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Plasma lutein and zeaxanthin and other carotenoids as modifiable risk factors for age-related maculopathy and cataract: the POLA Study.

PURPOSE: To assess the associations of plasma lutein and zeaxanthin and other carotenoids with the risk of age-related maculopathy (ARM) and cataract in the population-based Pathologies Oculaires Liées à l'Age (POLA) Study. METHODS: Retinal photographs were graded according to the international classification. ARM was defined by the presence of late ARM (neovascular ARM, geographic atrophy) and/or soft indistinct drusen (>125 microm) and/or soft distinct drusen (>125 microm) associated with pigmentary abnormalities. Cataract classification was based on a direct standardized lens examination at the slit lamp, according to Lens Opacities Classification System III. Plasma carotenoids were measured by high-performance liquid chromatography (HPLC), in 899 subjects of the cohort. RESULTS: After multivariate adjustment, the highest quintile of plasma zeaxanthin was significantly associated with reduced risk of ARM (OR=0.07; 95% CI: 0.01-0.58; P for trend=0.005), nuclear cataract (OR=0.23; 95% CI: 0.08-0.68; P for trend=0.003) and any cataract (OR=0.53; 95% CI: 0.31-0.89; P for trend=0.01). ARM was significantly associated with combined plasma lutein and zeaxanthin (OR=0.21; 95% CI: 0.05-0.79; P for trend=0.01), and tended to be associated with plasma lutein (OR=0.31; 95% CI: 0.09-1.07; P for trend=0.04), whereas cataract showed no such associations. Among other carotenoids, only beta-carotene showed a significant negative association with nuclear cataract, but not ARM. CONCLUSIONS: These results are strongly suggestive of a protective role of the xanthophylls, in particular zeaxanthin, for the protection against ARM and cataract.

Cataract↗

Suppression of androgen production by D-tryptophan-6-luteinizing hormone-releasing hormone in man.

Four male transsexual subjects were given a superactive luteinizing hormone-releasing hormone (LHRH) analogue, D-tryptophan-6-LHRH at daily doses of 100 micrograms for 3--6 mo. A decrease in beard growth, acne, and erectile potency was noted; the latter was documented objectively with the recordings of nocturnal penile tumescence episodes. Plasma testosterone and dihydrotestosterone levels fell to castrate values; basal prolactin and luteinizing hormone levels showed a small decline, whereas the acutely releasable luteinizing hormone was significantly suppressed. A rise of plasma testosterone from castrate to normal levels was demonstrable with the use of human chorionic gonadotropin. Discontinuation of treatment led to a normalization of erectile potency and plasma testosterone. The suppression of Leydig cell function by D-tryptophan-6-LHRH might have wide application in reproductive biology and in endocrine-dependent neoplasia (where it could replace surgical castration).

Adult↗

Uptake of low density lipoproteins by rat tissues. Special emphasis on the luteinized ovary.

The aim of this study was to determine how luteal cells of the hormone-primed (luteinized) ovary process low density lipoproteins (LDL). Ovary uptake of perfused 125I-LDL was assessed by tissue levels of radioactivity; the distribution of LDL protein in cells was assessed on autoradiograms of the fixed tissue; and the level of stimulation of steroidogenesis, as well as degradation of LDL protein, was assessed on effluent perfusion samples. Human LDL ligand used in these studies was rigorously defined biochemically and physiologically. Homologous (rat) LDL was used as a special ligand control. Other tissue controls included the use of perfused or in vivo-infused luteinized ovaries from animals pretreated to reduce circulating lipoprotein levels, perfused ovaries from a second hormone-primed model, perfused liver from estrogen-treated rats, and isolated and cultured cells from the same ovarian tissues used in the perfusion experiments. The results show that perfused LDL promptly stimulates steroidogenesis. However, the labeled protein moiety of the LDL is not interiorized by the luteal cells, nor is there evidence of LDL protein degradation in the effluent samples. In contrast, internalization of the ligand occurs when luteal cells are incubated with the ligand in vitro. We have observed also that uptake of the 125I-LDL by the ovary can be displaced equally well by excess unlabeled LDL or HDL3. Overall, these experiments suggest that in the intact luteinized ovary, LDL binds to the same sites on the cell surface where HDL "binds," and that LDL cholesterol must be obtained by these steroid hormone-producing cells by a mechanism that does not require internalization of the intact lipoprotein particle.

Animals↗

Glucagon-like peptide-1 stimulates luteinizing hormone-releasing hormone secretion in a rodent hypothalamic neuronal cell line.

To examine the influence of the putative satiety factor (GLP-1) on the hypothalamo-pituitary-gonadal axis, we used GT1-7 cells as a model of neuronal luteinizing hormone- releasing hormone (LHRH) release. GLP-1 caused a concentration-dependent increase in LHRH release from GT1-7 cells. Specific, saturable GLP-1 binding sites were demonstrated on these cells. The binding of [125I]GLP-1 was time-dependent and consistent with a single binding site (Kd = 0.07+/-0.016 nM; binding capacity = 160+/-11 fmol/mg protein). The specific GLP-1 receptor agonists, exendin-3 and exendin-4, also showed high affinity (Ki = 0.3+/-0.05 and 0.32+/-0.06 nM, respectively) as did the antagonist exendin-(9-39) (Ki = 0.98+/-0.24 nM). At concentrations that increased LHRH release, GLP-1 (0.5-10 nM) also caused an increase in intracellular cAMP in GT1-7 cells (10 nM GLP-1: 7.66+/-0.4 vs. control: 0.23+/-0.02 nmol/mg protein; P < 0.001). Intracerebroventricular injection of GLP-1 at a single concentration (10 microg) produced a prompt increase in the plasma luteinizing hormone concentration in male rats (GLP-1: 1.09+/-0.11 vs. saline: 0.69+/-0.06 ng/ml; P < 0.005). GLP-1 levels in the hypothalami of 48-h-fasted male rats showed a decrease, indicating a possible association of the satiety factor with the low luteinizing hormone levels in animals with a negative energy balance.

Animals↗

Co-localization of secretogranins/chromogranins with thyrotropin and luteinizing hormone in secretory granules of cow anterior pituitary.

We investigated the co-localization in secretory granules of secretogranins/chromogranins, thyrotropin, and luteinizing hormone in ultra-thin frozen sections of cow anterior pituitary by double immunoelectron microscopy, using specific antibodies and protein A-gold particles of different sizes. The distribution of secretogranin II, chromogranin A, and chromogranin B (secretogranin I) was largely similar. In cells containing secretory granules of relatively small size (100-300 nm) and low electron density (identified as thyrotrophs and gonadotrophs by immunolabeling for the respective hormone) and in cells containing both small (170-250 nm) and large (300-500 nm) secretory granules of low electron density (also identified as gonadotrophs), all three secretogranins/chromogranins were detected in most if not all granules, being co-localized with the hormone. In cells containing both relatively large (400-550 nm), electron-dense granules and small, less electron-dense secretory granules (150-300 nm), identified as somatomammotrophs by double immunolabeling for growth hormone and prolactin, all three secretogranins/chromogranins were predominantly detected in the subpopulation of small, less electron-dense granules containing neither growth hormone nor prolactin. Interestingly, this granule subpopulation of somatomammotrophs was also immunoreactive for thyrotropin and luteinizing hormone. These data show that somatomammotrophs of cow anterior pituitary are highly multihormonal, in that the same cell can produce and store in secretory granules up to four different hormones and, in addition, the three secretogranins/chromogranins. Moreover, selective localization of the secretogranins/chromogranins together with thyrotropin and luteinizing hormone in a subpopulation of secretory granules of somatomammotrophs indicates the preferential co-packaging of the secretogranins/chromogranins and these hormones during secretory granule formation.

Animals↗

Binding of an iodinated substance P analogue to cultured anterior pituitary prolactin- and luteinizing hormone-containing cells.

Several lines of anatomic, biochemical, and pharmacological evidence suggest that the neuropeptide substance P has a direct action on cells of the anterior pituitary lobe via a specific neurokinin-1 receptor. In the present study we confirmed this association by combining Bolton-Hunter iodinated substance P-receptor autoradiography with immunocytochemistry on cultured anterior pituitary cells. Radiolabeled substance P was bound to living cell cultures at 0 degrees C, and after a brief wash the cultures were fixed and processed immunocytochemically for prolactin and luteinizing hormone. A large proportion of cultured anterior pituitary cells possessed substance P binding sites. When receptor autoradiography was combined with immunocytochemistry, it was evident that both prolactin- and luteinizing hormone-immunoreactive cells were labeled with radiolabeled substance P. However, a small proportion of the radioligand-labeled cells were not stained by the immunocytochemical procedure, suggesting that additional cell types possess substance P receptors. The present study presents morphological evidence that substance P binds to prolactin- and luteinizing hormone-containing cells of the anterior pituitary lobe. Therefore, it is likely that substance P has a direct action on mammotrophs and gonadotrophs.

Animals↗

Hyperinsulinemia and stromal luteinization of the ovaries in postmenopausal women with endometrial cancer.

Ovarian secretion of testosterone and androstenedione is increased in postmenopausal women with endometrial cancer, and insulin stimulates ovarian stromal androgen synthesis in vitro. We undertook this study to investigate whether women with endometrial cancer have increased serum immunoreactive insulin levels. Ten postmenopausal women with endometrial carcinoma and 10 postmenopausal women without cancer who matched the cancer patients in age, years since menopause, and percentage of ideal body weight were studied. The women with endometrial cancer had significantly higher fasting serum insulin levels than the normal women [mean, 187 +/- 26 (+/- SE) vs. 55 +/- 11 pmol/L; P less than 0.01]. The cancer patients had significantly higher insulin responses after glucose administration than normal women (sum of 1, 2, and 3 h postglucose values, 5545 +/- 1526 vs. 1444 +/- 156 pmol/L; P less than 0.02), even though their glucose responses were similar. Nests of luteinized cells, which were positive for testosterone by immunoperoxidase staining, were found in the ovarian stroma of 8 of the women with endometrial cancer, but in only 1 of those without cancer (P less than 0.01). Specific high affinity insulin receptors were demonstrable in the stroma of the postmenopausal ovaries. These results suggest that the frequency of stromal luteinization is increased in women with endometrial cancer and that insulin may play a role in the pathogenesis of this luteinization.

Adenocarcinoma↗

Regulation of alpha 2-macroglobulin by luteinizing hormone and prolactin during cell differentiation in the rat ovary.

Alph alpha 2-macroglobulin (alpha 2M), a protease inhibitor which also binds growth factors and cytokines, is temporally expressed in association with remodelling phenomena in the ovary: ovulation and luteinization. Specific hormonal, cellular, subcellular, and molecular events regulating alpha 2M mRNA and protein have been analyzed during follicular growth, ovulation, and luteinization using complementary in vivo and in vitro models. Data demonstrate that alpha 2M mRNA and protein are synthesized in thecal cells of developing follicles in response to low levels of LH. Conversely, alpha 2M mRNA and protein are only synthesized by granulosa cells of follicles that have been stimulated to luteinize either in vivo by the LH surge or in vitro by FSH and testosterone and are also exposed to PRL. The obligatory requirement for PRL is specific; associated with increased numbers of PRL-binding sites; mediated by time-dependent appearance of alpha 2M in the endoplasmic reticulum (12 h), Golgi apparatus (24 h), and secretion vesicles (48 h); and involves in part increased transcription of the alpha 2M gene.

Animals↗

Prostaglandin D2 stimulates secretion of luteinizing hormone from pituitary gland in vitro.

The effect of prostaglandin D2 on the release of luteinizing hormone was studied in a superfusion system by superfusing human pituitary gland. Perfusion with 30 micrograms of prostaglandin D2 induced a significant increase of luteinizing hormone secretion. This is the first evidence of a direct effect of prostaglandin D2 on the secretion of luteinizing hormone from the human pituitary gland. This finding suggests the possible role of prostaglandin D2 in human reproductive function.

Adult↗

Effect of restraint stress on the preovulatory luteinizing hormone profile and ovulation in the rat.

Plasma profiles of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured during restraint stress on the day of pro-oestrus; these profiles were considered in relation to ovulation rate on the next day. Rats bearing a permanent jugular vein cannula were subjected to restraint, which was started 0, 1 or 2 h before the presumed onset of the LH surge and ended just before the beginning of the dark period. Exposure to restraint resulted in a suppression of the secretion of both gonadotrophins on the day of pro-oestrus. Suppression of the LH surge was virtually complete (plasma LH < or = 0.2 ng/ml) in 15 out of 32 stressed rats, and the ovaries of these rats contained graafian follicles with oocytes in germinal vesicle stage. In these rats, the LH surge did not occur 24 h later. In the remaining 17 rats, restraint resulted in a considerable suppression of the LH surge. Of these rats, five had an ovulation rate of 100% and four ovulated partially. In unruptured follicles of the latter, the oocyte had not resumed meiosis and the follicle wall was not luteinized. In the remaining eight rats with a reduced LH surge, ovulations had not occurred and graafian follicles were unaffected. The results of this study indicate that during pro-oestrus restraint stress suppresses and does not delay the release of preovulatory gonadotrophins. Partial suppression of LH by restraint does not result in induction of meiotic resumption without subsequent ovulation or in luteinized unruptured follicles.

Animals↗

Enhanced serum oestrogen levels and highly steroidogenic, luteinized atretic follicles in the ovaries of the Djungarian hamster (Phodopus sungorus) kept under a short photoperiod from birth.

OBJECTIVE: In contrast to the elaborate information available on the effects of the photoperiod on the testes of hamsters, little is known about the influence on their ovaries. This study aimed to describe the ovarian follicular development and steroid hormone production in Djungarian hamsters kept from birth under a short daylight regime. DESIGN AND METHODS: Female Djungarian hamsters (Phodopus sungorus) were kept under two different light regimes: (i) 16 h light:8 h darkness (long daylight; LD) and (ii) 4 h light:20 h darkness (short daylight; SD). They were killed at 28, 56 and 80 days after birth; blood and ovaries were collected. Ovaries were either fixed in Bouin's solution or frozen. Fixed material was dehydrated, embedded in paraffin, serially sectioned at 5 microm and stained with haematoxylin and eosin, whereafter all healthy and atretic follicles were classified and counted. 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) was histochemically demonstrated in 10 microm sections of frozen ovaries. Serum oestradiol-17beta and progesterone levels were determined by RIA. RESULTS: The numbers of healthy preantral and antral follicles were higher in LD than in SD hamsters. Antral follicles did not significantly differ in number during development in LD hamsters, but they were completely absent from 80-day-old SD animals. In LD animals the number of apoptotic preantral follicles dramatically increased with age. In SD animals the numbers of apoptotic antral follicles strongly decreased with age, whereas numerous non-apoptotic follicles with luteinized granulosa cells and a degenerated oocyte appeared, and in increasing numbers with age. During development, moderate 3beta-HSD activity was present in interstitial cells, theca cells of healthy follicles, and in both theca and granulosa cells of degenerating follicles. Strong enzyme activity was found in the hypertrophied granulosa cells of luteinized atretic follicles. Mean serum progesterone values varied from 2 to 6 nmol/l and were not different in LD and SD hamsters. Mean serum oestradiol levels varied from 132 to 542 and 325 to 2353 pmol/l in LD and SD hamsters respectively. The highest oestradiol levels were found in SD animals at day 28 of development. CONCLUSIONS: Folliculogenesis was dramatically disturbed in Djungarian hamsters raised under a short photoperiod. These animals developed high serum oestradiol levels and numerous luteinized atretic follicles with highly steroidogenic granulosa cells, which appear to be the source of the increased serum oestradiol levels.

3-Hydroxysteroid Dehydrogenases↗

Divergent optimum levels of lycopene, beta-carotene and lutein protecting against UVB irradiation in human fibroblastst.

Exposure of living organisms to UV light leads to photooxidative reactions. Peroxyl radicals are involved in the propagation of lipid peroxidation. Carotenoids are dietary antioxidants and show photoprotective effects in human skin, efficiently scavenging peroxyl radicals and inhibiting lipid peroxidation. Cultured human skin fibroblasts were used to examine the protective effects of the carotenoids, lycopene, beta-carotene and lutein on UVB-induced lipid peroxidation. The carotenoids were delivered to the cells using liposomes as the vehicle. The cells were exposed to UVB light for 20 min. Lycopene, beta-carotene and lutein were capable of decreasing UV-induced formation of thiobarbituric acid-reactive substances at 1 h to levels 40-50% of controls free of carotenoids. The amounts of carotenoid needed for optimal protection were divergent at 0.05, 0.40 and 0.30 nmol/mg protein for lycopene, beta-carotene and lutein, respectively. Beyond the optimum levels, further increases of carotenoid levels in cells led to prooxidant effects.

Carotenoids↗

High-performance thin-layer chromatographic analysis of lutein and beta-carotene in Cerithidia californica (Gastropoda) infected with two species of larval trematodes.

High-performance thin-layer chromatography (HPTLC) analysis was done on lutein and beta-carotene in the digestive gland-gonad complex (DGG) and whole body of uninfected Cerithidia californica snails and those infected with the larval trematodes Mesostephanus appendiculatis or Euhaplorichis californiensis. HPTLC of the DGG extract on C-18 reversed-phase plates developed in petroleum ether-acetonitrile-methanol (1:2:2) mobile phase showed 2 identifiable pigment zones; the least polar zone had a retention factor (Rf) of 0.07, identical to a beta-carotene standard, and the more polar zone had an Rf of 0.41, identical to a lutein standard. Densitometric scanning of the pigment zones in sample versus standard chromatograms showed that the weight percent of lutein in the uninfected DGGs (3.4x10(-3)%) was significantly greater (P<0.05) than that of DGGs infected with either M. appendiculatis (0.35x10(-3)%) or E. californiensis (0.82x10(-3)%). Changes in beta-carotene in the infected DGGs were insignificant compared to the uninfected controls. However, the beta-carotene content of whole snails was significantly reduced (P<0.05) by infection with either trematode.

Animals↗

Absence of leptin expression and secretion by human luteinized granulosa cells.

Whether leptin is secreted by the human ovary is not known. The available data on leptin gene (ob gene) expression by human granulosa cells are conflicting. The aim of the present study was first to re-examine the expression of leptin messenger RNA (mRNA) by human granulosa cells and second to investigate if these cells have the ability to secrete leptin in cultures. Human luteinized granulosa cells were obtained from normal women undergoing in vitro fertilisation treatment after ovarian stimulation and follicle aspiration. The expression of ob gene was studied by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) both in primary granulosa cells treated immediately after oocyte recovery and in cells cultured up to 24 h under baseline and hormonally stimulated conditions (FSH: 100 ng/ml, LH: 100 ng/ml). ob mRNA transcripts were not detected in luteinized granulosa cells, while they were present in adipose tIssue cDNA. Actin gene expression was detected in all studied samples. Using a sensitive radioimmunoassay (lower limit of detection 0.05 ng/ml), leptin was undetectable in the culture media at all points during the 72 h cultures, while at the same time significant amounts of oestradiol and progesterone were produced particularly after the addition of androstendione (1 microM) to the incubation media. These results demonstrate for the first time that leptin is not secreted by human luteinized granulosa cells in cultures. From a physiological point of view, this may contribute to the development of the optimal follicular environment for oocyte maturation during the preovulatory period.

Base Sequence↗

Inhibitory action of porcine follicular fluid upon granulosa cell luteinization in vitro: assay and influence of follicular maturation.

Culture medium 199 supplemented with follicular fluid from 1-2 mm antral porcine follicles inhibited spontaneous luteinization of granulosa cells from preovulatory porcine follicles in vitro. Three characteristics of luteinization were inhibited: morphological transformation, progesterone secretion, and accumulation of cyclic AMP in response to LH. The last was inhibited more effectively by culture media containing 50% follicular fluid than by media containing 20% follicular fluid. The inhibitory actions of the follicular fluid were not altered by charcoal or petroleum ether extraction. Follicular fluid from large follicles (6-12 mm) did not exhibit any of these inhibitory actions. These observations may indicate the presence of a luteinization inhibitor in the fluid of small follicles which (1) is lost by the time the follicle reaches the preovulatory stage, or (2) is overcome by a stimulatory agent which may accumulate as the follicle grows.

Animals↗

Activation induced by luteinizing hormone of type II protein kinase dependent on cyclic adenosine monophosphate and phosphorylation of soluble proteins in porcine granulosa cells.

The present experiments were designed to study whether exogenous LH could elicit acute cyclic AMP-mediated activation of cyclic AMP-dependent protein kinase and phosphorylation of cellular protein in intact porcine granulosa cells. Incubation of porcine granulosa cells (from 3 to 5 mm diameter follicles) with 2 microgram luteinizing hormone/ml (LH) caused a significant rise of cellular cyclic AMP content within 2 min of the addition of LH. The increase was dose-dependent and occurred between doses of 0.2 and 2.0 microgram LH/ml. Luteinizing hormone also caused a time- and dose-dependent dissociation of the type II cyclic AMP-dependent protein kinase isozyme in porcine granulosa cells. Luteinizing hormone (0.05--2 microgram/ml) significantly dissociated the cyclic AMP-dependent protein kinase between 2 and 30 min after stimulation. The protein kinase dissociation was a specific effect of LH and was not elicited by either adrenocorticotrophic hormone or prolactin. During the period of LH-induced protein kinase activation, several soluble granulosa cell proteins, ranging in molecular weights from about 43 000 to 99 000, became phosphorylated in a time-dpeendent and hormone-specific manner. The results suggest that cyclic AMP-mediated activation of granulosa cell type II cyclic AMP-dependent protein kinase may be a prerequisite in the short-term molecular action of LH leading to LH-specific phosphorylation of several soluble granulosa cell proteins of an as yet unidentified function.

Animals↗

Detection and functional characterisation of the transcription factor peroxisome proliferator-activated receptor gamma in lutein cells.

A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPARgamma, a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPARgamma in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPARgamma in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1.5-fold of the basal level) to an endogenous ligand of PPARgamma, 15-deoxy-delta12,14 prostaglandin J2 (15-dPGJ2) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPARgamma level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPARgamma proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPARgamma was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPARgamma in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ2, suggesting that 15-dPGJ2 exerts its effect on steroidogenic activity via PPARgamma and that the 15-dPGJ2-PPARgamma system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.

Analysis of Variance↗

Changes of luteinizing hormone and progesterone for dairy cows after gonadotropin-releasing hormone at first postpartum breeding.

Gonadotropin-releasing hormone administered at breeding enhances fertility of dairy cows, so a study was designed to evaluate the mechanism for enhanced fertility following administration of gonadotropin-releasing hormone at first postpartum breeding. Twenty-four cows were assigned randomly to one of two treatments, 100 micrograms of gonadotropin-releasing hormone intramuscular or saline vehicle intramuscular at insemination. Blood samples for luteinizing hormone assay were taken at 2-h intervals prior to breeding and .5-h intervals for 3 h after insemination. Composite morning milk samples for progesterone assay were collected for 30 days after insemination or until next estrus. Cows given gonadotropin-releasing hormone had higher luteinizing hormone concentrations in blood serum following treatment than cows given saline, 13.2 versus 3.0 ng/ml. There was no relationship between luteinizing hormone and subsequent conception. Progesterone for cows that became pregnant was higher throughout sampling days. Mean progesterone concentrations were 4.6 versus 2.2 ng/ml in pregnant and nonpregnant cows during the first 4 days after insemination. Cows treated with gonadotropin-releasing hormone that conceived had higher progesterone than other cows, and that was evident at the first 4 days postbreeding.

Animals↗