Effect of the dietary delivery matrix on vitamin D3 bioavailability and bone mineralisation in vitamin-D3-deficient growing male rats.

This study assessed bioavailability and utilisation of vitamin D3 in two feeding trials using young, growing Sprague-Dawley male rats. Trial one fed animals standard AIN-93G diet (casein protein) containing no vitamin D3 and goat or cow skimmed milk supplemented with vitamin D3. Trial two fed animals modified dairy-free AIN-93G diet (egg albumin) containing no vitamin D3 and goat or cow skimmed or full-fat milk supplemented with vitamin D3. Control groups received AIN-93G diets with or without vitamin D, and water. At 8 weeks of age, blood samples were collected for vitamin and mineral analysis, and femurs and spines were collected for assessment of bone mineralisation and strength. In both trials, analyses showed differences in bioavailability of vitamin D3, with ratios of serum 25-hydroxyvitamin D3 to vitamin D3 intake more than 2-fold higher in groups drinking supplemented milk compared with groups fed supplemented solid food. Bone mineralisation was higher in groups drinking supplemented milk compared with groups fed supplemented solid food, for both trials (P<0·05). There was no difference in the parameters tested between skimmed milk and full-fat milk or between cow milk and goat milk. Comparison of the two trials suggested that dietary protein source promoted bone mineralisation in a growing rat model: modified AIN-93G with egg albumin produced lower bone mineralisation compared with standard AIN-93G with casein. Overall, this study showed that effects of vitamin D3 deficiency in solid diets were reversed by offering milk supplemented with vitamin D3, and suggests that using milk as a vehicle to deliver vitamin D is advantageous.

Identification of goat milk powder by manufacturer using multiple chemical parameters.

Concentrations of multiple elements and ratios of stable isotopes of carbon and nitrogen were measured and combined to create a chemical fingerprint of production batches of goat whole milk powder (WMP) produced by different manufacturers. Our objectives were to determine whether or not differences exist in the chemical fingerprint among samples of goat WMP produced at different sites, and assess temporal changes in the chemical fingerprint in product manufactured at one site. In total, 58 samples of goat WMP were analyzed by inductively coupled plasma-mass spectrometry as well as isotope ratio mass spectrometry and a suite of 13 elements (Li, Na, Mg, K, Ca, Mn, Cu, Zn, Rb, Sr, Mo, Cs, and Ba), δ(13)C, and δ(15)N selected to create the chemical fingerprint. Differences in the chemical fingerprint of samples between sites and over time were assessed using principal components analysis and canonical analysis of principal coordinates. Differences in the chemical fingerprints of samples between production sites provided a classification success rate (leave-one-out classification) of 98.1%, providing a basis for using the approach to test the authenticity of product manufactured at a site. Within one site, the chemical fingerprint of samples produced at the beginning of the production season differed from those produced in the middle and late season, driven predominantly by lower concentrations of Na, Mg, K, Mn, and Rb, and higher concentrations of Ba and Cu. This observed temporal variability highlights the importance of obtaining samples from throughout the season to ensure a representative chemical fingerprint is obtained for goat WMP from a single manufacturing site. The reconstitution and spray drying of samples from one manufacturer by the other manufacturer enabled the relative influence of the manufacturing process on the chemical fingerprint to be examined. It was found that such reprocessing altered the chemical fingerprint, although the degree of alteration varied among samples and individual elements. The findings of this study support the use of trace elements and stable isotope ratios to test the authenticity of goat WMP, which can likely be applied to other dairy goat products. This approach could be used test to the factory of origin (and potentially batch of origin) of products in the supply chain, thus providing the ability to audit the supply chain and monitor for fraudulent activity.

Nutritional adequacy of goat milk infant formulas for term infants: a double-blind randomised controlled trial.

The safety and nutritional adequacy of goat milk infant formulas have been questioned. The primary aim of the present study was to compare the growth and nutritional status of infants fed a goat milk infant formula with those of infants fed a typical whey-based cow milk infant formula. The secondary aim was to examine a range of health- and allergy-related outcomes. A double-blind, randomised controlled trial with 200 formula-fed term infants randomly assigned to receive either goat or cow milk formula from 2 weeks to at least 4 months of age was conducted. A cohort of 101 breast-fed infants was included for comparison. Weight, length and head circumference were measured at 2 weeks and 1, 2, 3, 4, 6 and 12 months of age. Nutritional status was assessed from serum albumin, urea, creatinine, Hb, ferritin, and folate and plasma amino acid concentrations at 4 months. Z-scores for weight, length, head circumference and weight for length were not different between the two formula-fed groups. There were differences in the values of some amino acids and blood biomarkers between the formula-fed groups, but the mean values for biomarkers were within the normal reference range. There were no differences in the occurrence of serious adverse events, general health, and incidence of dermatitis or medically diagnosed food allergy. The incidence of parentally reported blood-stained stools was higher in the goat milk formula-fed group, although this was a secondary outcome and its importance is unclear. Goat milk formula provided growth and nutritional outcomes in infants that did not differ from those provided by a standard whey-based cow milk formula.

Comparison of the compositions of the stool microbiotas of infants fed goat milk formula, cow milk-based formula, or breast milk.

The aim of the study was to compare the compositions of the fecal microbiotas of infants fed goat milk formula to those of infants fed cow milk formula or breast milk as the gold standard. Pyrosequencing of 16S rRNA gene sequences was used in the analysis of the microbiotas in stool samples collected from 90 Australian babies (30 in each group) at 2 months of age. Beta-diversity analysis of total microbiota sequences and Lachnospiraceae sequences revealed that they were more similar in breast milk/goat milk comparisons than in breast milk/cow milk comparisons. The Lachnospiraceae were mostly restricted to a single species (Ruminococcus gnavus) in breast milk-fed and goat milk-fed babies compared to a more diverse collection in cow milk-fed babies. Bifidobacteriaceae were abundant in the microbiotas of infants in all three groups. Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium bifidum were the most commonly detected bifidobacterial species. A semiquantitative PCR method was devised to differentiate between B. longum subsp. longum and B. longum subsp. infantis and was used to test stool samples. B. longum subsp. infantis was seldom present in stools, even of breast milk-fed babies. The presence of B. bifidum in the stools of breast milk-fed infants at abundances greater than 10% of the total microbiota was associated with the highest total abundances of Bifidobacteriaceae. When Bifidobacteriaceae abundance was low, Lachnospiraceae abundances were greater. New information about the composition of the fecal microbiota when goat milk formula is used in infant nutrition was thus obtained.

Whey-Adapted versus Natural Cow's Milk Formulation: Distinctive Feeding Responses and Post-Ingestive c-Fos Expression in Laboratory Mice.

The natural 20:80 whey:casein ratio in cow's milk (CM) for adults and infants is adjusted to reflect the 60:40 ratio of human milk, but the feeding and metabolic consequences of this adjustment have been understudied. In adult human subjects, the 60:40 CM differently affects glucose metabolism and hormone release than the 20:80 CM. In laboratory animals, whey-adapted goat's milk is consumed in larger quantities. It is unknown whether whey enhancement of CM would have similar consequences on appetite and whether it would affect feeding-relevant brain regulatory mechanisms. In this set of studies utilizing laboratory mice, we found that the 60:40 CM was consumed more avidly than the 20:80 control formulation by animals motivated to eat by energy deprivation and by palatability (in the absence of hunger) and that this hyperphagia stemmed from prolongation of the meal. Furthermore, in two-bottle choice paradigms, whey-adapted CM was preferred against the natural 20:80 milk. The intake of the whey-adapted CM induced neuronal activation (assessed through analysis of c-Fos expression in neurons) in brain sites promoting satiation, but importantly, this activation was less pronounced than after ingestion of the natural 20:80 whey:casein CM. Activation of hypothalamic neurons synthesizing anorexigenic neuropeptide oxytocin (OT) was also less robust after the 60:40 CM intake than after the 20:80 CM. Pharmacological blockade of the OT receptor in mice led to an increase in the consumption only of the 20:80 CM, thus, of the milk that induced greater activation of OT neurons. We conclude that the whey-adapted CM is overconsumed compared to the natural 20:80 CM and that this overconsumption is associated with weakened responsiveness of central networks involved in satiety signalling, including OT.

Compositional and functional characteristics of goat milk and relevance as a base for infant formula.

Goat milk has a long history of use for human nutrition. There are a number of studies investigating the technofunctional properties and nutritional value of goat milk for production of consumer products such as cheese, yoghurts, and Ultra-high temperature (UHT) milks. Although fresh goat milk was traditionally used for feeding young children, use of goat milk for commercial production of formula for infants or young children has only been considered in the scientific literature since 2000s. In this review, the nutritional science relating to goat milk is discussed from the perspective of infant nutrition. A critical analysis of the scientific data concerning the composition and functional characteristics of goat milk that pertain to infant nutrition is included. From this overview, three key conclusions are possible: (1) there is a range of compositional data to support the application of goat milk for infants, provided it is fortified; (2) goat milk has the potential to influence the gastrointestinal environment differently to cow milk; and (3) the nutritional adequacy of fortified goat milk formula for newborn infants has been confirmed in clinical trials.

Adjustment of Whey:Casein Ratio from 20:80 to 60:40 in Milk Formulation Affects Food Intake and Brainstem and Hypothalamic Neuronal Activation and Gene Expression in Laboratory Mice.

Adjustment of protein content in milk formulations modifies protein and energy levels, ensures amino acid intake and affects satiety. The shift from the natural whey:casein ratio of ~20:80 in animal milk is oftentimes done to reflect 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; these data parallel animal model findings. It is unknown whether the adjustment from the 20:80 to 60:40 ratio affects appetite and brain processes related to food intake. In this set of studies, we focused on the impact of the 20:80 vs. 60:40 whey:casein content in milk on food intake and feeding-related brain processes in the adult organism. By utilising laboratory mice, we found that the 20:80 whey:casein milk formulation was consumed less avidly and was less preferred than the 60:40 formulation in short-term choice and no-choice feeding paradigms. The relative PCR analyses in the hypothalamus and brain stem revealed that the 20:80 whey:casein milk intake upregulated genes involved in early termination of feeding and in an interplay between reward and satiety, such as melanocortin 3 receptor (MC3R), oxytocin (OXT), proopiomelanocortin (POMC) and glucagon-like peptide-1 receptor (GLP1R). The 20:80 versus 60:40 whey:casein formulation 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. We conclude that the shift from the 20:80 to 60:40 whey:casein ratio in milk affects short-term feeding and relevant brain processes.

The acute-phase protein serum amyloid A3 is expressed in the bovine mammary gland and plays a role in host defence.

The serum amyloid A protein is one of the major reactants in the acute-phase response. Using representational difference analysis comparing RNA from normal and involuting quarters of a dairy cow mammary gland, we found an mRNA encoding the SAA3 protein (M-SAA3). The M-SAA3 mRNA was localized to restricted populations of bovine mammary epithelial cells (MECs). It was expressed at a moderate level in late pregnancy, at a low level through lactation, was induced early in milk stasis, and expressed at high levels in most MECs during mid to late involution and inflammation/mastitis. The mature M-SAA3 peptide was expressed in Escherichia coli, antibodies made, and shown to have antibacterial activity against E. coli, Streptococcus uberis and Pseudomonas aeruginosa. These results suggest that the mammary SAA3 may have a role in protection of the mammary gland during remodelling and infection and possibly in the neonate gastrointestinal tract.

Nε-carboxymethyllysine in nutritional milk formulas for infants.

Production of infant formulas involves high temperature processing for microbiological safety. However, heat processes generate Advanced Glycation End-products (AGEs), including Nε-carboxymethyllysine (CML) formed between lysine and lactose. Formulas manufactured from cow or goat milk, with or without whey adjustment, or hydrolysates of cow whey proteins, were tested for CML levels using a commercially available ELISA kit. CML concentrations ranged from 2 to 210 µg/g protein in formulas containing intact proteins. Median CML concentrations were up to 3-fold greater in formulas containing 60% whey protein compared with 20% whey protein, for both cow and goat formulas. Goat milk formulas contained 7 to 12-fold less CML than cow milk formulas. Formulas made from intact proteins contained lower CML compared to formulas using whey hydrolysates. Western immunoblotting techniques detected higher CML levels in whey proteins compared with casein. This study showed whey addition to infant formula significantly contributes to CML levels.

Gastric digestion of cow and goat milk: Peptides derived from simulated conditions of infant digestion.

Infant formula products are predominantly manufactured using cow milk protein; goat milk also provides a suitable protein source. In this study, we directly compared cow and goat milk protein digestion using pH and enzyme conditions to simulate infant gastric conditions. Generated peptides, identified using liquid chromatography coupled to a mass spectrometer, show both similarities and differences in cow and goat milk post-digestion profiles. The majority of peptides were from casein proteins, 50% representing ß-casein, with many peptides unique to each species. Low or no peptides for ß-Lactoglobulin and α-Lactalbumin, respectively, suggest these proteins were highly resistant to infant gastric digestion, as reported by others. Minor milk proteins, comprising 5% of peptides, were represented by different proteins from cow and goat. Peptides with known bioactivities were also observed, both in common and unique to each species. Together these data may explain reported differences in digestion characteristics of cow and goat milk.

Palatability of Goat's versus Cow's Milk: Insights from the Analysis of Eating Behavior and Gene Expression in the Appetite-Relevant Brain Circuit in Laboratory Animal Models.

Goat's (GM) and cow's milk (CM) are dietary alternatives with select health benefits shown in human and animal studies. Surprisingly, no systematic analysis of palatability or preference for GM vs. CM has been performed to date. Here, we present a comprehensive investigation of short-term intake and palatability profiles of GM and CM in laboratory mice and rats. We studied consumption in no-choice and choice scenarios, including meal microstructure, and by using isocaloric milks and milk-enriched solid diets. Feeding results are accompanied by qPCR data of relevant genes in the energy balance-related hypothalamus and brain stem, and in the nucleus accumbens, which regulates eating for palatability. We 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. We conclude that, while both GM and CM are palatable, GM is preferred over CM by laboratory animals, and this preference is driven by central mechanisms controlling eating for pleasure.

Gastric digestion of cow and goat milk: Impact of infant and young child in vitro digestion conditions.

Many infants and young children are fed nutritional milk formulas. Although products are commonly based on cow milk, goat milk provides an alternative. We directly compared digestion of cow and goat milk proteins, varying pH, enzyme concentrations and incubation times to simulate infant and young child gastric conditions. Protein digestion and peptide formation were evaluated using electrophoresis and chromatography. Digestion of higher molecular weight whey proteins increased with decreased pH and higher enzyme concentrations of young child gastric digestion conditions compared to infant conditions. ß-lactoglobulin was poorly digested under all gastric digestion conditions. Caseins reacted to pH changes differently compared to whey proteins, with less digestion of casein at pH 3.0 than at pH 5.0. Caseins from goat milk tended to be more efficiently digested compared to caseins from cow milk and peptide profiles from goat milk were distinct from cow milk.

Digestive Responses to Fortified Cow or Goat Dairy Drinks: A Randomised Controlled Trial.

Fortified milk drinks are predominantly manufactured from bovine (cow) sources. Alternative formulations include those prepared with hydrolysed bovine milk proteins or from alternate bovidae species, such as caprine (goat) milk. Currently, there is little data on protein digestive and metabolic responses following ingestion of fortified milk drinks. To examine the digestive and metabolic responses to commercially-available fortified milks, young adults (n = 15 males: 15 females), in a randomised sequence, ingested isonitrogenous quantities of whole cow-protein (WC), whole goat-protein (WG), or partially-hydrolysed whey cow-protein (HC), commercial fortified milks. Plasma amino acid (AA) and hormonal responses were measured at baseline and again at 5 h after ingestion. Paracetamol recovery, breath hydrogen, and subjective digestive responses were also measured. Postprandial plasma AA was similar between WC and WG, while AA appearance was suppressed with HC. Following HC, there was a negative incremental AUC in plasma branched-chain AAs. Further, HC had delayed gastric emptying, increased transit time, and led to exaggerated insulin and GLP-1 responses, in comparison to whole protein formulas. Overall, WC and WG had similar protein and digestive responses with no differences in digestive comfort. Contrastingly, HC led to delayed gastric emptying, attenuated AA appearance, and a heightened circulating insulin response.

Differentiation of Bifidobacterium longum subspecies longum and infantis by quantitative PCR using functional gene targets.

BACKGROUND: Members of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota (B. longum subsp. longum; B. longum subsp. infantis). B. longum subspecies are not differentiated in many studies, so that their prevalence and relative abundances are not accurately known. This may largely be due to difficulty in assigning subspecies identity using DNA sequences of 16S rRNA or tuf genes that are commonly used in bacterial taxonomy. METHODS: We developed a qPCR method targeting the sialidase gene (subsp. infantis) and sugar kinase gene (subsp. longum) to differentiate the subspecies using specific primers and probes. Specificity of the primers/probes was tested by in silico, pangenomic search, and using DNA from standard cultures of bifidobacterial species. The utility of the method was further examined using DNA from feces that had been collected from infants inhabiting various geographical regions. RESULTS: A pangenomic search of the NCBI genomic database showed that the PCR primers/probes targeted only the respective genes of the two subspecies. The primers/probes showed total specificity when tested against DNA extracted from the gold standard strains (type cultures) of bifidobacterial species detected in infant feces. Use of the qPCR method with DNA extracted from the feces of infants of different ages, delivery method and nutrition, showed that subsp. infantis was detectable (0-32.4% prevalence) in the feces of Australian (n = 90), South-East Asian (n = 24), and Chinese babies (n = 91), but in all cases at low abundance (<0.01-4.6%) compared to subsp. longum (0.1-33.7% abundance; 21.4-100% prevalence). DISCUSSION: Our qPCR method differentiates B. longum subspecies longum and infantis using characteristic functional genes. It can be used as an identification aid for isolates of bifidobacteria, as well as in determining prevalence and abundance of the subspecies in feces. The method should thus be useful in ecological studies of the infant gut microbiota during early life where an understanding of the ecology of bifidobacterial species may be important in developing interventions to promote infant health.

Amino acid composition determined using multiple hydrolysis times for three goat milk formulations.

The amino acid composition of goat milk formulations with varying protein and carbohydrate concentrations were determined. Proteins in goat milk infant formula, goat milk growing-up formula and goat whole milk powder were hydrolysed using multiple hydrolysis time intervals. A least-squares non-linear regression model was used to predict the free and protein bound amino acid concentrations. The amino acid composition of goat infant formula was compared with human milk reference values. There was good agreement between the multiple hydrolysis and single 24-h hydrolysis methods for approximately one-half of the amino acids. Tryptophan, aspartic acid, threonine, tyrosine, isoleucine, valine, serine and alanine contents were underestimated by 10.6, 5.6, 5.6, 4.7, 4.4, 3.7, 3.7 and 3.6%, respectively, by the single 24-h hydrolysis. The study provides accurate reference data on the amino acid composition of goat milk powders. Goat milk infant formula has amino acids in amounts similar to human milk reference values, when expressed on a per-energy basis.

Composition of the non-protein nitrogen fraction of goat whole milk powder and goat milk-based infant and follow-on formulae.

The non-protein nitrogen fraction of goat whole milk powder and of infant and follow-on formulae made from goat milk was characterized and compared with cow milk powder and formulae. Goat milk infant formula contained 10% non-protein nitrogen, expressed as a proportion of total nitrogen, compared with 7.1% for cow milk formula. Goat follow-on formula contained 9.3% and cow 7.4% non-protein nitrogen. Urea, at 30%, was quantitatively the most abundant component of the non-protein nitrogen fraction of goat milk and formulae, followed by free amino acids at 7%. Taurine, glycine and glutamic acid were the most abundant free amino acids in goat milk powders. Goat milk infant formula contained 4 mg/100 ml total nucleotide monophosphates, all derived from the goat milk itself. Goat milk has a very different profile of the non-protein nitrogen fraction to cow milk, with several constituents such as nucleotides at concentrations approaching those in human breast milk.

Immune components of colostrum and milk--a historical perspective.

Key developments in the understanding of the immune functions of milk and colostrum are reviewed, focusing on their proteinaceous components. The topics covered include the immunoglobulins, immune cells, immunomodulatory substances, and antimicrobial proteins. The contributions of new technologies and the introduction of fresh approaches from other fields are highlighted, as are the contributions that mammary biology research has made to the development of other fields. Finally, a summary of some current outstanding questions and likely future directions of the field are given.

Effects of melatonin on the yield and composition of milk from grazing dairy cows in New Zealand.

The aim was to determine whether administration of melatonin would alter the yield and composition of milk from grazing dairy cows in summer. Twelve sets of spring-calving identical twin Friesian cows were used in the experiment. In late-November (late spring), one twin from each set was given slow-release melatonin implants behind the ears (108 mg melatonin/cow). Two further implantations occurred at 4-weekly intervals to maintain increased circulating concentrations of melatonin for 12 weeks. The other twin served as a control. Milk yield and composition were measured twice prior to treatment and then four times over the following 12 weeks. Concentrations of melatonin, prolactin and insulin-like growth factor 1 (IGF-1) were measured in blood plasma twice before treatment and then either seven (melatonin and prolactin) or three (IGF-1) further times during the experiment. Management procedures for all cows were similar and cows grazed a daily pasture allowance of approximately 30 kg DM/cow as their sole feed source. In melatonin-treated cows there was a decrease in mean concentrations of prolactin in plasma, but concentrations of IGF-1 did not change. Melatonin reduced milk yield by 6 weeks after treatment and by the end of the 12-week experimental period milk yield in melatonin-treated cows had fallen by 23%. Melatonin also reduced concentrations of lactose in milk, but increased concentrations of fat, protein and casein, changes that were broadly similar to those that occur in late lactation in seasonally calving dairy cows. Thus, the results suggest that some of the variation in the volume and quality of milk throughout the season in New Zealand dairy systems may be due to changes in photoperiod mediated by increased concentrations of plasma melatonin in association with decreased concentrations of plasma prolactin.

Effects of atropine on plasma amino acid profiles and on the synthesis of individual milk proteins in dairy cows fed with pasture.

Effects of atropine on blood plasma amino acid profile and on the yields and concentration of milk components were investigated in 12 Friesian cows in early lactation. Cows were housed indoors and fed with cut pasture ad libitum. Each cow received four treatments over 12 d during a replicated 4 x 4 Latin square experiment. Treatments were: control (saline); low dose (L; 30 microg atropine/kg body weight (BW)); medium dose (M; 40 microg atropine/kg BW); and 2 x L dose, 2 h apart (2 x L). On each of four treatment days, cows were milked at about 7.00, after which treatments were administered by subcutaneous injection. Cows were milked again at 2 h, 6 h and 10 h after injection. Milk samples were collected at each milking. Immediately after the 2 h milking, blood samples were drawn from each cow and the second injection was given for the 2 x L treatment. Atropine reduced hourly milk yield, and concentrations and hourly yields of total protein, casein, whey protein, alpha-casein, beta-casein, kappa-casein, beta-lactoglobulin and alpha-lactalbumin, but by differing amounts. Milk concentrations of bovine serum albumin and immunoglobulin G were increased by atropine, and overall yields of these proteins were mostly unchanged. Atropine lowered concentrations of most, but not all, amino acids in blood plasma, with essential amino acids reduced more than non-essential amino acids. Concentrations of alpha-amino N in whole blood, and glucose and insulin in blood plasma, fell after atropine injection. There was no difference between the L and M doses of atropine, but the 2 x L dose had greater effects on milk composition than the single doses. For yields of milk and milk components, the effect of the 2 x L dose was also more persistent. The results highlight the differential synthesis of individual milk proteins, and suggest that atropine might be useful for evaluating the mechanisms regulating milk protein composition.

Effects of stage of lactation and time of year on plasmin-derived proteolytic activity in bovine milk in New Zealand.

The objective of this study was to determine the effects of stage of lactation (SOL) and time of year on plasmin-derived proteolytic activity in the milk of pasture-fed dairy cows in New Zealand. Four herds of 20 Friesian cows were used, one herd calving in each of January, April, July and October. Cows grazed ryegrass/white clover pasture only, except during June (winter) when all cows received supplementary pasture silage. Milk samples were collected on four occasions during the year (spring, summer, autumn and winter) from each cow in milk, to give a total of three samples per cow (early, mid and late lactation; c. 30, 120 and 220 days after calving, respectively). Milk samples were analysed for plasmin-derived proteolytic activity. There was no effect of either SOL or time of year on plasmin activity and therefore yields of plasmin followed patterns in milk yield (highest in early lactation and in summer). There were effects of both SOL and time of year on plasminogen-derived and total plasmin plus plasminogen-derived activity, both of which were highest in late lactation and in spring. Changes in plasminogen-derived activity and total plasmin plus plasminogen-derived activity due to SOL were not only due to the decrease in milk yield associated with advancing lactation, because enzyme yields were also increased with advancing lactation. Similarly, effects of time of year on plasminogen-derived activity and total plasmin plus plasminogen-derived activity could not be attributed solely to concomitant changes in milk yield, and may be influenced by the variation in the quality and quantity of feed during the year inherent in a pasture-based dairy system. Effects of SOL on proteolytic activity were greater than, and independent of, effects of time of year.