Gelsolin expression in sheep milk somatic cells during lactation.

The identification of genes involved in phenotypes related to milk quality is important for both economic and health aspects in livestock production. The aim of this study was to assess the level of gelsolin gene expression in two breeds of dairy sheep - Sarda and Gentile - with pronounced differences in quantitative and qualitative milk traits. Gelsolin, a type of actin-modulating proteins is involved in the processes of actin remodeling during cell growth and apoptosis; therefore a role of this protein in mammary changes during lactation was here hypothesized. Individual milk samples were collected three times during lactation from 26 ewes of the two breeds. The differential gene expression of gelsolin in the two breeds and the three lactation times was estimated by quantitative PCR on RNA extracted from milk somatic cells. Correlations of gelsolin gene expression with milk yield and quality and days of lactation were also estimated. The results showed that gelsolin gene expression was significantly higher in the Sarda compared to the Gentile at each lactation stage, in agreement with the longer lactation duration and the higher daily milk yield of the first breed. Significant correlations of gelsolin gene expression were found with milk fat content in Sarda breed (-0.46, P<0.05). Gelsolin expression analysis confirmed the link between gelsolin gene function and milk fat content of sheep.

ClC-1 mutations in myotonia congenita patients: insights into molecular gating mechanisms and genotype-phenotype correlation.

KEY POINTS: Loss-of-function mutations of the skeletal muscle ClC-1 channel cause myotonia congenita with variable phenotypes. Using patch clamp we show that F484L, located in the conducting pore, probably induces mild dominant myotonia by right-shifting the slow gating of ClC-1 channel, without exerting a dominant-negative effect on the wild-type (WT) subunit. Molecular dynamics simulations suggest that F484L affects the slow gate by increasing the frequency and the stability of H-bond formation between E232 in helix F and Y578 in helix R. Three other myotonic ClC-1 mutations are shown to produce distinct effects on channel function: L198P shifts the slow gate to positive potentials, V640G reduces channel activity, while L628P displays a WT-like behaviour (electrophysiology data only). Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function. ABSTRACT: Myotonia congenita is an inherited disease caused by loss-of-function mutations of the skeletal muscle ClC-1 chloride channel, characterized by impaired muscle relaxation after contraction and stiffness. In the present study, we provided an in-depth characterization of F484L, a mutation previously identified in dominant myotonia, in order to define the genotype-phenotype correlation, and to elucidate the contribution of this pore residue to the mechanisms of ClC-1 gating. Patch-clamp recordings showed that F484L reduced chloride currents at every tested potential and dramatically right-shifted the voltage dependence of slow gating, thus contributing to the mild clinical phenotype of affected heterozygote carriers. Unlike dominant mutations located at the dimer interface, no dominant-negative effect was observed when F484L mutant subunits were co-expressed with wild type. Molecular dynamics simulations further revealed that F484L affected the slow gate by increasing the frequency and stability of the H-bond formation between the pore residue E232 and the R helix residue Y578. In addition, using patch-clamp electrophysiology, we characterized three other myotonic ClC-1 mutations. We proved that the dominant L198P mutation in the channel pore also right-shifted the voltage dependence of slow gating, recapitulating mild myotonia. The recessive V640G mutant drastically reduced channel function, which probably accounts for myotonia. In contrast, the recessive L628P mutant produced currents very similar to wild type, suggesting that the occurrence of the compound truncating mutation (Q812X) or other muscle-specific mechanisms accounted for the severe symptoms observed in this family. Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function.

Hypothalamic-pituitary-adrenal axis activation and immune regulation in heat-stressed sheep after supplementation with polyunsaturated fatty acids.

The aim of this study was to assess the effects of supplementation with polyunsaturated fatty acids from different sources on immune regulation and hypothalamic-pituitary-adrenal (HPA) axis activation in heat-stressed sheep. The experiment was carried out during the summer 2012. Thirty-two Comisana ewes were divided into 4 groups (8 sheep/group): (1) supplemented with whole flaxseed (FS); (2) supplemented with Ascophyllum nodosum (AG); (3) supplemented with a combination of flaxseed and A. nodosum (FS+AG); and (4) control (C; no supplementation). On d 22 of the experiment, cortisol concentrations in sheep blood were measured after an injection of ACTH. Cellular immune response was evaluated by intradermic injection of phytohemagglutinin (PHA) at 0, 15, and 30 d of the trial. Humoral response to ovalbumin (OVA) was measured at 0, 15, and 30 d. At 0, 15, and 30 d of the experiment, blood samples were collected from each ewe to determine production of T-helper (Th)1 cytokines (IL-12 and IFN-γ), and Th2 cytokines (IL-10, IL-4, IL-13), and concentrations of heat shock proteins (HSP) 70 and 90. Ewes supplemented with flaxseed alone had greater cortisol concentrations and a longer-lasting cell-mediated immune response compared with ewes in the control and other groups. Anti-OVA IgG concentrations increased in all groups throughout the trial, even though ewes in the FS+AG group had the lowest anti-OVA IgG concentrations at 15 d. The level of IL-10 increased in all groups throughout the experiment; the FS+AG group had the lowest IL-13 concentration at 15 and 30 d. The concentration of HSP 70 increased in AG ewes at the end of the experiment and decreased in FS ewes, whereas that of HSP 90 increased in FS ewes compared with FS+AG ewes. Flaxseed supplementation was found to influence in vivo HPA activation in heat-stressed sheep, resulting in increased cortisol concentrations, probably to meet increased energy demand for thermoregulation. Flaxseed supplementation also supported Th1 response via a complex cross-talk between IL-10, IL-12, and IFN-γ production.

The ACACA gene is a potential candidate gene for fat content in sheep milk.

No major gene has yet been reported in sheep that explains the variation of milk fat content. The coding region of the acetyl-CoA carboxylase alpha (ACACA) gene, which plays an important role in de novo fatty acid synthesis, had been investigated, but no non-synonymous mutations have been reported. In this study, the genomic regions encoding the three promoters of the ACACA gene were directly sequenced in 264 sheep of three different breeds, and 10 SNPs were identified. Allele frequencies of most SNPs significantly differed (P = 0.05-0.0001) between breeds. The SNPs that potentially altered either gene regulatory elements or putative binding sites of transcription factors were made evident through in silico analysis. The association analysis with milk traits, performed for one SNP of PIII (GenBank AJ292286, g.1330G>T), showed a significant allelic substitution effect (+0.33%, P < 0.0001 and +0.35%, P < 0.01) in the Altamurana and Gentile breeds respectively. Because this SNP was located in the binding site of the paired box protein transcription factors, which was shown to function as an efficient promoter element, and because PIII transcripts are expressed in the mammary gland, the SNP in PIII of the ACACA gene might affect the variation of fat content in sheep milk.

Improvement of renal function in epidermolysis bullosa patients after gluten free diet: two cases.

Epidermolysis bullosa (EB) is a rare inherited genetic disease characterized by an abnormal response of the skin and mucosa to mechanical trauma. Dystrophic EB (DEB) is very often associated with many extra cutaneous complications. Those complications involve either epithelial associated tissues or other organs. In particular, several renal complications have been described for DEB in the recessive form, such as amyloidosis, post-infection glomerulonephritis, upper and lower urinary tract obstruction and IgA-Nephropathy (IgAN). In the cases reported below we have two patients diagnosed with DEB that showed compromised renal function and proteinuria. The switch of the normal diet toward a gluten free diet resulted beneficial for both patients, since renal function was rescued and proteinuria cured. Moreover, a general health status improvement was recognised, given that nutritional condition was ameliorated and bone growing enhanced. Furthermore, in both patients the presence of autoantibodies anti-COL7 indicating an autoimmune form of the disease. Therefore, patients received low doses of betametasone useful to reduce inflammatory state and to control immune system function. In conclusion, our results prompt us to hypothesized that in these patients, due to the fragility of the intestinal mucosa, the absence in the diet of gluten may be beneficial.

Probiotics in milk replacer influence lamb immune function and meat quality.

This study was undertaken to assess the effect of milk replacer (MR) containing Lactobacillus acidophilus and a mix of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. longum on lamb immune response and on lamb meat quality. A 6-week-trial was conducted on 40 male Comisana lambs, divided into four groups, fed maternal milk (MM), MR, MR with L. acidophilus supplementation (MRL) and MR with a mix (1 : 1) of B. animalis subsp. lactis and B. longum subsp. longum supplementations (MRB). Lambs fed MR containing a mix of bifidobacteria showed the highest in vivo cellular immune response to phytohemagglutinin, whereas MM and MRB showed the highest antibody response to ovalbumin. At day 11 of the trial, MRL displayed the highest value of Interleukin-10; differences disappeared among groups subsequently. Blood cholesterol levels in lambs fed MR containing L. acidophilus was almost halved compared with that found in MM and MR groups. Meat from artificially reared lambs was characterized by trans-11 18:1 and total conjugated 18:2n-6, whereas meat from the dam-suckled lambs was characterized by 14:0, cis-9 14:1 and 16:0. Polyunsaturated to saturated fatty acid ratio was higher in meat of MR, MRL and MRB than in MM lambs. Meat from artificially reared lamb fed MR containing probiotics showed an improved fatty acid profile for human diet.

Development of affinity to the stockperson in lambs from two breeds.

The interactions between genetic and environmental factors on the development of lamb affinity to humans was assessed on 48 animals from two breeds (Gentile di Puglia and Comisana) and two treatments (Gentled and Not Gentled) producing 4 groups (GPg, GPng, Cg, Cng, respectively). The Gentile di Puglia and Comisana gentled animals were subjected to a period of training consisting of gently handling each lamb for 5 min three times a day for the first week and then twice a week for three additional weeks. The gentling procedure included both tactile and visual/auditory interactions. At 30-32 days of age lambs were subjected to three arena tests conducted in a novel environment: i) isolation test (each animal was exposed to a novel environment, and isolated from tactile and visual contact with conspecifics for 5 min), ii) stationary human test (as previously but a human sitting in a corner of the pen), and iii) pen-mates' test (each animal was tested in the presence of 2 pen-mates whose behaviour was not recorded). During the 13 training sessions lambs exhibited an increasing number of contacts with the human and a decreasing number of bleats (P<0.001), although Gentile di Puglia lambs interacted more with the human (P<0.001) and tended to bleat less (P<0.10) than Comisana lambs. Lambs vocalised more and climbed more when tested in isolation as compared with lambs tested either with the human or with the pen-mates (P<0.01) and they vocalised more in the presence of the human than with the pen-mates (P<0.001). A higher number of contacts with the human stimulus was observed in gentled animals (P<0.05). However, differences between gentled and not gentled animals were only significant in Gentile di Puglia subjects (P<0.01). GPng lambs displayed the longest ambulatory activity during the isolation test (P<0.05) and exhibited more climbing attempts during the isolation test as compared with the human or the pen-mates' tests (P<0.001). A higher cortisol level was shown by GPng lambs in comparison with Cng subjects during the stationary human test (P<0.01), whereas no differences were detected between the two gentled groups. Gentling determined an improvement of the quality of human animal relationship in more reactive breeds such as Gentile di Puglia sheep.

Relationship between cortisol response to stress and behavior, immune profile, and production performance of dairy ewes.

The existence of a relationship between cortisol levels, after an acute stress, and behavioral activities, immunological profile, and production performance in sheep was studied. An initial flock of 30 Comisana ewes was involved in the experiment, and each of the 30 ewes was individually subjected to an isolation test in a novel environment. Subsequently, from the initial flock, 2 groups of 8 Comisana ewes were each retrospectively selected, and the animals were divided, according to their cortisol concentration 10 min after the isolation test, into high cortisol (HC) ewes, having a peak of cortisol concentration >90 ng/mL (average: 119.3 ng/mL +/- 11.8), and low cortisol (LC) ewes having a peak of cortisol concentration <80 ng/mL (average: 52.4+/-11.8). During the isolation test, the behavior of each animal was video-recorded and behavioral activities were registered. Blood samples were collected before the isolation test, immediately after the test (10 min), and at 60, 120, 300 min, 24 h, and 48 h after the test to evaluate percentages of T-helper (CD4(+)) and T-cytotoxic (CD8(+)) cells, CD4(+)/CD8(+) ratio, and IL-1beta and IL-6 levels. The ewes were milked for 3 d after the isolation test to determine cortisol levels and IL-1beta and IL-6 concentrations in whey. Milk yield was recorded at each milking, and milk samples were analyzed for pH, nutritional parameters, renneting properties, and somatic cell count. During the isolation test, HC ewes exhibited a shorter duration of movement and fewer bleats than LC ewes. The average plasma IL-1beta concentration was higher in HC than in LC ewes. The average whey IL-1beta and IL-6 concentrations were higher in whey from HC ewes than in LC ewes. A positive correlation emerged between plasma and whey IL-1beta concentrations. The average CD4(+)/CD8(+) ratio in blood was lower in HC than in LC ewes. Time from isolation affected the CD4(+)/CD8(+) ratio: at 120 min, the CD4(+)/CD8(+) ratio increased compared with that at 10 min after isolation and then decreased until 300 min after isolation. On average, ewes with low cortisol concentrations showed higher milk production and lower SCC than ewes with high cortisol concentrations. Results suggest that plasma cortisol concentration is connected to the behavioral response and immune competence of dairy ewes and cytokine concentrations. Both whey IL-1beta and IL-6 can be considered reliable indicators of the magnitude of hypothalamic-pituitary-adrenal axis activation. The stress-induced changes in CD4(+)/CD8(+) ratio are critical for controlling disease incidence and planning appropriate vaccination programs. High reactivity of the hypothalamic-pituitary-adrenal axis is also associated with a reduction in milk production and an increased predisposition to develop intramammary inflammatory processes.

Ovine acyl CoA:diacylglycerol acyltransferase 1- molecular characterization, polymorphisms and association with milk traits.

The objective of this work was to characterize the complete coding region of the ovine acylCoA:diacylglycerol acyltransferase 1 (DGAT1) gene of three Italian sheep breeds: Sarda, Altamurana and Gentile di Puglia. Characterization was accomplished by direct sequencing of 8676 bp of the relevant DNA, including introns and partial 5' and 3' untranslated regions (UTRs). We detected five novel SNPs; one SNP (g.5553C>T) is located in intron 2, has similar frequencies in the three breeds and showed a negative association with milk fat content. More interesting is an SNP in the 5' UTR (g.127C>A), the occurrence of which is rare in the higher milk-fat breeds (Altamurana and Gentile di Puglia); it is located in the core sequence of Sp1, a putative binding site of transcription factors. This SNP showed a significant negative association with milk fat content in the Sarda sheep. Because DGAT1 plays a fundamental role in triacylglycerol synthesis, the novel detected SNP in the 5' UTR of the DGAT1 gene might explain, at least partially, the variation of fat content in the milk of Sarda sheep.

Changes occurring in immune responsiveness of single- and twin-bearing Comisana ewes during the transition period.

Changes induced by twin and single lambing in the immune response of 16 periparturient Comisana ewes were studied. Cell-mediated immune responses were evaluated by means of skin tests performed from 3 wk before and up to d 35 after parturition. At d 21 and 7 before lambing, the sheep received an intramuscular injection of the antigen keyhole limpet hemocyanin (KLH), to which the animals had not been previously exposed, to determine their humoral immune response. Starting 3 wk before lambing and up to d 35 postlambing, the ewes were sampled to determine the plasma concentrations of anti-KLH antibody (IgG), IL-6, and IL-1 beta. From parturition through d 35 postpartum, individual milk samples were collected for determination of anti-KLH IgG titers and IL-6 and IL-1beta concentrations by means of a capture ELISA. The number of lambs born affected IL-6 concentrations in ewe plasma; IL-6 secretion always was higher in ewes birthing twins than in single-lambing ewes. Apart from the number of lambs born, the concentrations of plasma IL-6 in ewes were higher at lambing than at d 21 antepartum and at d 35 postpartum. An interaction of number of lambs born x time of sampling was observed for plasma antibody titers to KLH. The IgG concentrations were significantly higher in single-bearing ewes than in twin-bearing ewes before parturition and were very similar across groups after parturition. A time effect was found for the cell-mediated immune response and for anti-KLH IgG concentrations in milk, such that at parturition, cellular responses were lowest, and the anti-KLH IgG concentration was highest. A significant correlation was found for IgG titers to KLH in plasma and milk. Results indicate that IL-6 concentrations in blood can be considered a reliable indicator of stress connected to lambing and that the mammary gland is a microenvironment unrelated to blood stream with respect to interleukins expression. In contrast, a relationship was found for the IgG secretions in milk and blood, which suggests that the assessment of humoral immune status may be combined with milking routine in dairy animals.

Effects of ventilation regimen on the welfare and performance of lactating ewes in summer.

A 6-wk trial was performed with thirty-six lactating Comisana ewes during the summer of 2001. The animals were divided into three groups of 12, which were designated low (LVR), moderate (MVR), and programmed (PROGR) ventilation regimens. In LVR and MVR rooms, fans provided 10 ventilation cycles of 12.5 and 25 min/h, respectively, whereas in the PROGR room, the fan was programmed to operate at 30 degrees C air temperature and 70% relative humidity. Mean ventilation rates were 33, 66, and 173 m3/h per ewe in LVR, MVR, and PROGR rooms. Air concentrations of microorganisms and dust and of gaseous pollutants were measured twice weekly. Respiration rate and rectal temperature were monitored throughout the trial at 1430. Behavioral traits of ewes were recorded once per week from 0930 to 1230. Cell-mediated immune response to phytohemagglutinin at d 3, 20, and 40 and humoral response to chicken egg albumin at d 11, 21, 30, and 40 were determined. At d 37, ewes were injected with 2 IU porcine ACTH/kg body weight(0.75) and subjected to blood sampling for evaluation of cortisol concentrations immediately before and 1, 2, and 4 h after ACTH injection. Milk yield was recorded daily. Individual milk samples were analyzed weekly for composition and renneting parameters and fortnightly for bacteriological characteristics. Averages of temperature-humidity index values were 78.9, 76.8, and 74.5 in LVR, MVR, and PROGR rooms, respectively. The LVR and MVR treatments resulted in higher NH3 and CO2 air concentrations than PROGR treatment (P < 0.05). The LVR and MVR ewes had higher rectal temperatures than PROGR ewes (P = 0.001). LVR animals also exhibited higher idling compared to PROGR (P < 0.01) and lower feeding times than MVR (P < 0.05) and PROGR animals (P < 0.01). Ewes under the LVR treatment displayed significant lower averages of antibody titers and higher plasma cortisol levels than PROGR (P < 0.01) and MVR ewes (P < 0.05) 60 min after ACTH injection. The LVR treatment resulted in lower yields of milk (P < 0.01) and reduced feed efficiency (P < 0.01) than PROGR treatment. Results suggest that a fan-ventilated system, providing ventilation cycles during the warmest hours of the day and the night at a mean ventilation rate of 66 m3/ewe per hour, may sustain the performance and welfare in lactating ewes raised in warm climates during summer. A ventilation regimen, programmed to operate over upper critical air temperature and relative humidity, may be economically unattractive under these conditions.

Effects of solar radiation and feeding time on behavior, immune response and production of lactating ewes under high ambient temperature.

A 6-wk trial was performed with 40 late-lactation Comisana ewes, which were either exposed to or protected from solar radiation and fed either in the morning (EXPM, PROM) or afternoon (EXPA, PROA) during summer in a Mediterranean climate. Behavioral traits of ewes were recorded once per week from 0800 to 2000 h. Rectal temperature (RT) and respiration rate (RR) were measured twice weekly at 1430 h. The phytohemagglutinin (PHA) skin test was performed to induce nonspecific delayed-type hypersensitivity at d 10, 20, and 32 of the experiment. Jugular blood samples were taken from ewes at the beginning and at d 21 and 42 of the experiment. Ewe milk yield was recorded daily. Individual milk samples were analyzed weekly for milk composition, coagulating properties, somatic cell count (SCC) and polymorphonuclear neutrophil leukocyte counts (PMNLC) and every 2 wk for bacteriological characteristics. Solar radiation and the interaction of solar radiation x time of feeding had significant effects on rectal temperatures. EXPM ewes had higher rectal temperatures than EXPA ewes, which in turn exhibited higher RT compared with PROM and PROA ewes. EXP groups also had significantly higher respiration rates than PRO groups. Immune response was lower in EXPM ewes at d 10 and in EXPM, EXPA, and PROM animals at d 20 compared with PROA ewes. Exposure to solar radiation resulted in decreased plasma concentrations of alanine amino-transferase, alkaline phosphatase, potassium, and magnesium, as well as in increased levels of nonesterified fatty acids and aspartate amino-transferase. Milk yield and composition were not changed by exposure to solar radiation and time of feeding, but the EXPM treatment resulted in lower yields of casein and fat and reduced clot firmness compared with the three other treatments. Milk SCC was similar across treatments, but PMNLC was higher in EXPM than in PROM and PROA milk. EXPM animals also had the greatest amounts of total and fecal coliforms and of Pseudomonadaceae as well as the highest number of mastitis related pathogens in their milk. Results suggest that provision of shaded areas can play a major role in helping lactating ewes to minimize the adverse effects of high ambient temperatures on thermal balance and energy and mineral metabolism. Changing the time of feeding to late afternoon may be beneficial to exposed ewes in lowering their heat loads during the warmest hours of the day, thereby reducing the detrimental impact of thermal stress on immune function and udder health.

Airspace effects on the yield and quality of ewe milk.

Three groups of 12 midlactating Comisana ewes were housed in separate rooms of the same building and assigned to treatments of low (LV, 4.1 m3), medium (MV, 5.6 m3), or high (HV, 7.3 m3) airspace/animal. The concentrations of airborne microorganisms in the experimental rooms were measured twice weekly at 0930 and 1630. Ewe milk yield was recorded daily. Individual milk samples were analyzed weekly for milk composition, coagulating properties, somatic cell concentration (SCC), and polymorphonuclear neutrophil leukocyte count (PMNLC), and fortnightly for bacteriological characteristics; samples with more than 10(6) somatic cells/ml were cultured for mastitis-related pathogens. The LV and MV treatments resulted in higher relative humidity and air concentrations of staphylococci than the HV treatment. Greater amounts of air mesophilic bacteria were also found in the LV than in the HV room. Ewes in the HV group gave greater yields of milk than those in the LV and MV groups. LV milk also had a lower casein content than HV milk. Significant interactions of treatment x time were found for milk protein and fat content as well as for clotting time and clot firmness, with LV milk having the poorest composition and deteriorated renneting ability during the last 3 wk of the trial. The HV ewes had lower SCC and PMNLC and psychrotroph counts in their milk than LV and MV ewes and smaller amounts of mesophilic bacteria and fecal coliforms than LV animals. Subclinical mastitis occurred in two ewes of the LV and one of the MV groups, while no cases were recorded in the HV group. Results suggest that airspace is a critical factor in dairy sheep housing and indicate that a volume allocation of less than 7 m3/animal may adversely affect the performance and health of the lactating ewe.

Effect of parity on milk yield, composition, somatic cell count, renneting parameters and bacteria counts of Comisana ewes.

Twenty-four Comisana ewes, with no history of mastitis, were included in this study, with eight ewes each in parities 1, 2 and 3. Groups were separately penned on straw litter and ewes were individually checked for yield, composition, renneting properties and bacteriological characteristics of milk from January, when separated from their lambs (50+/-3 days after lambing), to May. Samples with more than 3.5x10(5) somatic cells/ml were cultured for mastitis related pathogens. Milk yield was not significantly affected by parity. The P3 ewes had significantly higher milk protein, casein and fat contents compared to the P1 and P2 ewes. The P3 ewes also had improved renneting ability of milk as compared to the P1 ewes. Quality of milk decreased with lower lactations. The milk of P1 ewes had significantly greater amounts of mesophilic bacteria than the P2 and P3 ewes, as well as higher concentrations of psychrotrophs and total coliforms in their milk with respect to the P3 ewes. Somatic cell counts in milk and the prevalence of subclinical mastitis were not changed by parity, although mastitis infection set in progressively earlier as the number of lactations decreased. These results suggest that ewes in first or second lactation have a less favourable milk secretion status in relation to mastitis than ewes with a higher number of lactations. Milk yield and quality of younger ewes may be improved by offering feed rations that take into account this reduced capacity to mobilise body reserves. Also, most scrupulous control of sanitation of housing, equipment and personnel is necessary.

Effect of stocking density on ewes' milk yield, udder health and microenvironment.

Three groups of 12 lactating Comisana ewes were housed in separate rooms of the same building that contained straw-bedded pens with total areas of 12, 18 and 24 m2. The ewes were assigned to groups with different stocking densities: high (HSD, 1 m2/animal), medium (MSD, 1.5 m2/animal) or low (LSD, 2 m2/animal). The LSD treatment resulted in lower air concentrations of total microorganisms and coliform bacteria than the other two. Ewes in the LSD group gave greater yields of milk, total protein and fat than those in the other two groups. LSD treatment increased milk casein content and improved clotting time and clot firmness. LSD also resulted in ewes giving milk with lower somatic cell counts and smaller concentrations of mesophiles, psychrotrophs and faecal coliforms than MSD and HSD. Subclinical mastitis occurred in three ewes of the MSD groups and four ewes of the HSD group, while no cases were recorded in the LSD group. These results indicate that stocking density is a critical factor in dairy sheep housing and suggest that a space allocation < 2 m2/animal may adversely affect the performance and health of the lactating ewe.