinity of a sexually active male, an estrous female or staying elsewhere) before and after they had been tested for feminine and masculine sexual behavior. All naive androgen-treated groups showed a male-oriented preference, while the naive E2-treated females did not show any consistent partner preference. Sexual experience abolished the male-directed orientation of the androgen-treated groups while E2 treatment induced a male-directed orientation. E2-treated females spent significantly longer time elsewhere in both tests compared to the other groups which might be due to insufficient levels of E2. Feminine sexual behavior was seen after treatment with E2 or T but not after treatment with DHT or R 1881. It was concluded, 1) that the effect of treatment with an androgen on the partner preference behavior differs according to whether the females are virgins or sexually experienced, and 2) the effect of the hormone treatment on the partner preference behavior is independent of whether the hormone stimulates feminine sexual behavior.

Peripheral anosmia attenuates female-enhanced aggression in male rats.

It is well established that male rats with prior access to sexually active females show enhanced offensive aggression toward unfamiliar male intruders. The present study assessed the importance of the sense of smell for this facilitatory effect. It was found in 2 independent experiments that anosmia, induced peripherally by surgically removing the olfactory epithelium and cutting the olfactory nerves, reduced baseline levels of offensive aggression and significantly attenuated the female-enhanced aggression effect. It was also found that sexual performance of anosmic rats was context-dependent, in that it was more impaired in the homecage environment than in standard observation cages. In contrast to sham-operated males, the experimental animals showed no preference for estrous over anestrous females in a mate choice test. Anosmic males did not appear more fearful than controls, as assessed in a hyponeophagia test, but they showed less exploratory behavior (rearing and head-dipping) in the hole-board test, and less rearing activity in automated activity boxes.

Sexual orientation in male rats prenatally exposed to ethanol.

Previous studies have shown that prenatal ethanol exposure causes feminization of the male offspring, as evidenced by display of female sexual response (lordosis), when mounted by a stud male. In the present study we examined whether or not the feminization induced by prenatal ethanol exposure also affected a different aspect of sexually motivated behavior, namely, the approach towards a receptive female normally displayed by male rats. The testing apparatus consisted of an open-field arena with two small boxes in which were placed the stimulus animals, in one box a male rat, in the other a receptive female. The partition between the stimulus and the experimental animals consisted of a metal net allowing both animals to see and smell each other without actual physical contact. The tendency to approach the receptive female or the male was assessed by the proportion of the observation period the experimental male spent near the receptive female or the male rat, respectively. The experiment was performed on the adult male offspring of mothers consuming a liquid diet containing 5% ethanol, giving rise to a daily ethanol intake of about 14 g/kg. One group of control mothers was given a liquid diet without alcohol but isocaloric with the alcohol-containing diet. Another control group had free access to water and lab chow. The results showed that male offspring of both control groups devoted 29% of the observation period near the receptive female as compared to 13% near the male. The ethanol-exposed males on the other hand devoted as much time, 20%, to the male as to the receptive female.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of environmental events immediately after birth on postnatal testosterone secretion and adult sexual behavior in the male rat.

A rise in plasma testosterone (T) levels occurs in male rats during the first 2 hr after birth which is of importance for the process of sexual differentiation. To study the influence of environmental factors on the postnatal T surge and sexual development, newborn male rats were subjected to various treatments immediately after cesarean delivery including cooling, ether anesthesia, and mother-infant separation. In adulthood, the animals were observed for masculine and feminine sexual behavior. Males anesthetized at 0 hr showed elevated levels of feminine sexual behavior and impaired masculine sexual behavior. Pups subjected to cooling or mother-infant separation showed slightly prolonged intromission latencies, but otherwise normal levels of feminine sexual behavior. Significantly elevated plasma T levels were found in intact pups 2 hr after birth but not in pups subjected to cooling or ether anesthesia. Significantly higher levels of T were observed in pups subjected to cooling 4 hr after birth, suggesting a delay of the T surge. The most pronounced impairing effects were seen in the defeminization process, but the masculinization process also is affected by ether anesthesia. It was concluded that ether anesthesia immediately after birth may permanently interfere with the sexual development by suppressing the neonatal T surge.

Sexual orientation, proceptivity, and receptivity in the male rat as a function of neonatal hormonal manipulation.

The influence of neonatal androgen on the potential to exhibit feminine sexual behavior was investigated. Male rats castrated on Day 0 but not those castrated on Day 4 or later showed hop/darting, ear wiggling, and lordotic behavior in response to treatment with estrogen and progesterone in adulthood at a frequency equal to that of females. Neonatal treatment with testosterone propionate (1 mg/rat for 4 days) abolished the capacity to show these behaviors. In subsequent experiments, involving castration of male rats at 0 or 4 hr after cesarean delivery, the effect of the postnatal surge of testicular secretions on the expression of female sexual behavior was investigated. No differences were seen in the frequency of hop/darting, ear wiggling, and receptivity between males castrated immediately or 4 hr after delivery. In a preference test where the experimental male could choose between an estrous female and a sexually active male, the neonatally castrated males preferred the company of a male when treated with estrogen and progesterone. The implantation of testosterone resulted in a preference for an estrous female. It was concluded that testicular secretions in the newborn male influence adult sexual orientation and suppress the ability to show proceptive and receptive behaviors.