Effect of parity on milk yield, composition, somatic cell count,
renneting parameters and bacteria counts of Comisana ewes
A. Sevi
a,*, L. Taibi
b, M. Albenzio
a, A. Muscio
a, G. Annicchiarico
b aIstituto di Produzioni e Preparazioni Alimentari, FacoltaÁ di Agraria di Foggia, via Napoli, 25, 71100 Foggia, ItalybIstituto Sperimentale per la Zootecnia, via Napoli, 71020 Segezia-Foggia, Italy
Accepted 11 October 1999
Abstract
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 (503 days after lambing), to May. Samples with more than 3.5105somatic cells/ml were cultured for mastitis related pathogens. Milk yield was not signi®cantly affected by parity. The P3 ewes had signi®cantly 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 signi®cantly 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 ®rst 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.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Ewes; Parity; Milk yield; Mastitis; Milk composition
1. Introduction
Findings of several researchers on the effect of parity on yield and quality of ewe milk are not consistent. Casoli et al. (1989), Hatziminaoglou
et al. (1990) and Ubertalle et al. (1990) have observed increased milk yields as the numbers of lactations advanced, whereas Dell'Aquila et al., (1993) did not ®nd any signi®cant effect of parity on productive levels of ewes.
A progressive increase of milk protein and fat contents with increasing number of lactations has been reported by Casoli et al. (1989) and Dell'Aquila et al. (1993), but the opposite trend has been observed *Corresponding author. Tel.:39-881-714544;
fax.39-881-740211.
E-mail address: prodan.fgagr@isnet.it (A. Sevi)
by Ubertalle (1989). Wohlt et al. (1981) did not ®nd any in¯uence of parity on ewe milk constituents.
Con¯icting results may be due to other factors, breed, feeding, number of lambs suckled, manage-ment practices and climatic conditions, which can play a role when different parities are compared. Differences between parities may also be due to selection by breeders who allow only the best sheep to be in the ¯ock for more than two lactations.
Little is known about the effect of parity on ewe udder health. Bergonier et al. (1994) observed higher somatic cell counts in sheep milk with the advance-ment of number of lactations. Sandrucci et al. (1992) stated that higher SCC in older cows may be due to increased amounts of epithelial cells in milk rather than to higher leukocyte proportions (Wever and Emanuelson, 1989). However, studies on electronic microscopy of sheep milk state that there are just a few epithelial cells in this milk (Lee and Outteridge, 1981; Dulin et al., 1983).
The present study was undertaken to assess the effects of parity on milk yield, composition, somatic cell count, renneting parameters and bacteria counts of Comisana ewes.
2. Materials and methods
2.1. Experimental animals
Twenty-four Comisana ewes, with no history of mastitis, were used in this study, with eight ewes each in parities 1, 2 and 3 (P1, P2 and P3, respectively). All ewes lambed in mid-November and were separated from their lambs at 503 days after parturition. The animals were housed in a prefabricated building and groups were separately penned on straw litter. Each pen was provided with two mangers and a hay crib (feeder space0.45 m/animal). Ewes had free access to automatic water troughs.
Groups were homogeneous in terms of number of lambs suckled. At the commencement of the experi-ment, body weights were 51.192.62, 53.762.11 and 56.372.41 kg (meansSE), respectively, in the P1, P2 and P3 groups.
Ewes were fed a diet composed of pelletted con-centrate, vetch/oat hay and oat straw (40, 50 and 10% of total diet, respectively), which was offered as TMR
twice daily. The chemical composition of dry matter was determined by the AOAC (1990) methods and resulted in:15.4%crudeprotein, 2.1% fat,byetherextract,23.4% crude ®bre, 9.4% ash. Average daily DM intake was 2.360.28 kg per ewe during the study period.
2.2. Sampling and analyses of milk
Ewes were milked twice daily using pipeline milk-ing machines (Alpha Laval Agri, SE-147 21 Tumbas, Sweden). Daily milk yield was recorded by means of graduated measuring cylinders attached to individual milking units. Individual milk samples, consisting of proportional volumes of morning and evening milk, were taken after cleaning and disinfection of teats (0.7 alcohol) and discharging the ®rst streams of foremilk. Samples were collected in 200 ml sterile plastic con-tainers at fortnightly intervals through the lactation period of 5 months (from January to May) and taken to our laboratory under refrigeration. Upon arrival in the laboratory (30±60 min after collection) and prior to testing, milk samples were divided into two equal portions, one half for milk composition, somatic cell count and renneting parameter determination and the other half for bacteriological analyses. The following measurements were made: pH, total protein, fat and lactose content, using an I.R. spectrophotometer (Milko Scan 133B; Foss Electric, DK-3400 Hillerùd, Denmark) according to the IDF (1990) standard; casein content (IDF, 1964); somatic cell count (SCC), using a Foss Electric Fossomatic 90 cell counter (IDF, 1995); renneting parameters (clotting time, rate of clot formation and clot ®rmness after 30 min) using a Foss Electric Formagraph and the method of Zannoni and Annibaldi (1981); enumera-tion of mesophilic micro-organisms (IDF, 1991a), psychrotrophs (IDF, 1991b), total coliforms (IDF, 1985) and faecal coliforms on Violet Red Bile Agar (VRBA, I-20100 Biolife, Milan, Italy) incubated at 44.50.58C for 24 h.
mastitis-related pathogens. Presumptive Escherichia coliwas detected after a 24 h incubation on VRBA with MUG (4-methylumbelliferil-B-D-glucuronide)
(Hartman, 1989) at 44.50.58C. Staphylococci were detected after a 24 h incubation on Mannitol Salt Agar medium (Biolife) at 378C and subsequently identi®ed to species level, using the API-STAPH system (Bio-merieux, F-69280 Marcy l'Etoile, France). Strepto-cocci were determined on modi®ed Edwards' Aesculin medium (Oxoid, Basingstoke, RG24 09W, United Kingdom) at 378C; streptococcal isolates were then identi®ed to species level, using the API 20 Strep System (Biomerieux).Pseudomonas spp.were deter-mined using Pseudomonas Selective Agar (Oxoid); Pseudomonas aeruginosawas detected after a 3-day incubation on Pseudomonas agar F and Pseudomonas Agar P (Oxoid) at 32±378C. As proposed by Watkins et al. (1991), milk samples were recorded as bacter-iologically positive when at least 1000 cfu of the same type were isolated from 1 ml of milk. If two bacterial species were isolated, they were treated as a case of either species. If three or more bacterial species were cultured from a sample, the sample was considered to be contaminated (Fox et al., 1995). In addition, the polymorphous neutrophil leucocyte (PMNL) count was carried out according to the method proposed by Morgante et al. (1996), which provides the direct microscopic count of almost 100 cells in milk smears stained with May±GruÈnwald Giemsa. As proposed by Andrew et al. (1983), udders without clinical abnorm-alities and whose milk was apparently normal, with somatic cell counts greater than 3.5105, PMNL counts higher than 20% of total somatic cells and bacteriologically positive, were considered to have subclinical mastitis when the same bacterial species was isolated from milk samples at least in two of three consecutive samplings.
The body weights of the ewes were recorded fort-nightlyanteprandiumafter the machine milking in the morning.
At the end of May (1963 days after parturition), the ewes were moved from the experimental building for mating and samplings were stopped.
2.3. Statistical analysis
Data from ewes considered to have subclinical mastitis were excluded from statistical analysis. All
the variables were tested for normal distribution using the Shapiro and Wilk (1965) test and milk somatic cell and micro-organism counts were transformed into logarithm form to normalize their frequency distribu-tions before performing any statistical analysis. The data were subjected to an analysis of variance, using the GLM procedure of the SAS (1990) statistical software (1990); the model used for all variables was Yparityewe (parity)monthtime of samplingparitymonthparitymonthday of samplingerror, whereYis the milk yield data of each individual ewe; ewe (parity) the error term to test the effect of parity; month the effect of month through lactation (1,. . ., 5); `day of sampling' the effect of day for each month of lactation that milk data were analysed (1, 2). For all parameters, model effects were declared signi®cant atp< 0.05, unless otherwise sta-ted.
3. Results and discussion
3.1. Milk yield and quality
Milk yield was not signi®cantly affected by parity (Table 1), in agreement with previous reports (Del-l'Aquila et al., 1993). Increased milk yields with the advancement of number of lactations were found by other groups (Casoli et al., 1989; Ubertalle et al., 1990; Hatziminaoglou et al., 1990), whereas only a slight increase of milk production was observed in this study as the number of lactations advanced. However, when milk samplings were stopped, ewes still had a milk production greater than 1 kg/day. This would suggest that had the study period been prolonged further the slight differences found could have increased and become signi®cant.
decrease for both parameters as the lactation pro-gressed.
Many possible causes could be suggested to explain the increase in milk protein, casein and fat as the number of lactations progressed. Firstly, the increased body weight of the ewes with a greater number of lactations which leads to a greater availability of body
reserves for the synthesis of milk components. This hypothesis may be supported by the fact that the differences between the groups which diminished in late lactation when the contribution of body reserve to milk component synthesis was reduced (Grummer, 1991). Secondly, the greater development of the udder glandular tissue as the number of lactations rises could
Table 1
Least squares means of milk yield and composition of Comisana ewes in parities 1 (P1), 2 (P2) and 3 (P3)
Parities SE Level of significance
P1 P2 P3 PTe Mf PTM
Milk yield(kg/day)
January 1.25b 1.31b 1.32b
February 1.54a 1.54a 1.59a
March 1.48a 1.52a 1.57a
April 1.47a 1.51a 1.56a
May 1.31b 1.32b 1.39b
Mean 1.41 1.44 1.48 0.02 NS ** NS
Protein content(%)
January 5.72bc 5.90b 6.27a
February 5.43cd 5.33d 5.77b
March 5.31d 5.48c 5.80b
April 5.57c 5.65c 5.99ab
May 5.92b 6.03ab 6.15a
Mean 5.59b 5.68b 5.99a 0.03 * ** NS
Casein content(%)
January 4.68b 4.74ab 4.92a February 4.11c 4.07c 4.27b
March 4.07c 4.21c 4.41b
April 4.19c 4.24c 4.48b
May 4.59ab 4.68a 4.77a
Mean 4.32b 4.38b 4.57a 0.02 * ** NS
Fat content(%)
January 5.22c 5.82b 6.43ad February 4.68d 5.06c 5.70be
March 4.38d 5.09c 5.48e
April 5.34c 5.81b 6.01ae
May 6.06b 6.23d 6.66a
Mean 5.13c 5.60b 6.05a 0.07 * ** NS
Lactose content(%)
January 5.50a 5.35bc 5.37bd February 5.47a 5.37bc 5.35cd March 5.55a 5.40bc 5.37bd April 5.46a 5.29bc 5.32bd
May 5.38b 5.39c 5.28d
Mean 5.47a 5.36ab 5.33b 0.01 * ** NS
a,b,c,dMeans within the same rows or columns followed by different letters differ signi®cantly atp< 0.05. eParity.
fMonth.
also result in an increased synthesis of milk constitu-ents. Another possible explanation could be an improved ef®ciency of the homeorhetic dynamics involved in the partitioning of the nutrients for the processes of lactogenesis and galactopoiesis as the number of lactations increases (Hart, 1983). The existence of a greater ¯ow of nutrients into galacto-poiesis at the expense of other body tissues in older than in younger ewes is supported by the fact that, during the study period, average weight gains tended to decrease (p0.07) from the P1 to the P2 and the P3 group (118, 95 and 84 g/day, respectively).
Irrespective of parity, the contents of total protein, casein and fat were higher in April and May, as a consequence of reduced milk yield and increased de
novo synthesis of relatively more milk constituents due to the positive energy balance of the ewes in late lactation (Auldist et al., 1995).
The lactose content of milk decreased with increas-ing number of lactations and was signi®cantly higher (p< 0.05) in the P1 than in the P3 group. Also, the changes of lactose content in milk during the lacta-tion were opposite to those observed for protein, casein and fat contents, with the lowest lactose con-tents recorded during the last two months of the lactation period.
These results, which are in line with those found by other groups (Casoli et al., 1989) are not easy to interpret given that lactose concentration in milk tends to remain constant, at least in animals that are not
Table 2
Least squares means of renneting parameters of milk of Comisana ewes in parities 1 (P1), 2 (P2) and 3 (P3)
Parities SE Level of significance
P1 P2 P3 Parity Month ParityMonth
pH
January 6.70 6.71 6.68
February 6.67 6.66 6.66
March 6.71 6.67 6.65
April 6.61 6.61 6.59
May 6.60 6.58 6.58
Mean 6.65 6.64 6.63 0.02 NS NS NS
Clotting time(min)
January 22.0a 21.1a 20.4a
February 20.6bc 20.5ab 19.0ab
March 18.9c 18.0c 17.5c
April 20.5bc 19.7abc 18.7ab
May 19.4c 18.0c 17.4c
Mean 20.3 19.5 18.6 0.3 NS ** NS
Rate of clot formation(min)
January 4.7a 4.7a 4.3a
February 4.6a 4.4a 4.0a
March 4.0ac 3.7bc 3.3b
April 4.4a 4.1abc 3.9b
May 3.9ac 3.6bc 3.2b
Mean 4.3a 4.1ab 3.7b 0.09 * ** NS
Clot firmness(mm)
January 39.6c 41.5c 45.4c February 46.8bc 46.3bc 51.0ac
March 49.9b 52.1b 58.0ab
April 45.4bc 46.0bc 49.1ac
May 46.7bc 51.4abc 55.2a
Mean 45.7b 47.5ab 51.7a 1.23 * * NS
suffering from mastitis, as lactose is the main osmo-tically active component in the milk. Pulina (1990) interpreted this decline in milk lactose level as the result of a worsening in udder health as the number of lactations increased, but in this study at least this would not seem to be the case. What is evident is the link with fact that as the number of lactations advanced, the endocrine±metabolic status in ewes changed.
The renneting behaviour of milk substantially re¯ected the differences in milk casein and fat con-tents among parities as well as the variations of these parameters through lactation (Table 2). Rate of clot formation and clot ®rmness were improved (p< 0.01) in the P3 as compared to the P1 group, whereas the P2 group exhibited intermediate values for both para-meters. This could be expected, because clot
forma-Table 3
Somatic cell and bacteria count in milk of Comisana ewes in parities 1 (P1), 2 (P2) and 3 (P3)d
Parities SE Level of significance
P1 P2 P3 Parity Month ParityMonth
Somatic cell count
January 4.98 5.07 5.01
February 4.87 5.08 4.96
March 4.98 4.89 4.91
April 4.99 4.90 4.87
May 5.18 5.01 5.00
Mean 5.00 4.99 4.95 0.07 NS NS NS
Mesophile count
January 4.61b 4.51bc 4.58bc
February 4.51b 4.53bc 4.41bc
March 5.80a 5.48ad 5.49a
April 5.46a 5.06ab 4.47b
May 6.07a 5.41d 5.21ad
Mean 5.29a 4.99b 4.83b 0.06 * ** NS
Psychrotroph count
January 4.34c 4.33c 4.49bc
February 5.07a 5.07a 4.78bc
March 5.48a 5.11a 5.11bc
April 5.51a 5.51ab 5.02b
May 5.46a 5.07ab 4.77b
Mean 5.17a 5.01ab 4.83b 0.05 * ** NS
Total coliform count
January 3.34c 3.19c 3.15bc February 3.35c 3.53bc 3.39bc
March 3.97ac 3.70bc 3.55bc
April 4.01a 3.66abc 3.37b
May 4.42a 3.80b 3.54b
Mean 3.81a 3.57ab 3.40b 0.08 * * NS
Faecal coliform count
January 0.21 0.52 0.40
February 1.01 0.88 0.84
March 1.05 0.80 0.48
April 1.79 1.33 1.06
May 2.38 2.11 1.94
Mean 1.28 1.12 0.94 0.12 NS NS NS
a,b,cMeans within the same rows or columns followed by different letters differ signi®cantly atp< 0.05. dData are least square means of log 10 of smoatic cells and cfu x ml of milk.
tion involves the aggregation of casein micelles into a network within which the fat is entrapped (Dalgleish, 1993).
Also, pH and clotting time were ameliorated by the increasing number of lactations, but differences among groups were smaller and not signi®cant for these parameters.
The somatic cell count (SCC) in milk was not changed by parity (Table 3). Average values were acceptably low, ranging from 249 to 321103 somatic cell/ml of milk in parities 3 and 1, respec-tively. Irrespective of parity, the highest values were recorded in May and the lowest in February (671 and 203103somatic cell/ml of milk on average, respec-tively). SCC were higher than those found by Mor-gante et al. (1996) in Comisana ewes.
Conversely, the analysis of variance of the loga-rithmic values highlighted a marked effect of number of lactations on milk bacteria counts. The P1 ewes exhibited signi®cantly greater (p< 0.05) amounts of mesophilic bacteria than the P2 and P3 ewes (521 vs. 348 and 298103cfu/ml, respectively) as well as higher concentrations of psychrotrophs and total coli-forms in their milk with respect to the P3 ewes (377 vs. 163103cfu/ml and 19 vs. 7103cfu/ml, respec-tively). Although the P1 ewes had a higher micro-organism concentration in this milk, but there was no increase in milk SCC. This could suggest that the greater bacterial colonisation of the udder in these ewes could be attributed to a reduced ef®ciency in the natural defence mechanisms against the penetration and multiplication of bacteria in the udder (Varner and Johnson, 1983).
Increased concentrations of micro-organisms could play a role in protein and fat contents decrease in the milk yield of the P1 ewes, due to enhanced post-secretory lipolytic and proteolytic actions in milk by enzymes produced from the bacterial ¯ora (Higoshi and Hamada, 1975; Auldist et al., 1996; Sevi et al., 1999).
3.2. Incidence of subclinical mastitis
There were no cases of clinical mastitis during the study period. One case of subclinical mastitis (Table 4) was recorded in each group, but mastitis infection set in progressively earlier as the number of lactations decreased and this supports the hypothesis of less ef®cient natural defence mechanisms in younger ewes,
perhaps due to a less advanced development of the immune system or to greater susceptibility to envir-onmental stress (Bertoni, 1996). Exposure to bacteria is a prerequisite but the development of infection and mastitis is a balance between the natural defence mechanisms of the teat and mammary gland and the number and pathogenicity of the micro-organisms in contact with the entrance of the teat canal (Klastrup et al., 1987).
As a consequence, the number of bacteriologically positive milk samples was higher in the P1 group (n15) than in the P2 (n11) and P3 groups (n9). On the whole, bacteria or combinations of bacteria were isolated from 35 milk samples and environmental bacteria were largely predominant. Streptococci (especially Enterococcus spp.) consti-tuted the majority of bacteria isolated, since they were isolated from about 70% of the 35 bacteriologically
Table 4
Cases of subclinical mastitis, bacteriologically positive milk samples and bacteria isolated from milk of Comisana ewes in parities 1 (P1), 2 (P2) and 3 (P3)
Bacteria isolated and number of milk samples from which isolated
Escherichia coli 6 4 4
Pseudomonas spp. 2 2 2
Pseudomonas aeruginosa 2 1 1
Staphylococcus aureus 2 1 0
Staphylococcus xylosus 0 1 1
Staphylococcus hyicus 1 0 0
Other CN-staphylococci 3 3 1
Streptococcus agalactiae 0 0 0
Streptococcus pneumoniae 1 1 0
Enterococcus spp. 6 6 3
positive milk samples. E. coli, coagulase-negative (CN)-staphylococci andPseudomonasspp.were iso-lated from 14, 10 and 6 milk samples at 40, 29, 17%, respectively.S. aureusandP. aeruginosawere detected in four and three milk samples, respectively, whereasS. agalactiaewas not isolated from any milk sample.
4. Conclusions
Ewes milk yield was not changed by parity, but milk protein, casein and fat contents increased and rennet-ing ability of milk improved as the number of lacta-tions advanced. Somatic cell counts in milk were very similar in parities 1, 2 and 3 and groups had one case of subclinical mastitis each. However, bacteria counts in milk increased and mastitis infection set in pro-gressively earlier with lower numbers of lactations.
Further studies on this topic are required using larger ¯ocks. Nevertheless, our ®ndings indicate that the differences in milk composition linked to the number of lactations may depend on different endo-crine and metabolic status in ewes of varying ages. Also, most scrupulous control of sanitation of housing, equipment and personnel is necessary.
References
Andrew, R.J., Kitchen, B.J., Kwee, W.S., Duncalfe, F., 1983. Relationship between individual cow somatic cell count and the mastitis infection status of the udder. Aust. J. Dairy Technol. 38, 71±74.
AOAC, 1990. Of®cial Methods of Analysis, 15th ed. Association of Of®cial Analytical Chemists, Washington, DC, pp. 69±88. Auldist, M.J., Coats, S., Rogers, G.L., McDowell, G.H., 1995.
Changes in the composition of milk from healthy and mastitic dairy cows during the lactation cycle. Austr. J. Exper. Agric. 35, 427±436.
Auldist, M.J., Coats, S., Sutherlans, B.J., Mayes, J.J., McDowell, G.H., Rogers, G.L., 1996. Effects of somatic cell count and stage of lactation on raw milk composition and the yield and quality of cheddar cheese. J. Dairy Res. 63, 269±280. Bergonier, D., Van de Wiele, A., Arranz, J.M., Barillet, F.,
Lagriffoul, G., Concordet, G., Berthelot, X., 1994. Non-infectious factors affecting somatic cell count in dairy sheep and goat. In: Rubino, R. (Ed.), Somatic Cells and Milk Quality in Small Ruminants, pp. 139±167.
Bertoni, G., 1996. Environment feeding and milk quality. Informatore Agrario (Suppl.) 52, 5±41.
Casoli, C., Duranti, E., Morbidini, L., Panella, F., Vizioli, V., 1989. Quantitative and compositional variations of Massases sheep
milk by parity and stage of lactation. Small Rumin. Res. 2, 47±62.
Dalgleish, D.G., 1993. Bovine milk protein properties and the manufacturing quality of milk. Livst. Prod. Sci. 35, 75±93. Dell'Aquila, S., Pilla, A.M., Catillo, G., Scardella, G., Taibi, L.,
1993. Milk yield of Comisana, Delle Langhe, Massese, Sarda and crossbred ewes. Zootecnica Nutrizione Animale 14, 95±102. Dulin, A.M., Paape, M.J., Berkow, S., Hamosh, M., Hamosh, P., 1983. Comparison of total somatic cells and differential cellular composition in milk from cows, sheep, goats and humans. Federation Proceedings 42, 1331.
Fox, L.K., Chester, S.T., Hallberg, J.W., Nickerson, J.W., Pankey, J.W., Weawer, L.D., 1995. Survey of intramammary infections in dairy heifers at breeding age and ®rst parturition. J. Dairy Sci. 78, 1619±1628.
Fruganti, G.S., Ranucci, S., Tesei, B., Valente, C., 1985. Assessment of the health status of ewes udder throughout lactation. Clinica Veterinaria 108, 286±296.
Grummer, R.R., 1991. Effect of feeding on the composition of milk fat. J. Dairy Sci. 74, 3244±3257.
Hart, I.C., 1983. Endocrine control of nutrient partition in lactating ruminants. Proc. Nutr. Soc. 42, 181±194.
Hartman, P.A., 1989. The MUG (gluconidase) test forEscherichia coliin feed and water. In: Balows, A., Tilton, R.C., Turano, A. (Eds.), Rapid Methods and Automation in Microbiology and Immunology, Brixion Academic Press, Brescia, Italy, pp. 290± 308.
Hatziminaoglou, I., Geogordiudis, A., Karalazos, A., 1990. Factors affecting milk yield and proli®cacy of Karagouniko sheep in West Thessaly (Greece). Livst. Prod. Sci. 24, 181±186. Higoshi, H., Hamada, S., 1975. Proteolytic and lipolytic activities
of psychrotrophic bacteria originated from raw milk. J. Food Hyg. Soc. Japan 17, 1±47.
IDF, 1964. Determination of the casein content of milk. IDF Standard No. 29. International Dairy Federation, Brussels, Belgium.
IDF, 1985. Enumeration of coliforms. IDF Standard No. 73A. International Dairy Federation, Brussels, Belgium.
IDF, 1990. Determination of milk fat, protein and lactose content Ð Guide for the operation of mid-infra-red instruments. IDF Standard No. 141B. International Dairy Federation, Brussels, Belgium.
IDF, 1991a. Enumeration of micro-organisms Ð Colony count at 308C. IDF Standard No. 100B. International Dairy Federation, Brussels, Belgium.
IDF, 1991b. Enumeration of psychrotrophic micro-organisms Ð Colony count at 6.58C. IDF Standard No. 101A. International Dairy Federation, Brussels, Belgium.
IDF, 1995. Enumeration of somatic cells. IDF Standard No. 148A. International Dairy Federation, Brussels, Belgium.
Klastrup, O., Bakken, G., Bramley, J., Bushnell, R., 1987. Environmental in¯uences on bovine mastitis. Bulletin No. 217. International Dairy Federation, Brussels, Belgium. Lee, C.S., Outteridge, P.M., 1981. Leucocytes of sheep colostrum,
Morgante, M., Ranucci, S., Pauselli, M., Casoli, C., Duranti, E., 1996. Total and differential cell count in milk of primiparous Comisana ewes without clinical signs of mastitis. Small Rumin. Res. 21, 265±271.
Pulina, G., 1990. In¯uence of feeding on ewes milk quality. Informatore Agrario 37, 31±39.
Sandrucci, A., Tamburini, A., Rapetti, L., 1992. A study on the use of a linear score for the evaluation of milk somatic cell count. Proceedings of the 27th Simposio Internazionale di Zootecnia, SocietaÁ per il Progresso della Zootecnia, Milan, Italy, pp. 175±190. SAS, 1990 SAS/STAT User's Guide Version 6, 4th ed., Statistical
Analysis System Inst, Cary, NC.
Sevi, A., Massa, S., Annicchiarico, G., Dell'Aquila, S., Muscio, A., 1999. Effect of stocking density on ewes milk yield, udder health and micro-environment. J. Dairy Res., in press. Shapiro, S.S., Wilk, M., 1965. An analysis of variance test for
normality. Biometrika 52, 591±601.
Ubertalle, A., 1989. Factors affecting the quality of ewes milk. Proceedings of The Accademia Economico-agraria dei Geor-go®li, Stamperia Editoriale Parenti, Firenze, Italy, pp. 1±18.
Ubertalle, A., Bianchi, M., Errante, J., Battaglini, L.M., 1990. Proli®cacy and milk yield: phenotypic relationships in Delle Langhe ewes. Zootecnica Nutrizione Animale 16, 219±224. Varner, M.A., Johnson, B.H., 1983. In¯uence of
adrenocortico-tropin upon milk production, milk constituents, and endocrine measures of dairy cows. J. Dairy Sci. 66, 458±465.
Watkins, G.H., Burriel, A.R., Jones, J.E.T., 1991. A ®eld investigation of subclinical mastitis in sheep in Southern England. Br. Vet. J. 147, 413±420.
Wever, P., Emanuelson, U., 1989. Effects of systematic in¯uences and intramammary infection on differential and total somatic cell counts in quarter milk samples from dairy cows. Acta Vet. Scandin. 30, 465±474.
