Pierre-Guy Marnet

Human contact and the effects of acute stress on cows at milking

We examined the effects of novelty/isolation stress on cows at milking and whether human contact reduced the stress. Holstein cows (n=18) were observed during three experimental milkings following a balanced order: (1) control (C) - milked in usual place; (2) novelty/isolation stress (S) - milked alone in an unfamiliar room; (3) human contact (HC) - milked in unfamiliar room and brushed by a familiar person. Behavior and heart rate during milking, milk yield and residual milk following oxytocin injections were recorded, and blood samples assayed for oxytocin and cortisol. Cows defecated/urinated and vocalized more and made more steps during milking in the unfamiliar room and human contact prevented this increase. Cows kicked and lifted their legs less in the unfamiliar room, but human contact did not affect this. Both plasma cortisol concentrations and heart rates were higher when cows were milked in the unfamiliar room. Although human contact reduced heart rates during the initial period of isolation, heart rates during milking and cortisol concentrations were not affected by human contact. Milk yield was lower, residual milk higher, and oxytocin during milking was lower in the unfamiliar room but this was not changed by human contact. Cows milked alone in an unfamiliar room showed signs of acute stress and gave less milk due to higher residual milk and reduced oxytocin secretion. Human contact reduced some behavioral signs of agitation and heart rate, but had no effect on milk yield or hormonal responses. For dairy cows, human contact may not be sufficiently comforting to be able to reduce the endocrine response to novelty/isolation stress.

Regulation of milk ejection and milkability in small ruminants

Abstract In small ruminants, the secretion of oxytocin following stimulation of the mammary gland is necessary for complete removal of milk during suckling and during machine milking. The optimisation of milk ejection and complete milk removal is the result of neuroendocrine mechanisms, which induce alveolar contraction, and expulsion of secreted milk and its components, hormonal maintenance of lactation, and the ability to easily remove milk from the mammary gland as a function of its anatomy, morphology, and teat physiology.

In-depth investigation of protein adsorption on gold surfaces: Correlating the structure and density to the efficiency of the sensing layer

Protein A (PrA), mouse monoclonal anti-IgG antibody (SAb) and deglycosylated avidin (NAV) were adsorbed on gold surfaces to capture the model rabbit IgG and build three immunosensing platforms. The assembling of immunosensors, their specificity, and the receptor accessibility were monitored by polarization modulation reflection-absorption infrared spectroscopy (PM-RAIRS) and quartz crystal microbalance with dissipation measurement (QCM-D) at each step. Combining these two techniques allows us to compare both chemical and structural properties of the sensing layers with the former bringing chemical and semiquantitative information on the grafted protein layers, whereas the latter, in addition to the mass uptake, enables us to take the layer rigidity into account. Grafting of the three capture proteins to the transducer surfaces, covered with appropriate self-assembled monolayers, yielded protein layers with variable properties. NAV formed a dense and rigid molecular layer, likely containing protein aggregates, whereas the amount of PrA was below one monolayer resulting in a flexible layer. The amount of immobilized rabbit IgG was different for the three systems with the densest capture protein layer exhibiting the lowest binding capacity. The accessibility of antibodies on the resulting immunosensors measured by interaction with a secondary antirabbit IgG antibody was found to be closely dependent on their coverage as well as on the rigidity of the protein layer. The overall study provides in-depth information on three of the most common immunosensor recognition interfaces and demonstrates the crucial influence of both structure and density of the protein layer on the efficiency of the molecular recognition phenomena.

Oxytocin release and milk ejection parameters during milking of dairy ewes in and out of natural season of lactation

Abstract Two groups of 40 milking Lacaune ewes were monitored during their natural (spring) and off-season (autumn) lactation. Serial samples collected before, during and after milkings were used to study oxytocin release in relation to milk flow patterns recorded during milking. Overall, oxytocin release was significant in 92.5% of the ewes but only 56% of them exhibited clear alveolar milk ejection. Oxytocin is essential for milk ejection but no correlation between oxytocin ejection and milk fraction volumes of milking was detected. Baseline oxytocin and progesterone concentrations and the proportion of cisternal milk were significantly higher in autumn than in spring. It was concluded that clear alveolar emission of milk during milk flow recordings are not reliable indicators of milk ejection efficiency in high producing animals with large cisternal volumes. Likewise, the amount of oxytocin released during milking cannot be used to select the animals best adapted to machine milking or the best producers. Nevertheless, this study suggests that luteal oxytocin has a major role in milk transfer between milkings.

Effect of milking frequency on oxytocin release and milk production in dairy ewes

The experiment was conducted to investigate the effectiveness of milking stimulus on oxytocin release and to compare the effect of milking frequency on plasma levels of oxytocin and milk parameters. Twelve Lacaune ewes were subjected to six treatments (T1, T2, T3, T4, T5 and T7 daily milkings) during 6 days. At each milking, blood was sampled and plasma oxytocin levels were determined by enzyme immunoassay. Baseline levels of oxytocin were similar for all milking frequencies. The start of milking was followed by a significant increase in oxytocin levels for all milking frequencies. One daily milking induced significantly higher oxytocin levels than 2, 3, 4, 5 and 7 daily milkings. Milk yield was significantly increased between 4 (1787.0+/-141.5ml) and 7 (1780.0+/-53.6ml) daily milkings compared to 1 (1104.0+/-81.2ml) daily milking. Total concentration of milk protein did not change, but the total milk fat yield for 5 (73.0+/-2.0g/l) and 7 (72.8+/-1.4g/l) daily milkings were significantly higher than for 1 (58.1+/-4.3g/l) daily milking. This study confirmed milk yield gains caused by frequent milk ejection and also showed that oxytocin release was not a limiting factor for milk yield gain when daily milking frequency was increased.

Oxytocin stimulates secretory processes in lactating rabbit mammary epithelial cells

Oxytocin plays a major role in lactation mainly by its action on milk ejection via the contraction of myoepithelial cells. The effect of oxytocin on milk production and the presence of oxytocin receptors on different epithelial cells suggest that this hormone may play a role in mammary epithelial cells. To determine precisely the various roles of oxytocin, we studied localization of oxytocin receptors in lactating rabbit and rat mammary tissue and the influence of oxytocin on secretory processes in lactating rabbit mammary epithelial cells. Immunolocalization of oxytocin receptors on mammary epithelial cells by immunofluorescence and in mammary tissue by immunogold in addition to in situ hybridization showed that lactating rat and rabbit mammary epithelial cells expressed oxytocin receptors. Moreover, oxytocin bound specifically to epithelial cells. To determine whether oxytocin had an effect on lactating rabbit mammary epithelial cells, isolated mammary fragments were incubated in the presence or absence of 10−6 i.u. ml−1 of oxytocin. After 1 min of incubation with oxytocin, the morphology of epithelial cells and the localization of caseins and proteins associated with the secretory traffic suggested a striking acceleration of the transport leading to exocytosis, whereas the contraction of myoepithelial cells was only detectable after 7 min. Addition of 10−8 g ml−1 of atosiban before the addition of oxytocin prevented the oxytocin effect on secretory processes and on myoepithelial cell contraction. Addition of 10−6 i.u. ml−1 of vasopressin to the incubation medium did not mimic the stimulating effect of oxytocin on secretory traffic. These results show that lactating rabbit and rat mammary epithelial cells express oxytocin receptors and that oxytocin binds to these receptors. They strongly suggest that oxytocin has a dual effect on lactating mammary tissue: an acceleration of the intracellular transfer of caseins in mammary epithelial cells followed by the contraction of myoepithelial cells.

Bovine Teat Microbiome Analysis Revealed Reduced Alpha Diversity and Significant Changes in Taxonomic Profiles in Quarters with a History of Mastitis

Mastitis is a mammary gland inflammatory disease often due to bacterial infections. Like many other infections, it used to be considered as a host-pathogen interaction driven by host and bacterial determinants. Until now, the involvement of the bovine mammary gland microbiota in the host-pathogen interaction has been poorly investigated, and mainly during the infectious episode. In this study, the bovine teat microbiome was investigated in 31 quarters corresponding to 27 animals, which were all free of inflammation at sampling time but which had different histories regarding mastitis: from no episode of mastitis on all the previous lactations (Healthy quarter, Hq) to one or several clinical mastitis events (Mastitic quarter, Mq). Several quarters whose status was unclear (possible history of subclinical mastitis) were classified as NDq. Total bacterial DNA was extracted from foremilk samples and swab samples of the teat canal. Taxonomic profiles were determined by pyrosequencing on 16s amplicons of the V3-4 region. Hq quarters showed a higher diversity compared to Mq ones (Shannon index: ~8 and 6, respectively). Clustering of the quarters based on their bacterial composition made it possible to separate Mq and Hq quarters into two separate clusters (C1 and C2, respectively). Discriminant analysis of taxonomic profiles between these clusters revealed several differences and allowed the identification of taxonomic markers in relation to mastitis history. C2 quarters were associated with a higher proportion of the Clostridia class (including genera such as Ruminococcus, Oscillospira, Roseburia, Dorea, etc.), the Bacteroidetes phylum (Prevotella, Bacteroides, Paludibacter, etc.), and the Bifidobacteriales order (Bifidobacterium), whereas C1 quarters showed a higher proportion of the Bacilli class (Staphylococcus) and Chlamydiia class. These results indicate that microbiota is altered in udders which have already developed mastitis, even far from the infectious episode. Microbiome alteration may have resulted from the infection itself and or the associated antibiotic treatment. Alternatively, differences in microbiome composition in udders with a history of mastitis may have occurred prior to the infection and even contributed to infection development. Further investigations on the dynamics of mammary gland microbiota will help to elucidate the contribution of this endogenous microbiota to the mammary gland health.

Influence of vacuum level, pulsation rate and pulsator ratio on machine milking efficiency in local Greek goats

Abstract Effects of vacuum level, pulsation rate and pulsator ratio on machine milking efficiency in local Greek goats were studied in three separate experiments lasting 12, 10 and 11 weeks in three different years. After weaning (49 days) all goats were milked for 14 days at a vacuum level 44 kPa, a 90-pulsations/min and a 50:50 pulsator ratio (this 2-week interval was considered the familiarization period). In the first experiment, three groups (treatments) of goats were subjected to 70, 90 and 120 pulsations/min at milking. For the second experiment another three groups were milked with a vacuum level of 36, 44 and 52 kPa. For the third and final experiment, three pulsator ratios; 65:35, 50:50, 35:65 were compared in three groups. Results suggest that a faster rate (120 pulsations/min) and a higher vacuum level (52 kPa) decreased the percentages of machine milk (MM; from 60.6 to 55.1% for the rate and from 77.3 to 67.9% for the vacuum) and total machine milk (TMM) while increasing the percentage of machine stripped milk (MSM) and hand stripped milk (HSM; from 17.1 to 20.3% for the rate and from 5.7 to 10.0% for the vacuum). Milking time decreased as vacuum levels increased from 36 to 52 kPa and as pulsator ratios increased from 35:65 to 50:50 and 65:35. The pulsator ratio did not modify the percentage of the above milk fractions (MM, MSM, HSM and TMM) but did increase the average milking rate. Optimum conditions for machine milking of local Greek goats appears to be a vacuum level of 36–44 kPa, a pulsation rate of 70–90 and a pulsator ratio of 65:35.

Suppression of ovarian secretions before puberty strongly affects mammogenesis in the goat.

The objective of this study was to provide insight into the biological mechanisms underlying mammary development and the role of the ovaries in prepubertal caprine mammogenesis using a serial ovariectomy approach. Young Alpine goats were ovariectomized (Ovx) or sham-operated (Int) at three periods before puberty (G1=1 month, G2=2 month and G3=3 months of age) and one after puberty (G7=7 months of age). The goats were slaughtered at 9 months of age and mammary glands were removed. Ovariectomy performed at 1, 2 and 3 months of age caused a 50% reduction in DNA concentration, in mammary tissue taken from the parenchyma-stroma border region. Morphological analysis of mammary tissue sections indicated that the parenchymal structures of Ovx goats were negatively affected by ovariectomy. Goats ovariectomized before 2 months of age (Ovx-1 and Ovx-2) showed a significant decrease in the percent of cells proliferating in mammary glands of 9-month old goats (proliferating cell nuclear antigen expression and antigen Ki67-positive cell number). Also, goats ovariectomized at 1 and 2 months of age had reduced matrix metalloprotease 2 activity at 9 months of age. E-cadherin was strongly decreased in goats ovariectomized before 2 months of age (80 and 85% in Ovx-1 and Ovx-2 goats, respectively). Quantitative PCR analysis of transcripts encoding for oestrogen (ERα) and progesterone receptors (PR) and immunodetection of ERα showed that ovariectomy at 1 and 2 months of age strongly inhibited the transcription of ERα and PR in the mammary gland. We conclude that ovariectomy before 3 months of age markedly impaired parenchymal development. These findings suggest that prepubertal mammogenesis in goats depends on the ovaries to initiate mammary epithelial cell proliferation and mammary gland remodelling.

Evaluation of the Biological Activity of Some Hormones, Growth Factors and Drugs on Cultured Cells, Isolated from Animal and Human Reproductive Organs

This report is a brief review of our experience in developing fast, sensitive and inexpensive methods for evaluating the biological activity of hormones, growth factors, their analogues and drugs, using cell cultures. It demonstrates that the analysis of parameters such as proliferation, apoptosis, meiosis, the production of peptide and steroid hormones, growth factors, growth factor binding protein, prostaglandins, cyclic nucleotides, protein kinases and transcription factors by cells isolated from bovine, porcine, rabbit, chicken and human ovarian and oviduct cells, oocyte-cumulus complexes and embryos can be an efficient means of studying the effects, mechanisms of action and safety of various biologically active substances.