title: Goat’s vs cow’s milk consumption: Analysis of feeding behaviour, brain activation and gene expression in laboratory animals

authors: Wood, Erin Lavinia

subjects: Milk

abstract: Milk is a complex and highly nutritive food. In Western societies, cow’s milk (CM) is most commonly consumed, but recent years have generated interest in milk from other species, especially in goat’s milk (GM). Importantly, select physical and chemical properties of milk are species-dependent and – thus – so are the physiological consequences of consumption of milk sourced from specific species. For example, variation between GM and CM protein impacts digestibility and gastrointestinal processes. Consumption of GM vs CM differentially affects levels of blood hormones regulating energy balance. Furthermore, some conflicting results on acceptability of GM- and CM-based foods have been reported, and it is unclear to what extent habituation to a specific milk type underpins these parameters. To add to the confusion, CM and GM are typically consumed and, therefore, studied as modified milk products, with one of the typical compositional alterations being done to the protein fraction in which the natural 20:80 whey:casein ratio is changed to resemble the 60:40 ratio of human milk. One of the most fundamental gaps in our knowledge regarding CM vs GM relates to the acceptability, palatability and satiating properties of these milks and to appetite-controlling brain processes triggered by CM and GM consumption. Thus, in this doctoral project, I sought to examine whether GM and CM diets elicit unique feeding responses in laboratory rodents and whether the presumed appetite differences are associated with changes in neuronal activation and/or gene expression in key central regions regulating food intake. 
In Specific Aim 1 of the project, I conducted a comprehensive investigation of short-term intake and palatability profiles of GM- and CM-based liquid and solid diets in mice and rats. Consumption was studied in no-choice and choice scenarios, including meal microstructure. Feeding experiments were followed by qPCR analysis of expression of relevant genes in the energy balance-related hypothalamus and brain stem, and in the nucleus accumbens, which regulates eating for palatability. I found that GM and CM are palatable to juvenile, adult, and aged rodents. Given a choice, animals prefer GM- to CM-based diets. Analysis of meal microstructure using licking patterns points to enhanced palatability of and, possibly, greater motivation toward GM over CM. Most profound changes in gene expression after GM vs. CM were associated with the brain systems driving consumption for reward. The results allow me to conclude that, while both GM and CM are palatable, GM is preferred over CM by animals, and this preference is driven by central mechanisms controlling eating for pleasure.
In Specific Aim 2 of the thesis, I investigated the impact of whey enhancement in GM protein fraction on appetite and feeding-related brain processes. The shift from the natural whey:casein ratio of ~20:80 in animal milks is done to match the 60:40 ratio of human milk. Studies show that 20:80 versus 60:40 whey:casein milks differently affect glucose metabolism and hormone release. It is unknown whether the 20:80-to-60:40 ratio adjustment affects appetite and brain processes related to food intake. In this set of studies I focused on the impact of the 20:80 vs 60:40 whey:casein content in GM on food intake and feeding-related brain mechanisms in laboratory mice. I found that the 20:80 whey:casein GM formulation was consumed less avidly and was less preferred than the 60:40 GM in short-term choice and no-choice paradigms. The qPCR analyses in the hypothalamus and brain stem revealed that the 20:80 whey:casein GM intake upregulated genes involved in early termination of feeding and in an interplay between reward and satiety, such as MC3R, OXT, POMC and GLP1R. The 20:80 versus 60:40 whey:casein GM intake differently affected brain neuronal activation (assessed through c-Fos, an immediate-early gene product) in the nucleus of the solitary tract, area postrema, ventromedial hypothalamic nucleus and supraoptic nucleus. Overall, the findings show that whey enhancement in GM promotes overconsumption of GM in no-choice and choice scenarios and that this increased appetite for the 60:40 GM is reflected by changes in neuronal activation and gene expression relevant to feeding regulatory mechanisms. 
Specific Aim 2 results showing preference for whey-enhanced GM and corresponding changes in c-Fos and gene expression, do not predetermine whether the preference for the 60:40 milk would be retained if - instead of a highly palatable GM - a somewhat less preferred CM was used. Thus, in Specific Aim 3, I replicated the aforementioned feeding, gene expression and c-Fos analyses using CM with the 20:80 vs 60:40 whey:casein. I found that mice exhibited preference for the 60:40 over 20:80 whey:casein CM. This preference for the 60:40 CM was retained even when animals had simultaneous access to the 20:80 GM. Consumption of similar quantities of 20:80 CM vs 60:40 CM differently affected c-Fos in the paraventricular, dorsomedial, arcuate and lateral hypothalamic nuclei and in the nucleus of the solitary tract in the brain stem and relative gene expression (melanocortin and opioid transcripts). It can be concluded that the 60:40 whey:casein milks are more preferred regardless of the species from which the milk was derived, indicating that whey:casein ratio influences preference. Mechanistic commonalities in the whey:casein ratio changes in CM vs GM include the hindbrain neuronal activity changes. Differences in hypothalamic c-Fos and gene expression as well as differences in no-choice feeding paradigms indicate that milk type (GM vs CM) influences some aspects of feeding processes driven by the shift in the whey:casein ratio.
Overall, the data presented in this thesis indicate that GM is generally more preferred and it has higher acceptance than CM in laboratory animal models. This phenomenon is reflected by unique changes in feeding-related brain processes induced by GM vs CM. Whey enhancement increases preference toward milk and this effect on consumption is more profound than the effect of the species from which the milk was derived. In a broader context, one has to consider, however, that whey enhancement’s impact on feeding, brain activation and molecular responses might – if sustained over a longer time period - have metabolic consequences.

text: Goat’s vs cow’s milk consumption: Analysis of feeding behaviour, brain activation and gene expression in laboratory animals Milk is a complex and highly nutritive food. In Western societies, cow’s milk (CM) is most commonly consumed, but recent years have generated interest in milk from other species, especially in goat’s milk (GM). Importantly, select physical and chemical properties of milk are species-dependent and – thus – so are the physiological consequences of consumption of milk sourced from specific species. For example, variation between GM and CM protein impacts digestibility and gastrointestinal processes. Consumption of GM vs CM differentially affects levels of blood hormones regulating energy balance. Furthermore, some conflicting results on acceptability of GM- and CM-based foods have been reported, and it is unclear to what extent habituation to a specific milk type underpins these parameters. To add to the confusion, CM and GM are typically consumed and, therefore, studied as modified milk products, with one of the typical compositional alterations being done to the protein fraction in which the natural 20:80 whey:casein ratio is changed to resemble the 60:40 ratio of human milk. One of the most fundamental gaps in our knowledge regarding CM vs GM relates to the acceptability, palatability and satiating properties of these milks and to appetite-controlling brain processes triggered by CM and GM consumption. Thus, in this doctoral project, I sought to examine whether GM and CM diets elicit unique feeding responses in laboratory rodents and whether the presumed appetite differences are associated with changes in neuronal activation and/or gene expression in key central regions regulating food intake. 
In Specific Aim 1 of the project, I conducted a comprehensive investigation of short-term intake and palatability profiles of GM- and CM-based liquid and solid diets in mice and rats. Consumption was studied in no-choice and choice scenarios, including meal microstructure. Feeding experiments were followed by qPCR analysis of expression of relevant genes in the energy balance-related hypothalamus and brain stem, and in the nucleus accumbens, which regulates eating for palatability. I found that GM and CM are palatable to juvenile, adult, and aged rodents. Given a choice, animals prefer GM- to CM-based diets. Analysis of meal microstructure using licking patterns points to enhanced palatability of and, possibly, greater motivation toward GM over CM. Most profound changes in gene expression after GM vs. CM were associated with the brain systems driving consumption for reward. The results allow me to conclude that, while both GM and CM are palatable, GM is preferred over CM by animals, and this preference is driven by central mechanisms controlling eating for pleasure.
In Specific Aim 2 of the thesis, I investigated the impact of whey enhancement in GM protein fraction on appetite and feeding-related brain processes. The shift from the natural whey:casein ratio of ~20:80 in animal milks is done to match the 60:40 ratio of human milk. Studies show that 20:80 versus 60:40 whey:casein milks differently affect glucose metabolism and hormone release. It is unknown whether the 20:80-to-60:40 ratio adjustment affects appetite and brain processes related to food intake. In this set of studies I focused on the impact of the 20:80 vs 60:40 whey:casein content in GM on food intake and feeding-related brain mechanisms in laboratory mice. I found that the 20:80 whey:casein GM formulation was consumed less avidly and was less preferred than the 60:40 GM in short-term choice and no-choice paradigms. The qPCR analyses in the hypothalamus and brain stem revealed that the 20:80 whey:casein GM intake upregulated genes involved in early termination of feeding and in an interplay between reward and satiety, such as MC3R, OXT, POMC and GLP1R. The 20:80 versus 60:40 whey:casein GM intake differently affected brain neuronal activation (assessed through c-Fos, an immediate-early gene product) in the nucleus of the solitary tract, area postrema, ventromedial hypothalamic nucleus and supraoptic nucleus. Overall, the findings show that whey enhancement in GM promotes overconsumption of GM in no-choice and choice scenarios and that this increased appetite for the 60:40 GM is reflected by changes in neuronal activation and gene expression relevant to feeding regulatory mechanisms. 
Specific Aim 2 results showing preference for whey-enhanced GM and corresponding changes in c-Fos and gene expression, do not predetermine whether the preference for the 60:40 milk would be retained if - instead of a highly palatable GM - a somewhat less preferred CM was used. Thus, in Specific Aim 3, I replicated the aforementioned feeding, gene expression and c-Fos analyses using CM with the 20:80 vs 60:40 whey:casein. I found that mice exhibited preference for the 60:40 over 20:80 whey:casein CM. This preference for the 60:40 CM was retained even when animals had simultaneous access to the 20:80 GM. Consumption of similar quantities of 20:80 CM vs 60:40 CM differently affected c-Fos in the paraventricular, dorsomedial, arcuate and lateral hypothalamic nuclei and in the nucleus of the solitary tract in the brain stem and relative gene expression (melanocortin and opioid transcripts). It can be concluded that the 60:40 whey:casein milks are more preferred regardless of the species from which the milk was derived, indicating that whey:casein ratio influences preference. Mechanistic commonalities in the whey:casein ratio changes in CM vs GM include the hindbrain neuronal activity changes. Differences in hypothalamic c-Fos and gene expression as well as differences in no-choice feeding paradigms indicate that milk type (GM vs CM) influences some aspects of feeding processes driven by the shift in the whey:casein ratio.
Overall, the data presented in this thesis indicate that GM is generally more preferred and it has higher acceptance than CM in laboratory animal models. This phenomenon is reflected by unique changes in feeding-related brain processes induced by GM vs CM. Whey enhancement increases preference toward milk and this effect on consumption is more profound than the effect of the species from which the milk was derived. In a broader context, one has to consider, however, that whey enhancement’s impact on feeding, brain activation and molecular responses might – if sustained over a longer time period - have metabolic consequences.

year: 2021

title: Development of an HPLC method to analyze four veterinary antibiotics in soils and aqueous media and validation through fate studies

authors: Srinivasan, Prakash

subjects: HPLC

abstract: A simple, yet robust analytical method was developed to detect and quantify three sulfonamides (SA), namely sulfamethoxazole (SMO), sulfachloropyridazine (SCP), and sulfamethazine (SM), and a macrolide tylosin (TT) in aqueous (calcium chloride and leachate solutions) and solid (agricultural soils) matrices using high performance liquid chromatography and ultra violet detection at 290 nm (TT) and 275 nm (SA) respectively. Chromatography was performed using a Phenomenex Onyx Monolithic C-18 column for TT and a C-18 Luna column for sulfonamides as single analytes eluted isocratically with a mobile phase consisting of acetonitrile: trifluoroacetic acid: tetrahydrofuran in the ratio 22.5:68:9.5 for TT, 40:55:5 for SMO, 32:63:5 for SCP and 31:64:5 for SM (v/v) at 1.0 mL min(-1) and an injection volume of 20 mu L. A gradient method to detect all three sulfonamides in a single run was also developed. The soil residue analysis consisted of extraction with dichloromethane and pre-concentration steps as the aqueous phase was measured directly. The limits of detection at an S/N (signal: noise) ratio of 3 were 20.0 mu g L-1 and 50 mu g L-1 for all sulfonamides and tylosin respectively. The average recoveries for all sulfonamides and tylosin in aqueous matrices ranged from 95 to 105% across the six concentrations investigated. Recoveries from the soils were slightly lower for sulfonamides and tylosin. The isocratic method was used to determine the sorption and degradation of sulfonamides in soils, while the gradient method was used to determine degradation kinetics and leachate concentrations in soils and aqueous systems.

text: Development of an HPLC method to analyze four veterinary antibiotics in soils and aqueous media and validation through fate studies A simple, yet robust analytical method was developed to detect and quantify three sulfonamides (SA), namely sulfamethoxazole (SMO), sulfachloropyridazine (SCP), and sulfamethazine (SM), and a macrolide tylosin (TT) in aqueous (calcium chloride and leachate solutions) and solid (agricultural soils) matrices using high performance liquid chromatography and ultra violet detection at 290 nm (TT) and 275 nm (SA) respectively. Chromatography was performed using a Phenomenex Onyx Monolithic C-18 column for TT and a C-18 Luna column for sulfonamides as single analytes eluted isocratically with a mobile phase consisting of acetonitrile: trifluoroacetic acid: tetrahydrofuran in the ratio 22.5:68:9.5 for TT, 40:55:5 for SMO, 32:63:5 for SCP and 31:64:5 for SM (v/v) at 1.0 mL min(-1) and an injection volume of 20 mu L. A gradient method to detect all three sulfonamides in a single run was also developed. The soil residue analysis consisted of extraction with dichloromethane and pre-concentration steps as the aqueous phase was measured directly. The limits of detection at an S/N (signal: noise) ratio of 3 were 20.0 mu g L-1 and 50 mu g L-1 for all sulfonamides and tylosin respectively. The average recoveries for all sulfonamides and tylosin in aqueous matrices ranged from 95 to 105% across the six concentrations investigated. Recoveries from the soils were slightly lower for sulfonamides and tylosin. The isocratic method was used to determine the sorption and degradation of sulfonamides in soils, while the gradient method was used to determine degradation kinetics and leachate concentrations in soils and aqueous systems.

year: 2012

title: Rata, the Effect of Māori Land Law on Ahikāroa

authors: Whaanga, Mere Joslyn

subjects: Ahikāroa

abstract: Rata, the effect of Māori land law on ahikāroa is an examination of the special relationship a whānau of Ngāi Tahu (ki Wairoa) has had since 1868 with their ancestral land Tutuotekaha. The effects of Māori land law on traditional concepts of land tenure are analysed from the advent of the Native Land Court in the 1860s through to a modern shareholding in the Anewa Trust. An analysis of Te Ture Whenua Māori Act 1993 looks at the basis for the current legislation and how the principles are applied in the case study central to this thesis, an application to have Rata farm returned to the Whaanga whānau.

The research and writing is multi-disciplinary, combining history, Māori land law (ture) and Māori land lore (tikanga and traditional ecological knowledge). An auto-ethnographic approach is balanced by the commentary and judgements of the Māori Land Court and the Māori Appellate Court which are documented in the final section of the thesis.

This thesis questions the canons of Māori land ownership and management – consolidation, amalgamation and incorporation – and posits whether these forms of land holding have become yet another type of alienation.

The three main areas are
I) Ahikāroa
Chapters 2 and 3 examine traditional Māori land tenure; definitions of mana whenua, the obligations as well as the authority; and traditional ecological knowledge, agriculture and Māori land lore. The latter illustrates ways of knowing the land, how, why and when its resources were used. Members of the community of Iwitea were interviewed to provide accounts of usage of some of the traditional resources known to remain on the Tutuotekaha blocks.

II) From gardeners and gatherers to agricultural shareholders
Chapters 4, 5 and 6 examine the shift from traditional tenure and land usage to large-scale agricultural shareholdings. The changes as land passed through the Native Land Court from 1868 are contextualised within the upheaval and radical reconfiguring of Māori society in the Mahia and Wairoa areas as Pākehā settlement increased, and the region was engulfed by the Hauhau and Te Kooti conflicts. This section contains an overview of Māori land law from 1862 to 1987 and the ideological conflict between the colonisers and the colonised. As well, it looks at the influence of Sir James Carroll and Sir Āpirana Ngata, the reasons that Māori went into large-scale farming and how Ngata’s 1931 policies set the model for the incorporations and trusts that still hold most of the district’s Māori land today.

III) Te Ture Whenua Māori Act 1993, philosophy versus implementation
Chapters 7 through to 11 centre on Te Ture Whenua Māori Act 1993 and the application to the Māori Land Court to partition Rata farm from the Anewa Trust. The philosophy of the Act is examined, aided by Deputy Chief Judge Fox’s commentary. This section documents the processes of filing the application to partition, the discussions and outcomes of a judicial hearing, two Māori Land Court hearings and subsequent appeals to the Māori Appellate Court.

text: Rata, the Effect of Māori Land Law on Ahikāroa Rata, the effect of Māori land law on ahikāroa is an examination of the special relationship a whānau of Ngāi Tahu (ki Wairoa) has had since 1868 with their ancestral land Tutuotekaha. The effects of Māori land law on traditional concepts of land tenure are analysed from the advent of the Native Land Court in the 1860s through to a modern shareholding in the Anewa Trust. An analysis of Te Ture Whenua Māori Act 1993 looks at the basis for the current legislation and how the principles are applied in the case study central to this thesis, an application to have Rata farm returned to the Whaanga whānau.

The research and writing is multi-disciplinary, combining history, Māori land law (ture) and Māori land lore (tikanga and traditional ecological knowledge). An auto-ethnographic approach is balanced by the commentary and judgements of the Māori Land Court and the Māori Appellate Court which are documented in the final section of the thesis.

This thesis questions the canons of Māori land ownership and management – consolidation, amalgamation and incorporation – and posits whether these forms of land holding have become yet another type of alienation.

The three main areas are
I) Ahikāroa
Chapters 2 and 3 examine traditional Māori land tenure; definitions of mana whenua, the obligations as well as the authority; and traditional ecological knowledge, agriculture and Māori land lore. The latter illustrates ways of knowing the land, how, why and when its resources were used. Members of the community of Iwitea were interviewed to provide accounts of usage of some of the traditional resources known to remain on the Tutuotekaha blocks.

II) From gardeners and gatherers to agricultural shareholders
Chapters 4, 5 and 6 examine the shift from traditional tenure and land usage to large-scale agricultural shareholdings. The changes as land passed through the Native Land Court from 1868 are contextualised within the upheaval and radical reconfiguring of Māori society in the Mahia and Wairoa areas as Pākehā settlement increased, and the region was engulfed by the Hauhau and Te Kooti conflicts. This section contains an overview of Māori land law from 1862 to 1987 and the ideological conflict between the colonisers and the colonised. As well, it looks at the influence of Sir James Carroll and Sir Āpirana Ngata, the reasons that Māori went into large-scale farming and how Ngata’s 1931 policies set the model for the incorporations and trusts that still hold most of the district’s Māori land today.

III) Te Ture Whenua Māori Act 1993, philosophy versus implementation
Chapters 7 through to 11 centre on Te Ture Whenua Māori Act 1993 and the application to the Māori Land Court to partition Rata farm from the Anewa Trust. The philosophy of the Act is examined, aided by Deputy Chief Judge Fox’s commentary. This section documents the processes of filing the application to partition, the discussions and outcomes of a judicial hearing, two Māori Land Court hearings and subsequent appeals to the Māori Appellate Court.

year: 2012