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Effect of parity on milk yield, composition, somatic cell count, renneting parameters and bacteria counts of Comisana ewes
Small Ruminant Research 37 (2000) 99±107
Effect of parity on milk yield, composition, somatic cell count, renneting parameters and bacteria counts of Comisana ewes A. Sevia,*, L. Taibib, M. Albenzioa, A. Muscioa, G. Annicchiaricob a
Istituto di Produzioni e Preparazioni Alimentari, FacoltaÁ di Agraria di Foggia, via Napoli, 25, 71100 Foggia, Italy b Istituto 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 (50 3 days after lambing), to May. Samples with more than 3.5 105 somatic 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 *
Corresponding author. Tel.: 39-881-714544; fax. 39-881-740211. E-mail address: [email protected] (A. Sevi)
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
0921-4488/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 9 9 ) 0 0 1 3 3 - 9
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A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
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, management 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 advancement 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 50 3 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 space 0.45 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 experiment, body weights were 51.19 2.62, 53.76 2.11 and 56.37 2.41 kg (means SE), respectively, in the P1, P2 and P3 groups. Ewes were fed a diet composed of pelletted concentrate, 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 2.36 0.28 kg per ewe during the study period. 2.2. Sampling and analyses of milk Ewes were milked twice daily using pipeline milking 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 containers 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); enumeration 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 44.5 0.58C for 24 h. At the beginning of the experiment and on the day before each milk sampling, all the ewes were examined carefully to assess the absence of signs of clinical mastitis, such as fever, pain or gland swelling. A small quantity of milk was checked visually for signs of mastitis. As proposed by Fruganti et al. (1985), an aliquot of 0.01 ml from all milk samples containing more than 3.5 105 somatic cells/ml was cultured for
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
mastitis-related pathogens. Presumptive Escherichia coli was detected after a 24 h incubation on VRBA with MUG (4-methylumbelliferil-B-D-glucuronide) (Hartman, 1989) at 44.5 0.58C. 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 (Biomerieux, F-69280 Marcy l'Etoile, France). Streptococci 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 determined using Pseudomonas Selective Agar (Oxoid); Pseudomonas aeruginosa was 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 bacteriologically 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 abnormalities and whose milk was apparently normal, with somatic cell counts greater than 3.5 105, 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 fortnightly anteprandium after the machine milking in the morning. At the end of May (196 3 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
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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 distributions 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 Y parity ewe (parity) month time of sampling parity month parity month day of sampling error, where Y is 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 at p < 0.05, unless otherwise stated. 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 (Dell'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. In contrast, parity strongly affected the quality of ewes milk. Milk protein and casein contents increased (p < 0.05) as the number of lactations advanced, with the P3 ewes having higher protein and casein contents in their milk as compared to the P2 (p < 0.05) and P1 ewes (p < 0.01). The P3 ewes also had a higher fat content in their milk than P2 (p < 0.05), which in turn had a higher milk fat content than P1 (p < 0.05). A combined effect of parity month of lactation was observed for milk protein (p 0.08) and fat content (p 0.09), since differences among parities tended to
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A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
Table 1 Least squares means of milk yield and composition of Comisana ewes in parities 1 (P1), 2 (P2) and 3 (P3) Parities
Milk yield (kg/day)
Protein content (%)
Casein content (%)
Fat content (%)
Lactose content (%)
SE
Level of significance PT e
Mf
PT M
0.02
NS
**
NS
6.27a 5.77b 5.80b 5.99ab 6.15a 5.99a
0.03
*
**
NS
4.74ab 4.07c 4.21c 4.24c 4.68a 4.38b
4.92a 4.27b 4.41b 4.48b 4.77a 4.57a
0.02
*
**
NS
5.22c 4.68d 4.38d 5.34c 6.06b 5.13c
5.82b 5.06c 5.09c 5.81b 6.23d 5.60b
6.43ad 5.70be 5.48e 6.01ae 6.66a 6.05a
0.07
*
**
NS
5.50a 5.47a 5.55a 5.46a 5.38b 5.47a
5.35bc 5.37bc 5.40bc 5.29bc 5.39c 5.36ab
5.37bd 5.35cd 5.37bd 5.32bd 5.28d 5.33b
0.01
*
**
NS
P1
P2
P3
January February March April May Mean
1.25b 1.54a 1.48a 1.47a 1.31b 1.41
1.31b 1.54a 1.52a 1.51a 1.32b 1.44
1.32b 1.59a 1.57a 1.56a 1.39b 1.48
January February March April May Mean
5.72bc 5.43cd 5.31d 5.57c 5.92b 5.59b
5.90b 5.33d 5.48c 5.65c 6.03ab 5.68b
January February March April May Mean
4.68b 4.11c 4.07c 4.19c 4.59ab 4.32b
January February March April May Mean January February March April May Mean
a,b,c,d
Means within the same rows or columns followed by different letters differ signi®cantly at p < 0.05. Parity. f Month. * p < 0.05; ** p < 0.01. e
decrease for both parameters as the lactation progressed. 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
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
also result in an increased synthesis of milk constituents. 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 galactopoiesis 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 (p 0.07) 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
103
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 increasing 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 lactation were opposite to those observed for protein, casein and fat contents, with the lowest lactose contents 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 P1 pH
SE P2
P3
Level of significance Parity
Month
Parity Month
January February March April May Mean
6.70 6.67 6.71 6.61 6.60 6.65
6.71 6.66 6.67 6.61 6.58 6.64
6.68 6.66 6.65 6.59 6.58 6.63
0.02
NS
NS
NS
January February March April May Mean
22.0a 20.6bc 18.9c 20.5bc 19.4c 20.3
21.1a 20.5ab 18.0c 19.7abc 18.0c 19.5
20.4a 19.0ab 17.5c 18.7ab 17.4c 18.6
0.3
NS
**
NS
Rate of clot formation (min) January February March April May Mean
4.7a 4.6a 4.0ac 4.4a 3.9ac 4.3a
4.7a 4.4a 3.7bc 4.1abc 3.6bc 4.1ab
4.3a 4.0a 3.3b 3.9b 3.2b 3.7b
0.09
*
**
NS
39.6c 46.8bc 49.9b 45.4bc 46.7bc 45.7b
41.5c 46.3bc 52.1b 46.0bc 51.4abc 47.5ab
45.4c 51.0ac 58.0ab 49.1ac 55.2a 51.7a
1.23
*
*
NS
Clotting time (min)
Clot firmness (mm)
a,b,c *
January February March April May Mean
Means within the same rows or columns followed by different letters differ signi®cantly at p < 0.05. p < 0.05; ** p < 0.01.
104
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
The renneting behaviour of milk substantially re¯ected the differences in milk casein and fat contents 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 parameters. This could be expected, because clot forma-
suffering from mastitis, as lactose is the main osmotically 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.
Table 3 Somatic cell and bacteria count in milk of Comisana ewes in parities 1 (P1), 2 (P2) and 3 (P3)d Parities
Somatic cell count
Mesophile count
Psychrotroph count
Total coliform count
Faecal coliform count
a,b,c
SE
P1
P2
P3
January February March April May Mean
4.98 4.87 4.98 4.99 5.18 5.00
5.07 5.08 4.89 4.90 5.01 4.99
5.01 4.96 4.91 4.87 5.00 4.95
January February March April May Mean
4.61b 4.51b 5.80a 5.46a 6.07a 5.29a
4.51bc 4.53bc 5.48ad 5.06ab 5.41d 4.99b
January February March April May Mean
4.34c 5.07a 5.48a 5.51a 5.46a 5.17a
January February March April May Mean January February March April May Mean
Level of significance Parity
Month
Parity Month
0.07
NS
NS
NS
4.58bc 4.41bc 5.49a 4.47b 5.21ad 4.83b
0.06
*
**
NS
4.33c 5.07a 5.11a 5.51ab 5.07ab 5.01ab
4.49bc 4.78bc 5.11bc 5.02b 4.77b 4.83b
0.05
*
**
NS
3.34c 3.35c 3.97ac 4.01a 4.42a 3.81a
3.19c 3.53bc 3.70bc 3.66abc 3.80b 3.57ab
3.15bc 3.39bc 3.55bc 3.37b 3.54b 3.40b
0.08
*
*
NS
0.21 1.01 1.05 1.79 2.38 1.28
0.52 0.88 0.80 1.33 2.11 1.12
0.40 0.84 0.48 1.06 1.94 0.94
0.12
NS
NS
NS
Means within the same rows or columns followed by different letters differ signi®cantly at p < 0.05. Data are least square means of log 10 of smoatic cells and cfu x ml of milk. * p < 0.05; ** p < 0.01. d
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
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 321 103 somatic cell/ml of milk in parities 3 and 1, respectively. Irrespective of parity, the highest values were recorded in May and the lowest in February (671 and 203 103 somatic cell/ml of milk on average, respectively). SCC were higher than those found by Morgante et al. (1996) in Comisana ewes. Conversely, the analysis of variance of the logarithmic 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 298 103 cfu/ml, respectively) as well as higher concentrations of psychrotrophs and total coliforms in their milk with respect to the P3 ewes (377 vs. 163 103 cfu/ml and 19 vs. 7 103 cfu/ml, respectively). Although the P1 ewes had a higher microorganism 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 postsecretory 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,
105
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) Parities P1
P2
P3
January February March April May
0 1 1 1 1
0 0 1 1 1
0 0 0 1 1
Bacteriologically positive milk samples January February March April May
2 2 5 3 3
1 1 3 4 2
1 2 1 3 2
Cases of subclinical mastitis
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 Other streptococci 3 2 2
perhaps due to a less advanced development of the immune system or to greater susceptibility to environmental 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 (n 15) than in the P2 (n 11) and P3 groups (n 9). On the whole, bacteria or combinations of bacteria were isolated from 35 milk samples and environmental bacteria were largely predominant. Streptococci (especially Enterococcus spp.) constituted the majority of bacteria isolated, since they were isolated from about 70% of the 35 bacteriologically
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positive milk samples. E. coli, coagulase-negative (CN)-staphylococci and Pseudomonas spp. were isolated from 14, 10 and 6 milk samples at 40, 29, 17%, respectively. S. aureus and P. aeruginosa were detected in four and three milk samples, respectively, whereas S. agalactiae was 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 renneting ability of milk improved as the number of lactations 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 progressively 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 endocrine 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. Noninfectious 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 for Escherichia coli in 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, milk and involution secretion, with particular reference to ultrastructure and lymphocyte sub-popolations. J. Dairy Res. 48, 225±237.
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107 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 Georgo®li, Stamperia Editoriale Parenti, Firenze, Italy, pp. 1±18.
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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 adrenocorticotropin 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. Wohlt, J.E., Kleyn, D.H., Vandernoot, G.W., Selfridge, D.J., Novotney, C.A., 1981. Effect of stage of lactation, age of ewe, sibling status and sex of lamb on gross and minor constituents of Dorset ewe milk. J. Dairy Sci. 64, 2175±2184. Zannoni, M., Annibaldi, S., 1981. Standardisation of the renneting ability of milk by Formagraph. Scienza e Tecnica LattieroCasearia 32, 9±94.
Effect of parity on milk yield, composition, somatic cell count, renneting parameters and bacteria counts of Comisana ewes A. Sevia,*, L. Taibib, M. Albenzioa, A. Muscioa, G. Annicchiaricob a
Istituto di Produzioni e Preparazioni Alimentari, FacoltaÁ di Agraria di Foggia, via Napoli, 25, 71100 Foggia, Italy b Istituto 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 (50 3 days after lambing), to May. Samples with more than 3.5 105 somatic 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 *
Corresponding author. Tel.: 39-881-714544; fax. 39-881-740211. E-mail address: [email protected] (A. Sevi)
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
0921-4488/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 9 9 ) 0 0 1 3 3 - 9
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A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
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, management 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 advancement 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 50 3 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 space 0.45 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 experiment, body weights were 51.19 2.62, 53.76 2.11 and 56.37 2.41 kg (means SE), respectively, in the P1, P2 and P3 groups. Ewes were fed a diet composed of pelletted concentrate, 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 2.36 0.28 kg per ewe during the study period. 2.2. Sampling and analyses of milk Ewes were milked twice daily using pipeline milking 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 containers 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); enumeration 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 44.5 0.58C for 24 h. At the beginning of the experiment and on the day before each milk sampling, all the ewes were examined carefully to assess the absence of signs of clinical mastitis, such as fever, pain or gland swelling. A small quantity of milk was checked visually for signs of mastitis. As proposed by Fruganti et al. (1985), an aliquot of 0.01 ml from all milk samples containing more than 3.5 105 somatic cells/ml was cultured for
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
mastitis-related pathogens. Presumptive Escherichia coli was detected after a 24 h incubation on VRBA with MUG (4-methylumbelliferil-B-D-glucuronide) (Hartman, 1989) at 44.5 0.58C. 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 (Biomerieux, F-69280 Marcy l'Etoile, France). Streptococci 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 determined using Pseudomonas Selective Agar (Oxoid); Pseudomonas aeruginosa was 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 bacteriologically 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 abnormalities and whose milk was apparently normal, with somatic cell counts greater than 3.5 105, 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 fortnightly anteprandium after the machine milking in the morning. At the end of May (196 3 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
101
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 distributions 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 Y parity ewe (parity) month time of sampling parity month parity month day of sampling error, where Y is 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 at p < 0.05, unless otherwise stated. 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 (Dell'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. In contrast, parity strongly affected the quality of ewes milk. Milk protein and casein contents increased (p < 0.05) as the number of lactations advanced, with the P3 ewes having higher protein and casein contents in their milk as compared to the P2 (p < 0.05) and P1 ewes (p < 0.01). The P3 ewes also had a higher fat content in their milk than P2 (p < 0.05), which in turn had a higher milk fat content than P1 (p < 0.05). A combined effect of parity month of lactation was observed for milk protein (p 0.08) and fat content (p 0.09), since differences among parities tended to
102
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
Table 1 Least squares means of milk yield and composition of Comisana ewes in parities 1 (P1), 2 (P2) and 3 (P3) Parities
Milk yield (kg/day)
Protein content (%)
Casein content (%)
Fat content (%)
Lactose content (%)
SE
Level of significance PT e
Mf
PT M
0.02
NS
**
NS
6.27a 5.77b 5.80b 5.99ab 6.15a 5.99a
0.03
*
**
NS
4.74ab 4.07c 4.21c 4.24c 4.68a 4.38b
4.92a 4.27b 4.41b 4.48b 4.77a 4.57a
0.02
*
**
NS
5.22c 4.68d 4.38d 5.34c 6.06b 5.13c
5.82b 5.06c 5.09c 5.81b 6.23d 5.60b
6.43ad 5.70be 5.48e 6.01ae 6.66a 6.05a
0.07
*
**
NS
5.50a 5.47a 5.55a 5.46a 5.38b 5.47a
5.35bc 5.37bc 5.40bc 5.29bc 5.39c 5.36ab
5.37bd 5.35cd 5.37bd 5.32bd 5.28d 5.33b
0.01
*
**
NS
P1
P2
P3
January February March April May Mean
1.25b 1.54a 1.48a 1.47a 1.31b 1.41
1.31b 1.54a 1.52a 1.51a 1.32b 1.44
1.32b 1.59a 1.57a 1.56a 1.39b 1.48
January February March April May Mean
5.72bc 5.43cd 5.31d 5.57c 5.92b 5.59b
5.90b 5.33d 5.48c 5.65c 6.03ab 5.68b
January February March April May Mean
4.68b 4.11c 4.07c 4.19c 4.59ab 4.32b
January February March April May Mean January February March April May Mean
a,b,c,d
Means within the same rows or columns followed by different letters differ signi®cantly at p < 0.05. Parity. f Month. * p < 0.05; ** p < 0.01. e
decrease for both parameters as the lactation progressed. 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
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
also result in an increased synthesis of milk constituents. 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 galactopoiesis 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 (p 0.07) 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
103
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 increasing 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 lactation were opposite to those observed for protein, casein and fat contents, with the lowest lactose contents 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 P1 pH
SE P2
P3
Level of significance Parity
Month
Parity Month
January February March April May Mean
6.70 6.67 6.71 6.61 6.60 6.65
6.71 6.66 6.67 6.61 6.58 6.64
6.68 6.66 6.65 6.59 6.58 6.63
0.02
NS
NS
NS
January February March April May Mean
22.0a 20.6bc 18.9c 20.5bc 19.4c 20.3
21.1a 20.5ab 18.0c 19.7abc 18.0c 19.5
20.4a 19.0ab 17.5c 18.7ab 17.4c 18.6
0.3
NS
**
NS
Rate of clot formation (min) January February March April May Mean
4.7a 4.6a 4.0ac 4.4a 3.9ac 4.3a
4.7a 4.4a 3.7bc 4.1abc 3.6bc 4.1ab
4.3a 4.0a 3.3b 3.9b 3.2b 3.7b
0.09
*
**
NS
39.6c 46.8bc 49.9b 45.4bc 46.7bc 45.7b
41.5c 46.3bc 52.1b 46.0bc 51.4abc 47.5ab
45.4c 51.0ac 58.0ab 49.1ac 55.2a 51.7a
1.23
*
*
NS
Clotting time (min)
Clot firmness (mm)
a,b,c *
January February March April May Mean
Means within the same rows or columns followed by different letters differ signi®cantly at p < 0.05. p < 0.05; ** p < 0.01.
104
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
The renneting behaviour of milk substantially re¯ected the differences in milk casein and fat contents 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 parameters. This could be expected, because clot forma-
suffering from mastitis, as lactose is the main osmotically 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.
Table 3 Somatic cell and bacteria count in milk of Comisana ewes in parities 1 (P1), 2 (P2) and 3 (P3)d Parities
Somatic cell count
Mesophile count
Psychrotroph count
Total coliform count
Faecal coliform count
a,b,c
SE
P1
P2
P3
January February March April May Mean
4.98 4.87 4.98 4.99 5.18 5.00
5.07 5.08 4.89 4.90 5.01 4.99
5.01 4.96 4.91 4.87 5.00 4.95
January February March April May Mean
4.61b 4.51b 5.80a 5.46a 6.07a 5.29a
4.51bc 4.53bc 5.48ad 5.06ab 5.41d 4.99b
January February March April May Mean
4.34c 5.07a 5.48a 5.51a 5.46a 5.17a
January February March April May Mean January February March April May Mean
Level of significance Parity
Month
Parity Month
0.07
NS
NS
NS
4.58bc 4.41bc 5.49a 4.47b 5.21ad 4.83b
0.06
*
**
NS
4.33c 5.07a 5.11a 5.51ab 5.07ab 5.01ab
4.49bc 4.78bc 5.11bc 5.02b 4.77b 4.83b
0.05
*
**
NS
3.34c 3.35c 3.97ac 4.01a 4.42a 3.81a
3.19c 3.53bc 3.70bc 3.66abc 3.80b 3.57ab
3.15bc 3.39bc 3.55bc 3.37b 3.54b 3.40b
0.08
*
*
NS
0.21 1.01 1.05 1.79 2.38 1.28
0.52 0.88 0.80 1.33 2.11 1.12
0.40 0.84 0.48 1.06 1.94 0.94
0.12
NS
NS
NS
Means within the same rows or columns followed by different letters differ signi®cantly at p < 0.05. Data are least square means of log 10 of smoatic cells and cfu x ml of milk. * p < 0.05; ** p < 0.01. d
A. Sevi et al. / Small Ruminant Research 37 (2000) 99±107
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 321 103 somatic cell/ml of milk in parities 3 and 1, respectively. Irrespective of parity, the highest values were recorded in May and the lowest in February (671 and 203 103 somatic cell/ml of milk on average, respectively). SCC were higher than those found by Morgante et al. (1996) in Comisana ewes. Conversely, the analysis of variance of the logarithmic 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 298 103 cfu/ml, respectively) as well as higher concentrations of psychrotrophs and total coliforms in their milk with respect to the P3 ewes (377 vs. 163 103 cfu/ml and 19 vs. 7 103 cfu/ml, respectively). Although the P1 ewes had a higher microorganism 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 postsecretory 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,
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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) Parities P1
P2
P3
January February March April May
0 1 1 1 1
0 0 1 1 1
0 0 0 1 1
Bacteriologically positive milk samples January February March April May
2 2 5 3 3
1 1 3 4 2
1 2 1 3 2
Cases of subclinical mastitis
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 Other streptococci 3 2 2
perhaps due to a less advanced development of the immune system or to greater susceptibility to environmental 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 (n 15) than in the P2 (n 11) and P3 groups (n 9). On the whole, bacteria or combinations of bacteria were isolated from 35 milk samples and environmental bacteria were largely predominant. Streptococci (especially Enterococcus spp.) constituted the majority of bacteria isolated, since they were isolated from about 70% of the 35 bacteriologically
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positive milk samples. E. coli, coagulase-negative (CN)-staphylococci and Pseudomonas spp. were isolated from 14, 10 and 6 milk samples at 40, 29, 17%, respectively. S. aureus and P. aeruginosa were detected in four and three milk samples, respectively, whereas S. agalactiae was 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 renneting ability of milk improved as the number of lactations 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 progressively 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 endocrine 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. Noninfectious 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
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