Feasibility of combined upper and lower gastrointestinal endoscopic biopsy in the common marmoset (Callithrix jacchus) to evaluate gastrointestinal diseases.

BACKGROUND: Chronic gastroenteropathies, including gluten sensitivity and marmoset wasting syndrome, frequently occur in captive colonies of common marmosets (Callithrix jacchus). Early identification and diagnosis of affected animals are desirable. Endoscopic examination of the colon in marmosets is described, but the small intestine can harbor significant mucosal lesions not representing those in the colon. Evaluating the small intestine currently requires invasive surgical biopsies due to the small patient size, carrying a risk of severe complications. METHODS: Endoscopic intubation and multisite biopsy of the duodenum/proximal jejunum are demonstrated in 10 marmosets under general anesthesia. RESULTS: Esophagogastroduodenoscopy with colonoscopy efficiently aid in examining the gastrointestinal tract and obtaining an antemortem histologic diagnosis in marmosets with chronic gastrointestinal signs. CONCLUSIONS: This minimally invasive technique is feasible in marmosets. Future investigations into the pathogenesis of chronic gastroenteropathies will benefit from these data, leading to improved animal welfare and better individual and colony health management.

A new S. suis serotype 3 infection model in pigs: lack of effect of buprenorphine treatment to reduce distress.

BACKGROUND: Streptoccocus suis (S. suis) is a major porcine pathogen causing meningitis, septicemia, arthritis and endocarditis. These diseases severely impair welfare of pigs. Experimental studies in pigs are important to better understand the pathogenesis and to identify protective antigens, as so far there is no vaccine available protecting against various serotypes (cps). Due to the severity of disease, application of appropriate refinement strategies in experimental S. suis infections is essential to reduce distress imposed on the piglets without jeopardizing the scientific output. The objectives of this study were to evaluate buprenorphine treatment as a refinement measure and serum cortisol levels as a distress read out parameter in a new S. suis cps3 infection model in pigs. RESULTS: Intravenous application of 2 × 108 CFU of S. suis cps3 (sly+, mrp+) to 6-week-old piglets led to severe morbidity in approximately 50% of the animals. Main pathological findings included suppurative meningoencephalitis and arthritis as well as fibrinosuppurative endocarditis. Buprenorphine treatment (0.05 mg/kg every 8 h) did not prevent signs of severe pain, high clinical scores, moderate to severe pathologies or high levels of serum cortisol in single severely affected piglets. Significant differences in the course of leukocytosis, induction of specific antibodies and bactericidal immunity were not recorded between groups with or w/o buprenorphine treatment. Of note, clinically unobtrusive piglets showed serum cortisol levels at 2 and 5 days post infectionem (dpi) comparable to the levels prior to infection with cps3. Cortisol levels in serum were significantly increased in piglets euthanized due to severe disease in comparison to clinically unobtrusive pigs. CONCLUSIONS: Different clinical courses and pathologies are induced after intravenous challenge of piglets with 2 × 108 CFU of this S. suis cps3 strain. The chosen protocol of buprenorphine application does not prevent severe distress in this infection model. Important parameters of the humoral immune response, such as the level of IgM binding to S. suis cps3, do not appear to be affected by buprenorphine treatment. Serum cortisol is a meaningful parameter to measure distress in piglets experimentally infected with S. suis and to evaluate refinement strategies. In this intravenous model, which includes close clinical monitoring and different humane endpoints, clinics and cortisol levels suggest convalescence in surviving piglets within 5 days following experimental infection.

Simultaneous detection of three sex steroid hormone classes using a novel yeast-based biosensor.

A biosensor detecting estrogens, progestogens, and androgens in complex samples and in a single step is described. Three Arxula adeninivorans yeast strains were created, each strain producing a different recombinant human hormone receptor and a different fluorescent reporter protein. These strains were then mixed to create G1212/YRC102-hHR-fluo, the biological component of the biosensor. During incubation with G1212/YRC102-hHR-fluo, hormones present in a sample bind to their target receptor, which leads to the production of a specific fluorescent protein. Three fluorescence scans of the yeast suspension determine which fluorescence protein has been produced, thus revealing which hormone receptor (estrogen, progesterone, and androgen) has been activated by the hormones or hormone mimics present in the sample. The biosensor has similar sensitivities to the existing A. adeninivorans cell-based assays. The detection of the three hormone classes in one single experiment reduces the labor and time required to assay for the three hormone classes. The biosensor was also trialed with animal serum samples for the detection of progestogens, androgens, and estrogens and gave results that correlated well with ELISA analysis in case of progestogens. These results highlight the potential usefulness of the biosensor for comprehensive determination of hormone status in samples from veterinary origin. Biotechnol. Bioeng. 2017;114: 1539-1549. © 2017 Wiley Periodicals, Inc.

Blood pressure as prognostic marker for body condition, cardiovascular, and metabolic diseases in the common marmoset (Callithrix jacchus).

BACKGROUND: The increasing life span of Callithrix jacchus in combination with the occurrence of metabolic and age-dependent diseases requires improved health surveillance for this species. METHODS: The health status of 56 marmosets was studied using a non-invasive blood pressure (BP) device. Age-, weight-, and sex-dependent changes were analyzed. Four animals with striking BP findings had follow-up exams. RESULTS: Physiological and pathological BP values could be defined. BP positively correlated with age and weight, while no effect of sex could be found. Measurement time for female and older animals was shorter than for male and younger individuals. Further analysis of the suspicious patients revealed renal or hepatic diseases and cardiac alterations. CONCLUSION: The description of age and weight influences on BP delivers physiological and pathological values for common marmosets. This may contribute to the understanding of aging process and cardiology in this primate species.

The influence of gluten on clinical and immunological status of common marmosets (Callithrix jacchus).

BACKGROUND: Common marmosets (Callithrix jacchus) are susceptible to gastrointestinal diseases. Sensitivity to nutritional elements, for example gluten, has been suggested, but a serological screening has not been performed yet. METHODS: A gluten-containing diet was offered to 24 animals, followed by a gluten-free diet. During these diets, serum IgA antibodies to gliadin (AGA), tissue transglutaminase (tTG), deamidated gliadin (ADGA), and glycoprotein 2 (AGP2A) were determined. Body weight, diarrhea, and other clinical symptoms were recorded. RESULTS: Gluten increased AGA, tTG, and AGP2A concentrations in 13 of 24 animals. A significant decline of AGA and AGP2A was seen on gluten withdrawal. Positive (AGA, tTG) animals presented diarrhea more frequently on gluten-containing diet and showed significantly increased body weight on gluten-free diet compared to negative animals. CONCLUSION: Gluten ingestion caused gastrointestinal symptoms in common marmosets, which disappeared on gluten withdrawal. Considering the immunological response to both diets, gluten sensitivity seems to be most likely.

Testicular biopsy in psittacine birds (Psittaciformes): comparative evaluation of testicular reproductive status by endoscopic, histologic, and cytologic examination.

Knowledge of the reproductive cycle of male parrots is important for examining the male genital tract and for successful breeding, especially of endangered species. To evaluate different diagnostic methods and criteria concerning the classification of reproductive stages, we examined 20 testicular samples obtained at necropsy in psittacine birds of different species and testicular biopsy samples collected from 9 cockatiels (Nymphicus hollandicus) and 7 rose-ringed parakeets (Psittacula krameri) by endoscopy 4 times over a 12-month period. The testicular reproductive status was assessed histologically and then compared with the macroscopic appearance of the testicles and cytologic results. The histologic examination was nondiagnostic in 19 of 59 testicular biopsy samples. By contrast, the cytologic preparations were diagnostic in 57 of 59 biopsy samples. The results of the cytologic examination coincided with the histologic results in 34 of 38 biopsy samples and 18 of 20 necropsy samples. Macroscopic parameters displayed some differences between reproductive stages but provided an unreliable indication of the reproductive status. These results suggest that microscopic examination of a testicular biopsy sample is a reliable method for evaluating the reproductive status of male parrots and is preferable to the macroscopic evaluation of the testicle. Cytologic examination provides fast preliminary results, even when the histologic preparation is not sufficient for evaluation, but results may be erroneous. Thus, a combination of histologic and cytologic examination is recommended for evaluating testicular reproductive status.

Testicular biopsy in psittacine birds (Psittaciformes): impact of endoscopy and biopsy on health, testicular morphology, and sperm parameters.

Histologic examination of a testicular biopsy sample may be required to evaluate the reproductive status of male psittacine birds. The purpose of this study was to evaluate the viability of testicular sampling from live birds by assessing the impact on the birds' health, testicular integrity, and sperm quality. Testicular biopsy samples were obtained by endoscopy 4 times during 12 months from 9 cockatiels (Nymphicus hollandicus) and 7 rose-ringed parakeets (Psittacula krameri). Only 2 of 16 birds showed testicular cicatrization or divided testicular tissue after a single endoscopy. Further complications, such as damage to the air sacs or bleeding, predominantly occurred in subsequent endoscopies. In both species, endoscopy and testicular biopsy caused only minor or transient effects on sperm production and sperm quality. These results support that a single testicular biopsy is a viable method for evaluating the reproductive status of male psittacine birds.

Prenatal stress programs lipid metabolism enhancing cardiovascular risk in the female F1, F2, and F3 generation in the primate model common marmoset (Callithrix jacchus).

BACKGROUND: Many human diseases are modulated by intrauterine environment, which is called prenatal programming. This study investigated effects of prenatal glucocorticoids on the lipid metabolism of three filial generations of common marmosets. METHODS: Pregnant primates were treated with dexamethasone during pregnancy. Body weight and blood lipid parameters of adult female offspring (F1: n = 5, F2: n = 6, F3: n = 3) were compared with age-related female controls (n = 12). RESULTS: F1, F2, and F3 offspring showed significantly lower percentage of plasma n3 fatty acids than controls. F2 and F3 presented higher cholesterol levels, with significantly more LDL cholesterol, significantly less HDL triglycerides and an enhanced cholesterol/HDL cholesterol ratio. Body weight was not significantly affected. CONCLUSIONS: Prenatal dexamethasone led to higher amounts of cardiovascular risk factors and less protective parameters in female F1-F3 offspring. The intergenerational consequences suggest prenatal programming through epigenetic effects.

Evaluating the reproductive status of the male budgerigar (Melopsittacus undulatus).

Limited knowledge about male psittacine reproduction reduces the success of breeding programmes. Within the scope of fecundity assessment, classification of male sexual status is essential for effective conservation of the species. The aim of the present study was to investigate the testes of male budgerigars (Melopsittacus undulatus), as psittaciform model species to verify their reproductive status by morphological and immunocytochemical examination. Using light microscopy, gonadal samples were categorized resulting in three reproductive states (active, intermediate, non-active). Calculation of testes weights plus measurement of tubular and interstitial dimensions displayed significant (p ≤ 0.05) differences between all three reproductive stages. Lipids in the testicular tubules, analysed by Sudan black staining and fluorescence microscopy (DAPI(2) mode) were highly present in non-active status. Immunocytochemistry involved two different hydroxysteroid dehydrogenases (HSD), 3ß-HSD and 17ß-HSD-2, as markers for steroidogenesis, as well as steroid receptors for androgens (AR), oestrogen (ER) and progesterone (PR). Both HSDs and AR declined in non-active gonads compared to active and intermediate stages, with a positive signal in germ and somatic cells of testis and epididymis. ER and PR were detected in testicular and epididymal cells, similarly expressed in all three stages. The proliferation rate of germ cells in the testicular tubules, obtained by Ki67, differed significantly in active (38.67%), intermediate (32.40%) and non-active (6.01%) status. According to this morphological study, we have been able to establish markers for the reproductive staging of psittacine testes. This knowledge will be useful to deepen reproductive biology in budgerigars.

Investigation of Body Development in Growing Holstein Heifers With Special Emphasis on Body Fat Development Using Bioelectrical Impedance Analysis.

This study analyzed skeletal development, body condition, and total body fat development of growing heifers. A total of 144 female primiparous Holstein cattle from four commercial dairy farms with different degrees of stillbirth rates were examined during the rearing period. This included measurements in body condition, fat tissue, metabolic, and endocrine factors. Pelvic measurements and the sacrum height were analyzed to assess skeletal development. The body condition was classified via body condition scoring, bioelectrical impedance analysis (BIA), back fat thickness measurements, and the body mass. For the first time, BIA was used as an appropriate method to evaluate the fat tissue content of cattle throughout the rearing period. This analysis technique can be performed on heifers aged 8-15 months. Throughout that period, the fat content decreased while the skeletal development increased. In addition, high free fatty acid concentrations in serum of the animals with high frame development were found, supporting our hypothesis that stored energy of body fat deposits is used for skeletal growth. Furthermore, we were able to demonstrate complex endocrine relationships between fat metabolism and skeletal growth by using specific markers, such as leptin, insulin growth factor-1 (IGF-1), and estradiol (E2). Food analysis showed high crude protein (CP) levels in the total mixed ration above recommendation for daily protein intake of all farms. However, there was a positive correlation between CP and the body frame measurements in our study. In summary, we established a novel regression formula for BIA analysis ("BIA-Heine") in heifers to evaluate the body composition throughout different ages and physiological stages in the development of heifers. This special formula allows the evaluation of fat tissue without a whole-body analysis and therefore provides an innovative technique for animal welfare support.

Luteotrophic effects of relaxin, chorionic gonadotrophin and FSH in common marmoset monkeys (Callithrix jacchus).

In early pregnant primates, relaxin (RLX) is highly upregulated within the corpus luteum (CL), suggesting that RLX may have an important role in the implantation of the blastocyst. Therefore, the aim of the present study was to investigate the local effects of RLX and gonadotrophins on the maintenance of the CL using an in vitro microdialysis system. CLs of common marmoset monkeys were collected by luteectomy during different stages of the luteal phase and early pregnancy. Each CL was perfused with either Ringer's solution alone or Ringer's solution supplemented with either porcine RLX (250, 500 and 1000 ng/ml) or gonadotrophins (50 IU/ml). Application of RLX provoked a significant luteal response of progesterone (P(4)) and oestradiol (E(2)) secretions during the mid-luteal phase (500 ng/ml: P(4) 54+/-42%, E(2) 24+/-11%; 1000 ng/ml: E(2) 16+/-13%), and especially during the late luteal phase (250 ng/ml: P(4) 53+/-10%; 500 ng/ml: P(4) 44+/-15%; 1000 ng/ml: P(4) 62+/-15%, E(2) 18+/-7%). The effects of RLX on steroid secretion were irrespective of the RLX dosages. While treatment with human chorionic gonadotrophin did not affect luteal steroid or RLX secretion, the application of FSH resulted in a significant increase in the secretion of both P(4) (20+/-8%) and E(2) (37+/-28%), and a prominent rise in RLX during early pregnancy. In conclusion, our results demonstrate that RLX and FSH have a luteotrophic function in the marmoset monkeys; moreover, FSH has a function beyond its traditional role just as a follicle-stimulating hormone.

[Cortisol concentration before, during and after sham foot trimming in German Holstein cows - the suitability of different matrices]. / Kortisolkonzentration bei Deutsch-Holstein-Kühen vor, während und nach der Klauenbehandlung im Durchtreibestand ­ Eignung verschiedener Matrizes.

OBJECTIVE: The objective of this study was to determine whether changes in cortisol concentration measured in blood serum (KoB) of cows exposed to an acute stressor shows a correlation to cortisol concentrations in saliva (KoS), tears (KoT) and milk (KoM) as well as the concentration of cortisol metabolites in feces (KoK). MATERIALS AND METHODS: In 10 healthy German Holstein cows, sham foot trimming (sKB) including the movement/fixation of the cows through/in a foot trimming chute was used as a model for acute stress. KoB, KoS, KoT, KoM and KoK were measured once a day for 10 days. During sKB, performed on day 4, KoB and KoT were measured at the initiation of foot trimming (minute 0) as well as 15, 25, 30, 40, 50, 60 and 80 minutes later. Additionally, KoK was measured 480, 540, 600 and 660 minutes after the start of sKB. RESULTS: KoB and KoT increased during sKB and reached a maximum at 60 minutes followed by a decrease. KoK increased after sKB and reached a maximum at 660 minutes. There was a significant correlation between KoB and KoT, KoK and KoB and a trend towards a significant correlation between KoK and KoT during sKB. KoB and KoT were significantly correlated (area under the curve, minute 0-10, p = 0.04). KoB decreased significantly from day 1 to day 4 (p < 0.01). On day, 5 KoB (p = 0.03) and KoK (p < 0.01) were significantly higher. KoS and KoT served as good proxies for KoB throughout the study, and KoK and KoB exhibited similar profiles. There were several differences between the profiles of KoM and KoB. During the 10-day measurement period, a significant positive correlation was detected between KoB and KoS (p = 0.002) as well as between KoB and KoT (p = 0.002). CONCLUSIONS AND CLINICAL RELEVANCE: The collection of tear fluid and saliva for determination of cortisol concentrations are non-invasive alternatives to blood sampling. Calm handling of cows may reduce their stress reaction and thus improve animal welfare.

The hormonal induction of cervical remodeling in the common marmoset monkey (Callithrix jacchus).

Controversy still exists regarding the involvement of relaxin (RLX) in cervical reorganization throughout parturition in the human, despite its well-known role in facilitating extensive extracellular matrix (ECM) remodeling in diverse organs. Therefore, the aim of the present study was to examine the influence of RLX and estrogen (E2) on the cervical tissue of the common marmoset monkey. Two experimental designs were used: 1) in vivo analysis of the intracervical diameter under locally applied RLX and 2) ovariectomized (ov) marmosets were treated systemically with either recombinant human (rh) RLX, E2 or rhRLX+E2 to examine their action on the cervix. In vivo-locally applied rhRLX induced a distinct and significant widening of the cervix (before: 4.8+/-1.1 mm versus after: 5.7+/-0.9 mm in diameter; P<0.030, MV+/-S.E.M.). This widening effect was most pronounced in animals without previous pregnancies. In vitro investigation of cervical tissue showed significantly increased wet weights after all three hormone treatments (E2: 0.27+/-0.07 g, RLX: 0.25+/-0.04 g, E2+RLX: 0.30+/-0.11 g; all P<0.05; MV+/-S.E.M.) versus controls (0.10+/-0.04 g). Furthermore, morphological changes such as loosening of the connective tissue structure and decline in collagen content, an increase in the number of eosinophils, increased the expression of matrix metalloproteinases (MMP1) and MMP2, as well as gene and protein expression of the RLX receptor RXFP1 could be detected in the cervical tissue after all hormone treatments, compared with controls. In summary, RLX has a potent widening effect on the cervix of the common marmoset monkey. Although E2 is not required for this RLX effect, a combined application of E2 and RLX induced the most prominent cervical ripening.

Chemical cues of female fertility states in a non-human primate.

An increasing number of studies suggest that olfaction is important for communication throughout the order of primates. Callitrichids, in particular, have well-developed olfactory systems and use anogenital glands to produce scent marks. Behavioural studies have shown that male common marmosets (Callithrix jacchus) distinguish between odours from the peri-ovulatory and luteal phase of females. However, large gaps remain in understanding the chemical underpinnings of olfactory cues. To investigate whether chemical cues vary with female fertility and reproductive quality, our study combined behavioural bioassays with chemical analyses of the anogenital odours of female common marmosets using gas chromatography-mass spectrometry. We found that cycle states, age and parity have an impact on chemical profiles and further identified affected chemical substances. Our results confirm and expand on previous behavioural evidence for cues of fertility. Our results indicate that cycle-related substances likely act as chemical cues. Males could use such olfactory fertility cues to optimize their mating effort and thereby increase their paternity certainty. This certainty could enhance paternal care for their infants. The results of our study open a promising avenue to find the metabolic pathways from which chemical cues of fertility arise and to unravel their importance during primate evolution in future comparative studies.

Gender and gonadal status differences in zona reticularis expression in marmoset monkey adrenals: Cytochrome b5 localization with respect to cytochrome P450 17,20-lyase activity.

Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p<0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by Western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p<0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive.

Different methods for volatile sampling in mammals.

Previous studies showed that olfactory cues are important for mammalian communication. However, many specific compounds that convey information between conspecifics are still unknown. To understand mechanisms and functions of olfactory cues, olfactory signals such as volatile compounds emitted from individuals need to be assessed. Sampling of animals with and without scent glands was typically conducted using cotton swabs rubbed over the skin or fur and analysed by gas chromatography-mass spectrometry (GC-MS). However, this method has various drawbacks, including a high level of contaminations. Thus, we adapted two methods of volatile sampling from other research fields and compared them to sampling with cotton swabs. To do so we assessed the body odor of common marmosets (Callithrix jacchus) using cotton swabs, thermal desorption (TD) tubes and, alternatively, a mobile GC-MS device containing a thermal desorption trap. Overall, TD tubes comprised most compounds (N = 113), with half of those compounds being volatile (N = 52). The mobile GC-MS captured the fewest compounds (N = 35), of which all were volatile. Cotton swabs contained an intermediate number of compounds (N = 55), but very few volatiles (N = 10). Almost all compounds found with the mobile GC-MS were also captured with TD tubes (94%). Hence, we recommend TD tubes for state of the art sampling of body odor of mammals or other vertebrates, particularly for field studies, as they can be easily transported, stored and analysed with high performance instruments in the lab. Nevertheless, cotton swabs capture compounds which still may contribute to the body odor, e.g. after bacterial fermentation, while profiles from mobile GC-MS include only the most abundant volatiles of the body odor.

The common marmoset monkey as a model for implantation and early pregnancy research.

This chapter describes methods used to investigate implantation in the common marmoset monkey, Callithrix jacchus. A reverse-transcriptase polymerase chain reaction-strategy with which to detect transcripts for steroid receptors and enzymes involved in estradiol biosynthesis is described, and an immunohistochemistry approach for detecting proteins within the implantation site is presented.

A promoter that acquired p53 responsiveness during primate evolution.

The tumor suppressor p53 activates the transcription of human PIG3 through direct interaction with a polymorphic microsatellite sequence, (TGYCC)(n). Here, the evolution of this p53-responsive element was recapitulated. Comparison between primate species revealed that the PIG3 promoter acquired this sequence element in its full length only in Hominoidea (apes and humans), whereas the number of TGYCC repeats is far lower in monkeys. Accordingly, only the PIG3 promoters from Hominoidea respond efficiently to p53, whereas those from monkeys respond poorly or not at all. In parallel, the PIG3 gene was strongly induced by p53 in human and chimpanzee cells but was unaffected by p53 in cells derived from a common marmoset monkey. Thus, a novel p53 target gene appeared as recently as during the evolution of primates. This suggests that mechanisms of tumor suppression are subject to ongoing evolution in humans and their closest relatives.

Relaxin peptides are new global players.

Relaxin (RLX) has come of age. From being one of the earliest hormones described with a very specific function in parturition, recent research has now shown that it is involved in a variety of roles, from endometrial differentiation during embryo implantation, to being a response factor in infarct and wound situations. It ameliorates fibrosis, and might also be involved in tumour growth and progression. And it is not alone: two other closely related peptide hormones have recently been identified, one specific for the brain, the other with roles in testicular descent and ovarian apoptosis. Finally, the recent cloning of the RLX receptors now provides the basis for a new molecular pharmacology for these peptide hormones, and preliminary studies suggest that their signal transduction is both interesting and unusual.

Insulinaemic and glycaemic responses to a second meal of a fibre- or starch-enriched compound feed in healthy horses.

This study aimed to investigate the effect of a second meal of a fibre- or starch-enriched compound feed on glycaemic and insulinaemic responses in healthy horses. In a cross-over design, horses were fed either a starch-enriched compound feed (SCF) or a fibre-enriched compound feed (FCF). On days of blood collection, test diets were fed in the morning (0800 h, first meal) and a second meal was fed 510 min after the first meal was finished (second meal). Significantly higher glycaemic and insulinaemic responses were associated with SCF compared with FCF. Feeding FCF for the second meal yielded similar moderate glycaemic and insulinaemic responses compared with the first meal. Feeding SCF as a second meal yielded significantly reduced glycaemic and insulinaemic responses from the first meal. In practice, evaluating glycaemic and insulinaemic responses of a single meal may not sufficiently describe the overall impact if more than one meal is fed per day.