PhD Yannick Le Cozler
email: yannick.lecozler@agrocampus-ouest.fr

Yves Gallard

PhD; Frédéric Dessauge

Jean-Rémy Peccate

Jean-Marie M Trommenschlager

Luc Delaby

PhD Kristen Sejrsen
email: kr.sejrsen@agrsci.dk

J R Peccatte

Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter two (W2), in a 36 mo of age at first calving strategy

Keywords: feed restriction, winter, rearing, heifers, breed, longevity feed restriction, winter, rearing, heifers, breed, longevity

Two experiments were performed simultaneously to test the hypothesis that restricted growth patterns during winter two (W2) affect further performance of dairy heifers born during winter one (W1) first calving at 36 mo of age. A total of 158 dairy heifers born during three successive winters (1994 to 1997) were considered at two experimental herds (INRA, Le Pin au Haras and Mirecourt) located in two different parts of France. Breeds of animals were Holstein (Ho) and Normande (No) at le Pin, and Holstein (Ho) and Montbeliarde (Mo) at Mirecourt. At the beginning of W2 (348-365 d of age on average), animals were housed and subject to a restricted feeding strategy to show an average daily gain (ADG) of 600, 400 or 200 g/d at Le Pin au Haras, and 500, 200 and -200 g/d at Mirecourt, respectively, during W2. During pasture seasons, management and feeding were similar for all animals. As expected, body weight differed between treatments at the end of W2 (P < 0.05), but it was similar when first calving at 36 mo of age. Overall fertility in heifers and cows was not affected by feeding treatments. In both herds, performance during the first lactation was generally not affected by feeding treatments, with the exception of the 600 g/d group of heifers at Le Pin showing lower milk performance calculated on a 4% basis and a deteriorated milk composition (P < 0.05) when all lactations were considered. However, when calculated on a productive life day basis, they had the best milk production on a 305 basis (P < 0.05). Of the 158 animals that started the experiment, 41% were culled because of reproductive disorders (troubles of reproduction, n = 22 or empty, n = 45), probably because of the seasonal calving system. Other differences noted between herds were mainly due to breeds involved, with Mo cows having lower milk performance on average (-800 kg per lactation, P < 0.001) but a better longevity (4.5 vs. 2.4 lactations at culling, respectively) when compared with Ho animals. No cows exhibited also lower milk performance in comparison with Ho animals on a 305 d length basis (P < 0.001), but this was not counterbalanced by any improved longevity (3.0 and 2.5 lactations at culling, respectively). These results indicate that, in a 36 mo of age at first calving strategy, it is possible to voluntary reduce ADG during a short growth period without any major effects on performance at short and long terms.

Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 A.J. Heinrichs PhD Senior Scientist, Department of Dairy and Animal Science, Pennsylvania State University ajh@psu.edu One of tbhe world leaders specialist in dairy heifers

Introduction

Seasonal calving systems are generally based on grazing optimisation, and as a result, feeding strategy is closely connected to the seasons. In such systems, breeding is also generally seasonal, and first calving usually occurs at 24 or 36 mo of age. First calving at 36 mo of age strategy is not rare in European dairy systems based on seasonal feeding strategy and using either late-(e.g., Normande) or earlymaturing (e.g., Holstein) breeds. This strategy is aimed at reducing labour and feeding costs [START_REF] Hoch | Mécanismes et conséquences de la croissance compensatrice chez les ruminants[END_REF]. Indeed, as daily gain from birth to farrowing is lower in a 36 mo of age at 1 st calving strategy than in a 24 mo of age at 1 st calving strategy, diets with less concentrate and more roughage can be used in the former Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 3 system. However, these arguments could also be tackled by additional extra costs (12 months of supplementary maintenance more, delayed return to investment…).

In case of unfavourable summer for hay and/or silage production, fodder availability during following winter could be however limited, thus leading to feed deficits for the entire herd. To preserve milk production of adults, heifers may be faced to a severe feed restriction, and consequently, may have very low or even negative growth performance during this period. However, these animals generally exhibit catch-up growth performance while grazing in comparison with normal fed heifers during the same period (Le [START_REF] Cozler | A comparative study of three growth profiles during rearing in dairy heifers: effect of feeding intensity during two successive winters on performances and longevity[END_REF]. Moreover, such alternated low to high feeding allowance could be of interest [START_REF] Park | Role of ompensatory growth in lactation: a stair step nutrient regime modulates differentiation and lactation bovine mammary gland[END_REF][START_REF] Ford | Nutritionally directed compensatory growth enhances heifer development and lactation potential[END_REF] to elicit compensatory development and increase lactation potential. In general, the less average daily gain (ADG) during winter is, the highest the compensatory increase observed thereafter will be. In a 36 mo of age at 1 st calving system, another strategy in unfavourable conditions could be to preserve all dairy cows and some of the growing heifers (i.e., generally those close to be inseminated), but assert a severe feed restriction for the younger reared animals (i.e., those around 12 mo of age). To our knowledge, the impacts of such a severe feeding and rearing strategy on latter performance are not known. The present experiment aimed at studying the effect of different patterns of growth restriction during winter 2 (W2) of rearing on performance and longevity of different breeds of heifers born during winter 1 (W1), and first calving at 36 mo of age.

Material and methods

Two experiments were performed simultaneously at two different experimental herds, located either in the western (Experiment 1) or in the eastern (Experiment 2) parts of France. Herds used different feeding strategies and management, as well as different breeds. Common rearing strategy is summarized on Figure1. All animals included ion these experiments were born during three successive winters (1994 to 1997).

Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science,244245246247248249250251252253254 1).

Animals were reared according to procedures detailed by [START_REF] Cozler | A comparative study of three growth profiles during rearing in dairy heifers: effect of feeding intensity during two successive winters on performances and longevity[END_REF].

Briefly, all heifers were reared similarly from birth until the end of the first grazing period (S1). Thereafter (winter 2, W2), they were housed and fed indoors with a restricted feed allowance to achieve ADG of either 600 (n = 26, H), 400 (n = 25, M) or 200 (n = 27, L) g/d during this period. The 600 g/d growth group was considered to be close to the standard condition used in dairy herds when first calving occurred at 36 mo of age. Diets were mainly composed of corn silage, straw, rapeseed meal, urea and minerals (Table 2). At the end of W2, all heifers followed similar rearing until calving. Pregnant heifers joined the cow herd three weeks approximately before the expected date of calving, to facilitate their adaptation to new housing (e.g., stable, automatic doors) and feeding conditions.

After calving and during winter (November to the end of March), all cows were continuously housed indoors, with deep straw bedding and a grooved concrete-floor alley along the feeding table. They were offered free access to corn silage, and a complement (cereals, sugar pulp beat, and soybean meal) was added according to individual milk yield. The lactation ration was formulated to meet requirements based on individual production levels (INRA, 1988). During grazing seasons (April to November), all cows grazed pasture on a rotational management system [START_REF] Hoden | Simplified rotational grazing management of dairy cows: effects of rates of stocking and concentrate[END_REF]. When necessary, they received corn or grass silage with a complement to maintain a normal milk production. were used (Table 1). Animals were reared similarly to those in experiment 1, except some of the rearing procedures that were adapted due to feedstuff availability.

Animals were housed and fed indoors at the end of the first grazing season (S1).

Three feeding levels were used to achieve ADG of either 500 (n = 27, H), 200 (n = 27, M) or -200 (n = 26, L) g/d during winter 2 (W2). Several sources of roughage and concentrates were available and used (Table 2), to achieve experimental daily gain goals. Similarly to the 600 g/d ADG during W2 group of animals in experiment 1, the 500 g/d group was considered to be close to standard feeding used in dairy herds where first calving occurred at 36 mo of age in this experiment (Troccon et al., 1994).

Experimental diets were mainly composed of soybean, cereals (wheat or barley), urea and roughage, primarily straw, grass and alfalfa hay and /or grass silage.

Minerals were also added (Table 2). At the end of W2, all heifers followed similar treatments until calving: they were grazing during summer seasons and fed mainly with roughage (straw, grass silage, hay) and wheat or barley during winter 3 (W3).

During grazing seasons (S1, S2 and S3; April to the end of November), they rotationally grazed a perennial ryegrass sward. As for experiment 1, roughage were offered during summer when necessary.

Pregnant heifers joined the cow herd two weeks on average before the expected date of calving. Contrary to Experiment 1, no special facilities were used for calving.

After calving and during winter (November to the end of March), all cows were continuously housed indoors, with deep straw bedding and a grooved concrete-floor alley along the feeding table. As for growing heifers, ingredients could vary according to feedstuff available, providing that milking production was preserved. Cows were generally offered diets based on corn and/or grass silage, hay and/or straw, and a complement (cereals, sugar pulp beat, and/or soybean meal), together with minerals, was added. The ration was adapted according to individual milk yield, as the lactation ration was formulated to meet requirements based on individual production levels (INRA, 1988). During grazing season (April to November), all cows grazed pasture on a rotational management system. When necessary, they received hay, corn or grass silage with a complement to maintain a normal milk production.

Calving started at the beginning of October and ended in February, but around 90 % cows calved before the end of December. Reproductive period started in December and ended in mid-June for both heifers and cows.

Procedures for oestrus synchronisation in the 2 experiments

In both experiments, all heifers were inseminated after oestrus synchronisation during winter 3 (W3) of rearing, so that calving should occur at around three years of age. Cows were not served during the first 63 d (experiment 1) or 45 d (experiment 2) following calving. For heifers and cows, oestrus was synchronised using a progestin ear implant (norgestomet) in conjunction with an intramuscular estrogen injection (Crestar®, Intervet, Angers, France), without any consideration of ovarian activity. A second synchronisation was eventually performed on similar cows three weeks later. When delay from calving was shorter than 63 days at the second synchronisation (experiment 1), no treatment was applied to cows and these animals were served when detected in heat (20 to 25 % of all calving cows each year). After nine days of treatment, the ear implant was removed. Heifers and cows generally exhibited signs of oestrus within 24-96 h and then were inseminated. A test of the plasma progesterone level was performed 21 d after insemination to check for the onset of gestation. For animals that did not show any oestrus according to this test, an echographic inspection was performed to confirm pregnancy at 42 d after insemination on average, only in experiment 1. Cows and heifers coming back into heat were inseminated until the end of the reproductive season (June).

Registration and measurements

Individual BW was measured at birth and, on average, every three weeks thereafter.

Since the duration of winter differed between years of birth and between calendar years (from 126 to 168 d, Table 3), BW was corrected (by extrapolation) according to age, and daily or monthly weight gains were then calculated. During winter seasons (W2 and W3), daily feed allowances and refusals were recorded on a per-pen basis.

Informations regarding heifers and cows at calving were scored according to their degree of difficulty (1= no difficulty; 2= minor difficulty; 3= major difficulty or caesarean section). Newborn calves were identified and weighed within 24 h after birth. Reasons for culling heifers and cows were classified as (1) reproduction (failure to conceive or not pregnant), (2) calving difficulties, (3) disease, (4) accident, (5) mastitis, (6) inadequate performance (less than 20 kg milk/d during the first four weeks of lactation), (7) replacement and age, and (8) miscellaneous.

Calculations and statistical analysis

Analyses were performed on a herd basis. For cows that were culled within 100 d after calving, lactation number at culling was then (n+1), but their lactating performances were included in the data only until the end of parity n. This means that days in milk (DIM) > 100 counted for the current and previous lactations and DIM < 100 counted only toward all previous lactations. Milking production and performances were calculated on a 305-d basis for all animals as: 305 Milk (kg) = total milk produced (kg) x (385 / (lactation length (d) + 80)). This method is used in the French evaluation system as a reference and had been discussed by [START_REF] Barbat | Comparaison de 2 méthodes de précorrection des lactations courtes pour l'évaluation des reproducteurs laitiers[END_REF]. An identical procedure was used for calculating fat and protein yield. Milk yield also was Livestock Science,244245246247248249250251252253254, DOI : 10.1016/j.livsci.2010.11.018 8 calculated on a 4% fat content basis, using the following formula: 4% Milk (kg) = total milk produced (kg) x (0.4 + 0.015 x fat concentration (g/kg)). Growth performance data were analysed on a per-animal basis, using statistical models that included the effect of treatment during winter 2 (H, M, and L), year of birth (1994/95, 1995/96 and 1996/97), breed (No, Mo and Ho) and their possible interactions were kept when significant.

Values of least squares means (breed or feeding treatment) were compared using pdiff tests in GLM options of SAS® statistics software (version 8.0; SAS Institute, Inc., Cary, NC). Other co-variables were tested and kept in the model when necessary.

Analysis performed on milk and longevity data included year of lactation. For data concerning reproduction, health and culling, the CHISQ option of the FREQ procedure and the LOGISTIC procedure were used (SAS software). Survival analysis based on the number of cows was studied with the LIFEREG procedure. For all tests, level of significance was 0.05. P. Values less than 0.10 are discussed as trends. 3).

Results

Age

Body growth and development

Observed ADG did not exactly match those expected (Table 3). The highest differences were noted for heifers born during winter 1995/1996 for treatment H (+29% on average in the two herds) and treatment M (especially at Mirecourt where Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. ( 2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science,244245246247248249250251252253254, DOI : 10.1016/j.livsci.2010.11.018

9

ADG was 98% greater than that expected). Moreover, heifers born during winter 1996/97 had generally a lower ADG than expected for treatment L and M treatments at Mirecourt. In all cases, observed ADG was highly variable among animals (i.e., coefficient of variation (CV) ranged from 77 to 176 g/d, Table 3).

As expected, animal growth was altered during W2 in both herds, with L heifers showing a stronger depression in their growth profile (Figure 1). At the end of the experimental period (480 d on average), the L, M and H heifers weighed 323, 339 and 369 kg, respectively, at Le Pin (P < 0.05) and 297, 346 and 369 kg at Mirecourt, respectively (P < 0.05). An increase in growth performance was observed when animals turned to pasture at the end of W2. The most severely restricted animals during W2 had the highest BW gains during this period (P < 0.01). However, BW at 600 d of age still ranged from L < M < H groups (i.e., 420, 437 and 448 kg at Le Pin, and 387, 424 and 436 kg at Mirecourt for L, M and H heifers, respectively; P < 0.05).

On the opposite, BW at calving was not different between treatments or breeds at Le Pin, whereas H heifers were heavier than L heifers at Mirecourt (P < 0.05). In this latter herd, BW of M animals were intermediary to those observed in L or H heifers.

At this stage, no difference in BW was noted between breeds, whatever the herd considered.

Animal survival, health and culling

Thirty-one heifers were culled during rearing, mainly because they did not become pregnant before the end of the reproductive period. The percentage of culled animals before first calving was not affected by treatment during W2, and did not differ between breeds. Main reason for culling was reproductive disorders but no significant difference was noted between breeds or treatments. Mirecourt (4.0, 3.2 and 3.2 for L, M and H cows, respectively) or at Le Pin (2.8, 3.0 and 2.4, respectively). Whereas age at culling was lower for H cows than M cows at Le Pin (1675 and 2034 d, respectively, P < 0.05), there was no difference in the same trait at Mirecourt. However, in this latter herd, Mo cows had a higher parity (4.5 vs. 2.4) and were older (2363 vs. 1749 d) at culling than Ho cows (P < 0.01), whereas there were no breed-associated differences in the same traits at Le Pin.

Reproductive and productive performances

Overall fertility in heifers was not affected by any of the treatments. Similarly, fertility rates at 1 st AI did not differ (averaging 48% at Le Pin, and 50% at Mirecourt, data not shown). There were no breed-associated differences in the percentage of successful gestations after one AI and of calving difficulties.

The duration of lactation did not differ between treatments or breeds at Mirecourt (Table 4). However, it was significantly reduced for H heifers at Le Pin when considering overall lactations. During lactation one, feeding treatment did not affect productive performances, with the exception of fat production that was the lowest (P < 0.05) in H cows at le Pin only (-18 kg and -36 kg, when compared to L and M treatment, respectively). Most performances were also affected by breeds, with Ho cows having better results than other breeds in the two experimental herds.

Especially, Ho primiparous cows produced 1160 kg more milk on a 305 days basis than primiparous No cows at Le Pin (P < 0.01), and the difference was + 462 kg in favour of the formers compared to Mo cows at Mirecourt (P < 0.05). The same conclusions can be drawn for milk yield calculated on a 4 % basis at Le Pin (P < 0.05), but breed-associated difference on this trait was greater than the previous one (milk production on a 305 d basis) at Mirecourt (+ 914 kg in favour of Ho cows).

Similarly, overall mean milk yield per lactation and per cow was not affected by the feeding treatments ( 11 breed-associated differences were observed on all parameters (P < 0.05), except lactation length. The differences were in favour to Ho cows, and were generally more marked at Le Pin than at Mirecourt. This resulted especially in a greater milk yield calculated on a 4% or a 305 d basis for Ho animals than for No and Mo ones (P < 0.01). As far as milk composition is concerned when all lactations were pooled, a significant effect of treatments during W2 was observed at Le Pin only. In this herd, H cows produced less fat and protein than L and M cows that produced milk with a similar biochemical composition. Protein and fat productions were also higher for Ho cows in comparison of No cows (Le Pin, P < 0.01) or Mo ones (Mirecourt, P < 0.01).

In addition, lactation rank had a positive impact on all parameters (P < 0.05), whatever the herd considered.

When analyses were done on productive day of life basis (Table 5), treatment had a slight effect on milk production calculated on 305 days at Le Pin only, with H cows showing the best results compared to L and M ones (20.2 vs. 17.8 kg on average, P < 0.05). Breed affected all performance calculated per day of productive life, with the exception of milk on a 305 d basis at Mirecourt. Protein and fat in the milk were greater for Ho cows than No and Mo animals (P < 0.05). In both herds, lactation rank at culling had a positive effect (P < 0.05) on these parameters, and year of birth also affected the results at le Pin mainly (Table 5).

Regardless of feeding treatment during W2, regression analysis however revealed that a 30-days difference in BW at first calving resulted in a 250 kg milk decrease during first lactation (P < 0.01; data not shown). No significant relationships were noted between age or BW at calving and milk yield production for higher parities.

Discussion

First calving at 36 mo of age allows the use of less concentrate and/or forage, which usually leads to a reduced daily gain during housing winter periods. The compensatory increase in growth obtained during grazing seasons needs therefore 12 to be optimized to compensate for extra-additional costs such as more heifers in the farm, 12 additional months of maintenance, delay in the return-on-investment and a slow genetic progress (Le [START_REF] Cozler | A comparative study of three growth profiles during rearing in dairy heifers: effect of feeding intensity during two successive winters on performances and longevity[END_REF]. One of the main interests of present study then consists on the comparison of the immediate and long-term responses to strategies to reduce ADG during a short period. Another interest of present investigation was the possibility to perform such trials in two herds geographically localized in two different areas of France, and with differences in both feedstuff and breeds of animals. Climate-related difference in the duration of winter housing between the two herds also resulted in differences on treatment durations that might have affected the final results. As reported in other experiments and previously discussed (Le [START_REF] Cozler | Effect of feeding regime, growth intensity and age at first insemination on performances and longevity of Holstein heifers born during autumn[END_REF][START_REF] Cozler | A comparative study of three growth profiles during rearing in dairy heifers: effect of feeding intensity during two successive winters on performances and longevity[END_REF], year of birth also affected overall performance per day of productive life, likely because the chemical composition of diets estimated from the ingredients slightly varied from year to year. This was especially the case when feeding regimes were adapted to the feedstuff availability, as it happened at Mirecourt.

Elicited growth patterns

At 6 mo of age, mean BW of heifers corresponded to 24 to 29 % of mature BW (i.e., 650 to 750 kg depending of the breeds) and was attained following an ADG of 817, 766 and 772 g/d for Ho, Mo and No heifers, respectively. These values were close to those recommended by Troccon et al. (1994). Many studies have investigated possible effects of ADG prior to puberty on subsequent performances (Le [START_REF] Cozler | Rearing strategy and optimizing first calving targets in dairy heifers: a review[END_REF] for a review). They generally recommended a high growth rate during the first 6 mo of age to ensure optimal body development, even when animals first calved at 36 mo of age. Thereafter, a subsequent decrease in growth rate is generally recommended to prevent excessive fatness and bad reproductive and productive performances (Troccon et al., 1994). This was realized in the current study with ADG growth which depends on the stage when it occurs [START_REF] Ford | Nutritionally directed compensatory growth enhances heifer development and lactation potential[END_REF][START_REF] Davis Rincker | Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age[END_REF]. Although compensatory growth has been proposed as an interesting tool to manage animals in a 24 mo of age at first calving, the interest of such a practice in a 36 mo of age at first calving strategy remains to be studied and proved.

Finally, a positive relationship between BW at calving and milk production at first lactation was observed in the present study, regardless of experimental treatments.

Similar relationships have been already reported by [START_REF] Lin | Multitrait estimation of relationships of first lactation yield to body weight changes in Holstein heifers[END_REF] and Waldo et al. (1998). Troccon and colleagues (1994) suggest that these relationships exist whatever the growth profiles are before lactation when first calving occurs at 36 mo of age. This is however opposite to what was stated in a 24 mo of age at first calving strategy [START_REF] Park | Role of ompensatory growth in lactation: a stair step nutrient regime modulates differentiation and lactation bovine mammary gland[END_REF][START_REF] Ford | Nutritionally directed compensatory growth enhances heifer development and lactation potential[END_REF], in which alternating low and high feeding rates during rearing have a positive impact on subsequent performance.

Culling, longevity and long-term performance

In the current study, long-term effects of a too slow growth rate during winter two of rearing were observed only in one herd (Le Pin). From a long-term point of vue, levels of production slightly differed between the two herds, with Ho animals from Le Pin usually having higher levels of production than those from Mirecourt. In complement, others studies [START_REF] Gaynor | Effects of prepubertal growth rate and diet on lipid metabolism in lactating Holstein cows[END_REF]Capuco et al., 1998) also indicated possible long-term effects of feed composition during rearing on later adult performance. This suggests that any recommendations for rearing strategies (nutrition, reproduction …) have to be adapted to the levels of animal production in the different herds.

At Le Pin, L and M treatments during W2 had a positive effect on longevity since the number of culling was lower when compared to H treatment. The 42 % of culling observed in this herd is closed to that previously observed, with reproductive disorder being the main reason for culling [START_REF] Cozler | A comparative study of three growth profiles during rearing in dairy heifers: effect of feeding intensity during two successive winters on performances and longevity[END_REF]. This was probably a When all lactations were considered, the L and M winter-feeding level had also positive effects on milk protein and fat contents as well as on 4% milk production per lactation, when compared to H winter feeding level at Le Pin only. However, if the possible effects of regimes during lactation on milk quality are well-known [START_REF] Hurtaud | Winter feeding systems and dairy cow breed have an impact on milk composition and flavour of two Protected Designation of Origin French cheeses[END_REF], such long-term effects of rearing treatments already observed [START_REF] Cozler | A comparative study of three growth profiles during rearing in dairy heifers: effect of feeding intensity during two successive winters on performances and longevity[END_REF] are still surprising but need further investigations. However, no more difference was noted between treatments when composition of milk and 4% milk production were calculated per day on productive life. Furthermore, L and M treatments led to a slight detrimental effect on 305 d-milk production per day of productive life. These results clearly show that the possible benefits or detrimental effects of a given rearing strategy during winter two should be different from first lactation and later ones. It points out the necessity of studies following animals during more than one lactation.

Lactation length also differed between treatments at Le Pin but not at Mirecourt. This probably resulted of herd policy, as discussed by [START_REF] Cozler | A comparative study of three growth profiles during rearing in dairy heifers: effect of feeding intensity during two successive winters on performances and longevity[END_REF]. Indeed, herd policy at Le Pin during the 1990s encouraged having high-performance animals (Holstein and Normande animals) and cows with inadequate lactating performances were often culled before 305 d of lactations. In opposition, at Mirecourt, herd policy was different and aimed at keeping Holstein with adequate performances (as for Le Pin), but the level of requested performances for Montbeliarde was in agreement with this breed, i.e., lower than for Holstein heifers.

Conclusion

Present results indicated that in a dairy system where first calving occurs at 36 mo of age, it is possible to reduce feed allowance of heifers aged around one year of age, without any major deleterious effects on subsequent performance. In a similar study Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. ( 2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 17 performed on beef heifers, [START_REF] Dozias | Influence du profil de croissance des génisses des troupeaux allaitants sur leurs performances ultérieures[END_REF] concluded similarly. In case of unfavourable weather conditions leading to limited fodder stock, a feed restriction occurring during a short period of rearing could be encouraged regardless of breed, providing that restriction was not too severe (i.e., during one and not two successive winters). During subsequent lactations, productive performances are lower in case of severe restriction, but because of a positive impact on culling rate, performances calculated on a day of productive life basis do not differ.

The positive effect on longevity questioned on the possible interest of sequential compensatory growth program, not only in an early age at first calving strategy, but also for animals first calving at 36 mo of age. In addition, studies on the impact (or not) of various growth profiles from a dynamic point of view are probably of interest, as suggested by [START_REF] Meyer | Random regression analyses using B-splines to model growth of Australian Angus cattle[END_REF] and need further development in the future. Heifers from different breeds first calved at 36 mo of age and during winter 2, heifers received experimental treatment in order to achieve different daily gains (600, 500, 400, 200 or -200 g/d) during this period. Ho: Holstein; No: Normande; Mo: Montbeliarde. High, medium and Low corresponded to expected ADG of 200, 400 and 600 g/g at Le Pin, and -200, +200 and +400 g/g at Mirecourt respectively. UFL: Unité Fourragère Lait (1 UFL = 7.115 MJ Net Energy); PDIN: Protein truly Digested in the small Intestine limited by Nitrogen supply; PDIE: Protein truly Digested in the small Intestine limited by Energy supply [START_REF] Jarrige | Ruminant nutrition: Recommended allowances and feed tables[END_REF].

Year of birth

4

  . , DOI : 10.1016/j.livsci.2010.11.018 Experiment 1: Holstein and Normande heifers Experiment 1 was performed at the INRA experimental farm of Le Pin au Haras (48°44'N; 0°08'W; 140-248 m above sea level, Normandy, France). It included a total of 78 heifers born during three successive calving winter seasons (W1, December to February) of either Normande (No; n = 34) or Holstein (Ho; n = 44) breeds (Table

  Calving usually occurred in special individual boxes. Calving period started at the beginning of December and ended in March. Because of the seasonal calving Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 5 strategy, the reproductive period started in March and ended in mid-June for both heifers and cows. Experiment 2: Holstein and Montbeliarde heifers The 2 nd experiment was performed at the INRA experimental farm of Mirecourt (48°18'N; 06°08'W; 261-378 above sea level, Eastern part of France). It included a total of 80 heifers born during three successive calving winter season (October to February, W1). Both Holstein (Ho; n = 42) and Montbeliarde (Mo; n = 38) animals

  Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age.

  at 1 st artificial insemination (AI) was around 800 d, so that experimental animals first calved at the average age of 1081 d. Small differences were noted between the two herds. Experimental treatments started at 348 and ended at 461 d of age at Le Pin, and 365 and 510 d of age at Mirecourt, respectively. Winter housing during year three started at 700 and 730 d of age at Le Pin and Mirecourt locations, respectively, and ended at 840 and 870 d, respectively. The duration of experimental treatment was then slightly lower at Le Pin (114 to 131 d, 124 d on average) than at Mirecourt (151 to 167 d, 160 d on average, Table

Figure 2

 2 Figure2considers only animals with a productive life, i.e., calving at least once. Only

  Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018

  361 and sexual development, animals fed L, M or H levels of feed during winter after the 362 first pasture season had similar lactation performance during first lactation, and no 363 interaction between treatment and breed was noted. These results are in agreement 364 with data from Troccon et al. (1997) and Le Cozler et al. (2010) who show no impact 365 of altered growth performance during two successive winters on first lactation 366 performance. Similarly to what was observed here for animals first calving at 36 367 months of age, others have also noted no effect of a limited feed intake up to puberty 368 on performance during the first lactation (Hoffman et al., 2007; Zanton and Heinrichs, 369 2007) on animals first calving at 24 mo of age. 370 In addition, present results did not show any effects of increased growth during 371 spring on milk performance during first lactation, as already reported (Le Cozler et 372 al., 2010). Ford and Park (2001) observed improved results when growth was altered 373 (using stair-step compensatory nutritional programs). In the previous study, 374 experiment was based on animals first calving at 24 mo of age and differences in age 375 (270 d in the current study vs. 180 d in the latter mentioned experiment) could also 376 explain the discrepancies. This might be due to the degree of catch-up mammary Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 15

  Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 16 possible effect of seasonal calving strategy where reproduction has to be performed in limited times and non-pregnant animals are usually culled.

  Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 20 Troccon, J.L., Coulon, J.B., Lescourret, F., 1994. Carrière des vaches laitières : caractérisation de la phase d'élevage en relation avec les performances ultérieures. INRA Prod. Anim. 7, 359-368. Troccon, J.L., Muller, A., Peccatte, J.R., Fargetton, M., 1997. Effet du niveau d'alimentation énergétique de génisses laitières de races Holstein et Normande jusqu'à l'âge de 14 mois sur les performances durant les périodes d'élevage et de lactation. Ann. Zootech. 46, 27-41. Van Amburgh, M.E., Galton, D.M., Bauman, D.E., Everett, R.W., Fox, D.G., Chase, L.E., Erb, H.N., 1998. Effects of three prepubertal body growth rates on performance of Holstein heifers during first lactation. J. Dairy Sci. 81, 527-538. Zanton, G.I., Heinrichs, A.J., 2005. Meta-analysis to assess effect of prepubertal average daily gain of Holstein heifers on first-lactation production. J. Dairy Sci. 88, 3860-3867. Zanton, G.I., Heinrichs, A.J., 2007. The Effects of Controlled Feeding of a High-Forage or High-Concentrate Ration on Heifer Growth and First-Lactation Milk Production. J. Dairy Sci. 90, 3388-3396. Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 21

Figure 2 .

 2 Figure 2. Growth of dairy heifers originated from two herds (Le Pin and Mirecourt) with different breeds and first calving at 36 mo of age.

Figure 3 .

 3 Figure 3. Percentage of survival dairy heifers from two different herds (Le Pin and Mirecourt), according to days in production.

  document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018

  Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018

Table 4 )

 4 . However H cows produced less milk 4% than L and M animals (-538 kg on average, P < 0.05) at Le Pin only. At Le Pin and Mirecourt,

Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018

2. Effects of feeding treatments on reproduction and production at first 337 lactation 338

  , 568 and 600 g/d from birth to first insemination for Ho, Mo and No heifers,In the present study, BW at the first successful insemination were higher (503 to 535 326 kg corresponding to 70 to 82 % of mature BW according to breeds) than commonlyThe present results indicate that deleterious ADG (200 g/d or less) around one year 339 of age had no or only limited effects on fertility of heifers first calving at 36 mo of age. or after puberty had been already discussed[START_REF] Cozler | Rearing strategy and optimizing first calving targets in dairy heifers: a review[END_REF].Puberty attainment was not directly assessed in the present study, but it can be 352 hypothesised that in early maturing breed such as Holstein, most heifers with an 353 ADG of 740 g/d closed to the recommendation for optimal ADG[START_REF] Sejrsen | High body weight gain and reduced bovine mammary growth: Physiological basis and implications for milk yield potential[END_REF]

	324 351
		respectively.
	327 354
	328 355	observed (450-500 kg at 27 mo of age and 55 to 60 % of mature BW, Troccon et al., Zanton and Heinrichs, 2005) before 240 d of age showed a first oestrus before the
	329 356	1994). However, it should be reminded that recommendations in a 36 mo of age at start of the experimental treatments (Le Cozler et al., 2009). This could explain the
	330 357	first calving strategy are generally higher than those for animals calving at a younger lack of treatment-associated differences in first lactation milk yield. But all late
	331 358	age. In addition, at Mirecourt, information on BW is available during entire lifespan of maturing animals (Montbeliarde and Normande breeds) probably did not have a first
	332 359	the herd and it indicates an average mature BW of 707 and 704 kg for Ho and Mo oestrus before experimental treatments. Indeed, according to Drogoul et al. (2005), if
	333 360	cows older than 1500 d, respectively. Based on these data, BW at 1 st calving attainment of puberty is generally around 9 to 10 mo of age in Ho heifers, it is two
	334	corresponded to 90 and 91.6 % of mature BW in Mo and Ho cows respectively in the months later in No heifers and even more for Mo animals. So, whatever the breed
	335	present experiment, which is finally closed to recommendations for animals first
		calving at 36 mo of age (85 to 90 %; Troccon et al., 1994).
	340
	341	In a 24 mo of age at first calving strategy, Troccon and Petit (1989) and Heinrichs
	342	(1996) concluded that very low ADG during rearing alters heifer fertility. However,
	343	increasing feed allowance during a brief period before ovulation (i.e., a so called
	344	"flushing procedure") may restore fertility rates. However, Le Cozler et al. (2010)
	345	found only limited effect of previous feed restriction during two successive winters on
	346	heifers' fertility. Taken together, it would be concluded that a preserved growth rate
		during winter should probably be favourable to preserve fertility for heifers first

Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (

2011

). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock

Science, 137 (1-3), 244-254

. , DOI : 10.1016/j.livsci.2010.11.018 13 of 610325 336 4.347 calving at 24 mo of age, but might not be necessary when first calving occurs later. 348 Results of present study showed that altered growth profiles around puberty had only 349 limited impact on subsequent productive performance. The importance of feed Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 14 strategy before

Table 1 .

 1 Number of heifers per breed by treatment and birth year, according to herd

Table 2 .

 2 Feed ingredients and correspondent calculated composition

Table 3 .

 3 Duration of experimental treatment and average daily gain, according to year of birth and herd

Table 4 .

 4 Overall performances according to treatment and breed

Table 5 .

 5 Overall performances per day of productive life, age and parity at culling, according to treatment and breed Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age.Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 Growth of dairy heifers originated from two herds with different breeds and Percentage of survival dairy heifers from two different herds, according to Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018 Figure 1. General design of the experiment used in both herds* Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018

	22

Table 1 . Number of heifers per breed by treatment and birth year, according to herd

 1

	Year of birth /				Treatment (1)
	treatment	Herd	-200	+200	400	500	600	Total
			Ho No
	Ho: Holstein; Mo: Montbeliarde; No: Normande
	(1): treatment: heifers born during winter 1 received feeding levels to reach average daily gain of either -200,
	+200, +400, +500 or + 600 g/d during winter 2

Mo Ho No Mo Ho No Mo Ho No Mo Ho No Mo 1994/1995

		Le Pin	-	-		6	3	-	6	3	-	-	-	-	6	3	-	27
		Mirecourt	5	-	4	5	-	4	-	-	-	5	-	4	-	-	-	27
	1995/1996	Le Pin	-	-	-	4	4	-	5	3	-	-	-	-	4	4	-	24
		Mirecourt	3	-	5	4	-	5	-	-	-	5	-	4	-	-	-	26
	1996/1997	Le Pin	-	-	-	5	5	-	4	4	-	-	-	-	4	5	-	27
		Mirecourt	5	-	4	5	-	4	-	-	-	5	-	4	-	-	-	27
	Total		13	-	13 29 12	13	15	10	-	15	-	12 14 12	-	158

Table Comment

 Comment

citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018

Table 2 . Feed ingredients and correspondent calculated composition

 2

Table 3 . Duration of experimental treatment and average daily gain, according to year of birth and herd

 3 Comment citer ce document : Le Cozler, Y. (Auteur de correspondance), Gallard, Y., Dessauge, F., Peccatte, J.-R., Trommenschlager, J.-M., Delaby, L. (2011). Performance and longevity of dairy heifers born during winter 1 (W1) and reared according to three growth profiles during winter 2 (W2) in a strategy based on first calving at 36 months of age. Livestock Science, 137 (1-3), 244-254. , DOI : 10.1016/j.livsci.2010.11.018

	Year of birth /				Duration of	W2 ADG per treatment*
	treatment	Herd	Start	End	experimental	High, H	Median, M	Low, L
					period, d
	1994/1995	Le Pin	01-12-95 05-04-96	126	609 (143) 373 (142)	266 (85)
		Mirecourt 14-11-95 25-04-96	163	436 (127)	151 (96)	-175 (176)
	1995/1996	Le Pin	25-11-96 05-04-97	131	748 (124) 496 (170)	207 (128)
		Mirecourt 07-11-96 07-04-97	151	667 (90)	396 (88)	-231 (137)
	1996/1997	Le Pin	09-12-97 02-04-98	114	699 (147) 368 (128)	167 (139)
		Mirecourt 13-11-97 29-04-98	167	420 (77)	179 (123)	-84 (92)
	* Winter 2 average daily gain: High, H: 600 and 500 g/d for Le Pin and Mirecourt herds, respectively; Median, M:
	400 and 200 g/d for Le Pin and Mirecourt herds, respectively; Low, L: 200 and -200 g/d for Le Pin and Mirecourt
				herds, respectively

Table 4 . Overall performances according to treatment and breed (1)

 4 **: P < 0.001; **: P < 0.01; *: P < 0.05; T: feeding treatment; B: breed (No=Normande; Ho=Holstein, Mo= Montbeliarde); L: lactation; Y: year of birth. Within a row of feeding treatment, mean values with a different letter are significantly different at P < 0.05.(1) Animals performed at least a 110 d lactation length: 119 out of 127 heifers that calved.

	Le Pin	Feeding treatment	Breed	Root	Statistical	Mirecourt	Feeding treatment	Breed	Root	Statistical
		200	400	600	Ho	No	MSE	significance		-200	200	500	Ho	Mo	MSE	significance
	1 st lactation								1 st lactation
	n	17	20	20	32	25	-	-	n	20	20	23	32	31	-	-
	Length, d	303	301	289	301	294	45		Length, d	308	303	311	319		71
	Milk yield, kg	6684	6944	6363	7311	6016	1143	B***	Milk yield, kg	6281	6244	6348	6734	5838	1556	B*
	Protein, kg	215	227	204	229	202	36.5	B*	Protein, kg	211	212	215	228		51.0	B*
	Fat, kg	265 ab	283 a	247 b	288	243	45.3	T*, B**	Fat, kg	272	273	270	291		67.2	B**
	Milk 4%, kg	6648	7016	6255	7240	6039	1103	B*	Milk 4%, kg	6595	6602	6589	7052	6138	1608	B*
	Milk 305d, kg	6720	6969	6608	7349	6183	710	B**	Milk 305d, kg	6235	6265	6175	6456	5994	808	B*
	all lactations								all lactations
	n	50	57	42	75	74	-	-	n	71	61	69	72		-
	Length, d	301 a	303 a	287 b	302	294	46	T*	Length, d	304	288	297	299		66
	Milk yield, kg	7178	7164	6790	7766	6322	1166	B*, L*	Milk yield, kg	6980	6668	6787	7155	6468	1518
	Protein, kg	229 a	231 a	214 b	240	210	39.5	T*, B***, L**	Protein, kg	230	225	226	237		51.4
	Fat, kg	288 a	290 a	263 b	304	257	48.9	T*, B**, L*	Fat, kg	300	296	286	312		69.2
	Milk 4%, kg	7192 a	7218 a	6667 b	7668	6383	1164	T*, B***, L***	Milk 4%, kg	7293	7113	7006	7539	6737
	Milk 305d, kg	7241	7192	7062	7822	6506	796	B***, L***	Milk 305d, kg	6984	6959	6919	7259	6650

*

Table 5 . Overall performances per day of productive life, age and parity at culling, according to treatment and breed (1)

 5 **: P < 0.001; **: P < 0.01; *: P < 0.05; T: feeding treatment; B: breed (No=Normande; Ho=Holstein, Mo= Montbeliarde); L: lactation; Y: year of birth. Within a row of feeding treatment, mean values with a different letter are significantly different at P < 0.05.(1) Animals performed at least a 110 d lactation length

	Mirecourt	Feeding treatment	Breed	Root	Statistical
		-200	200	500	Ho	Mo	MSE	significance
								-
	Protein, kg	0.55	0.57	0.55	0.58	0.54	0.07	B*, L*
	Fat, kg	0.72	0.74	0.70	0.76	0.69	0.10	B*, L*
	Milk 4%, kg	17.4	17.9	17.0	18.3	16.6	2.5	B*, L*
	Milk 305d, kg	16.1	17.9	16.4	17.4	16.3	4.2	Y**
	Parity at culling	4.0	3.2	3.2	2.4	4.5	1.96	B**
	Age, d	2116	1983	2069	1749	2363	850	B**

*

Acknowledgments

The authors wish to thank all the technical staff at the Dairy Research Stations at Le Pin au Haras (Normandy) and at Mirecourt (Vosges) for taking care of the animals, and Dr. F Gondret, for her valuable criticism during the preparation of the manuscript.


J Gorzecka

N C Friggens

C Ridder

H Callesen

A Universal Index of Uterine Discharge Symptoms from Calving to 6 Weeks Postpartum

In this paper, a new uterine discharge index (D-index) was created and tested. It was based on a principal component analysis (PCA) of clinical findings of classical uterine discharge symptoms and rectal temperature during the postpartum period of dairy cattle. The PCA analysis revealed how uterine discharge features relate to each other and how they cluster together possibly representing different degrees of uterine inflammation. The D-index was the result of the multivariate PCA-analysis, and the D-index gives a continuous value between 0 and 10. It was demonstrated that the same scale, i.e. the D-index, can be used without any adjustment from 1 to 6 weeks post-calving. It is valid for any type of uterine discharge without defining the type of infection or differentiating between infection and contamination. The D-index was tested using the uterine involution data. Uterine involution was significantly delayed in the high-D-index group of cows. Similarly, in the test with all cows, involution was progressively delayed and the rate of involution of the pregnant horn was slowed down with the increase of the D-index values. It is concluded that the D-index can be a new practical, universal, tool for improved management of dairy cows in the postpartum period under commercial conditions.

Introduction

Uterine inflammation is an important risk factor in dairy cattle reproduction, because it is associated with lower conception rates, increased intervals from calving to first service or to conception and culling of more animals for failure to conceive [START_REF] Borsberry | Periparturient diseases and their effect on reproductive performance in five dairy herds[END_REF][START_REF] Leblanc | Defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows[END_REF]. Besides local inflammatory effects in the uterus, the condition results in disturbances to ovarian function and to signalling from the hypothalamus and pituitary gland, which may cause negative changes in ovarian activity and the quality of oestrous cycle [START_REF] Battaglia | Endocrine alterations that underlie endotoxininduced disruption of the follicular phase in ewes[END_REF][START_REF] Sheldon | Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle[END_REF][START_REF] Sheldon | Postpartum uterine health in cattle[END_REF][START_REF] Herath | Ovarian follicular cells have innate immune capabilities that modulate their endocrine function[END_REF][START_REF] Miller | Arcanobacter Pyogenes Pyolysin Stimulates Bovine Endometrial Cell Prostaglandin Secretion[END_REF].

From a clinical point of view, i.e. as a basis for treatment and profylaxis, it is important to be able to characterize the relationship between degree of uterine inflammation and fertility. However, in practice, there are problems in assessing this relationship, and improving our ability to quantify degree of uterine inflammation would thus be a valuable step towards dealing with its impact on fertility.

Uterine infection can be diagnosed by bacteriological, cytological or histological examination, but these methods are not useful under field conditions where diagnosis and eventual treatment have to be performed immediately. Therefore, evaluation of uterine discharge collected from the vagina is considered as an effective, but also simple and non-invasive method in the field [START_REF] Bretzlaff | Rationale for treatment of endometritis in the dairy cow[END_REF][START_REF] Leblanc | Defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows[END_REF][START_REF] Sheldon | Defining postpartum uterine disease in cattle[END_REF]. It has been shown that uterine discharge reflects well the bacterial uterine infection and immune response [START_REF] Williams | Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle[END_REF] and that the presence of purulent discharge in the vagina is associated with the presence of pathogenic bacteria in the uterus [START_REF] Dohmen | The relationship between bacteriological and clinical findings in cows with subacute ⁄ chronic endometritis[END_REF][START_REF] Huszenicza | Uterine bacteriology, resumption of cyclic ovarian activity and fertility in postpartum cows kept in large-scale dairy herds[END_REF][START_REF] Williams | Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle[END_REF]. The types of uterine infection, based mainly on the uterine discharge, have been described in the literature and reviewed by [START_REF] Sheldon | Defining postpartum uterine disease in cattle[END_REF].

For assessing the infection's severity, scoring systems have been described that include some of the features of the discharge, which are frequently correlated, such as presence of pus, blood, smell and sometimes additional information concerning size and position of the uterus and cervix. However, these scoring systems are not uniform and are also limited to certain time periods after calving or certain types of uterine infection, for instance applying only to endometritis [START_REF] Murray | Bovine endometritis: comparative efficacy of alfaprostol and intrauterine therapies, and other factors influencing clinical success[END_REF][START_REF] Dohmen | The relationship between bacteriological and clinical findings in cows with subacute ⁄ chronic endometritis[END_REF][START_REF] Sheldon | Comparison of three treatments for bovine endometritis[END_REF][START_REF] Huszenicza | Uterine bacteriology, resumption of cyclic ovarian activity and fertility in postpartum cows kept in large-scale dairy herds[END_REF][START_REF] Williams | Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle[END_REF]. Furthermore, these systems aim to identify specific classes of infection (healthy, subclinical and clinical); they do not seek to quantify the underlying degree of inflammation.

In our study, a multivariate approach (principal components analysis, PCA) was used as a method for investigating general biological patterns of the discharge features in the postpartum period. The PCA method is particularly useful for situations where it is difficult or even not possible to measure something directly -such as 'degree of uterine inflammation', and it is also suitable for dealing with correlated data.

The main purpose of the study was to use this analysis to develop a universal index of uterine inflammation clinical symptoms, which is continuous and not limited by time after calving and type of uterine infection.

Materials and Methods

Animals

The data collection was done from January to December 2008 in the herd at the Danish Cattle Research Center in Foulum, Denmark. In total, 126 dairy cows in lactation 1 to 5 were examined: 86 Danish Holstein and 40 Danish Jersey. The cows were kept in 2 groups of Danish Holstein and 1 group of Danish Jersey in a loose housing system and they were milked by VMS (Voluntary Milking System, DeLaval, Tumba, Sweden). The average milk yield was 9640 kg ECM, and the cows were fed ad libitum with a total mixed ration containing 50% roughage (approx. 50 ⁄ 50 corn silage and grass silage) and 50% standard dairy concentrate.

Clinical examination

The cows were examined once weekly by the same person, starting from week 1 until week 6 after calving. All the examinations included (i) measuring rectal temperature, (ii) transrectal palpation of uterus and ovaries (presence of ovarian cysts was recorded), (iii) transrectal ultrasound scanning of the uterus and (iv) collecting uterine discharge.

At each ultrasound scanning, faeces from the rectum was first removed, and the uterine involution rate was determined based on measurement of the diameter of the uterine cervix (in the middle of the cervix) and the diameter of both uterine horns (5 cm from the bifurcation). This was measured using built-in calipers in the ultrasound scanner (Aloka,Co.Ltp, Japan) with a 5.0 MHz linear-array transducer.

Uterine discharge was collected from the vagina by hand with rectal glove and lubricant, after cleaning of the vulva with paper towels. Each sample of collected discharge was photographed and scored separately for five features (Table 1): proportion of pus, amount, consistency, smell and colour (content of blood).

Modelling and statistical analysis

Scoring system for uterine discharge The scores for uterine discharge symptoms used in the clinical examinations do need, a priori, to correlate with the severity of uterine infection as experienced by the cow. Even if such a correlation exists, it may not relate linearly to the scoring system scale. Thus, rather than assume some form of relation between the different score classes, it was assumed that they were independent. All score classes (Table 1) were therefore transformed into class-variables. For example, 'consistency' score 1 was transformed into 'watery' or 'not watery'. Thus, the single consistency-variable, which had been given values from 1 to 4 (watery, semi-viscous, mucus-like, dense), was transformed into four binary class-variables and each cow (sample) will have the value 1 (one) in only one of these classes and 0 (zero) in the rest of the classes.

Transforming all score-variables resulted in obtaining class-variables (Fig. 1): pus = 'pus0', 'pus1', 'pus2', 'pus3'; amount= 'amount0', 'amount1', 'amount2', 'amount3'; consistency = 'watery', 'semi-viscous', 'mucus-like', 'dense'; colour = 'no blood', 'pink', 'red', 'brown', 'black'. Only the variable 'smell' was not changed, because it already had numeric values 0 (no) and 1 (yes). Finally, the 'dense' consistency was excluded from the scale because of too few cases (only two cases in the whole study) that could falsely influence relations in the PCA model [START_REF] Esbensen | Multivariate data analysis-in practice[END_REF].

Missing values in the database

When the variable amount = 0, the rest of the discharge features were missing per definition, and these observations were therefore excluded from the PCA model. These animals were assumed to be healthy, but this rule would not concern cows with pyometra, where a uterus filled with pus is associated with closed cervix and a lack of discharge in the vagina [START_REF] Sheldon | Defining postpartum uterine disease in cattle[END_REF]. However, in the present study no pyometra cases were found.

Principal component analysis

The collected data of uterine discharge symptoms and rectal temperature were analyzed using LATENTIX 2.00 (Latent5, Copenhagen, Denmark, http://www.latentix.com), which is a chemometric software for multivariate data analysis, including principal components analysis (PCA), frequently used for explorative data structure modelling. PCA reveals interrelationship between all measurements (variables) and observations (objects) simultaneously and decomposes the data in order to detect and model latent structures ('hidden phenomena') [START_REF] Esbensen | Multivariate data analysis-in practice[END_REF].

Before creating the PCA model, the data were autoscaled to mean 0 and SD 1. Using these scaled data the software calculated loadings coefficients for each variable and for each principal component (Table 2). Loading coefficients, i.e. describing the relationship between all variables, were used to calculate the scores, i.e. the position of all observations in the PCA model.

Preparation for the main analysis

In the first analysis a loadings plot for principal components 1 and 2 was made, representing relations 'brown' and 'black' is indicated between all 17 new class-variables (Fig. 1). As seen from Fig. 1, the colour-variables 'red', 'black' and 'brown' are closely correlated. From the point of view of PCA modelling, these colour-variables have a similar meaning in the model. From the clinical point of view it was often difficult to distinguish between dark brown and black discharge and between red and brown discharge, which was often a mixture of these colours. Therefore, the colour-variables 'red', 'black' and 'brown' were transformed into one variable: 'brownish'. This simplification did not change the relations between other variables in the model (Fig. 2).

Index of uterine discharge symptoms (D-index)

An index of severity of discharge symptoms (D-index) was created on the basis of the first principal component, by multiplying the original measured variables with p1* coefficients. The p1* coefficients are p1 coefficients adjusted for the autoscaling of the data.

After this calculation the index ranged from )3 to 4, like in the scores plot (Fig. 3). Therefore, in order to simplify the scale and for convenience, it was linearly transformed into a 0-10 range (Fig. 8). For this range new 'discharge coefficients' (D-coefficients) were calculated (Table 3). When summed D-coefficients of the original measured variables (and adding the offset) resulted in the D-index, which ranges from 0 to 10.

Testing of the D-index

The uterine involution process was estimated by regressing the diameters of the cervix and the previously pregnant uterine horn on time from calving using MINITAB 9 software (http://www.minitab.com). In order to test the D-index, two tests were made:

Extreme animals: Groups of extreme cases were extracted: one group of 12 cows with discharge symptoms index 0-2 during the whole period of examinations, and one group of 17 cows with discharge symptoms index 8-10 in at least two successive examinations. All of these examinations occurred during the first 3 weeks after calving. The first group, called low-D-index was assumed to have a better uterine health than the second group, called high-D-index. The regression equation ln(Y) = ln(a) + b • t + k.inf + l • inf • t was used to estimate the influence of uterine infection on the rate of uterine involution in two described groups (low-D-index = 0 and high-D-index = 1), where Y is the diameter of either the pregnant horn or cervix.

All animals: The next test of uterine involution was performed using all animals. The maximum value of the discharge index within 21 days after calving was used as a constant value of uterine inflammation for each cow.

The regression equation ln

(Y) = ln(a) + b • t + k • maxD-index + l • maxD-index • t

Results

Interpretation of the PCA model In Fig. 3 (lower panel), the naturally occurring relationships between the uterine discharge scores revealed by the PCA are shown for the two main principal components, 1 and 2, that together accounted for 42.5% of a total variance. Symptoms that are close together are highly correlated (e.g. smell and pus3). Uterine discharge scores that are at opposing ends of a PC (e.g. pus0 and pus3 on PC#1) indicate features that differentiate different types of discharge and different severity of uterine inflammation clinical symptoms. In this context, PC#1 is the component with the maximum variance and differentiated symptoms into 'clean uterus' and 'not clean uterus' groups. This might be interpreted as 'healthy' uterus (with small amount of normal mucus) and 'sick' uterus (with high scores of discharge symptoms). The distributions of the individual scores from each cow observation are shown in the upper panel of Fig. 3. Coefficients for the first two principal components are presented in Table 3. The remaining coefficients for further PCs and the percent of variance, which they accounted for, are presented in Table 2.

Time factor in the model

In Fig. 4 a scores plot is presented, which is shaded according to days from calving (DFC). The observations with high values of PC#1 tended to occur closer to the calving, especially within the first 3 weeks after calving. In order to evaluate whether the relationship between discharge scores was affected by DFC, a DFCvariable was added to the PCA model. Adding this variable did not impair the model; neither did it affect the relations between the other variables or the percent of explained variation. It just showed that the proportion of cows having 'not clean uterus' decreased with time (Fig. 5 -note that due to the rotational ambiguity of PCA, the sign of the loadings is reversed). In order to further check the stability of the model across time from calving, the data were divided into two time intervals: weeks 1-3 and weeks 4-6 (Fig. 6). Objects in weeks 1-3 are spread proportionally in the space of the model. Objects in weeks 4-6 are more clustered in the left part of the model, which suggests that the uterus is more 'clean' in this period of time.

The model based only on data from weeks 1-3 was used to predict scores for weeks 4-6. The comparison of predicted scores with the model is a measure of the goodness of fit of the sample to a particular principal component model. The software calculates confidence limits for the scores applying the Student's t-distribution [START_REF] Wise | PLS_Toolbox 2.1 for use with MATLAB(TM)[END_REF]. As can be seen in Fig. 7, all predicted objects from week 4-6 remain in the ellipse, which shows the 95% confidence interval, demonstrating the stability of the model across time [START_REF] Jackson | A User's guide to Principal Components[END_REF][START_REF] Esbensen | Multivariate data analysis-in practice[END_REF]). Therefore, it was decided to create only one common discharge index for all 6 weeks after calving.

Index of uterine discharge symptoms (D-index)

The index of discharge symptoms was created on the basis of the first principal component and ranges from 0 to 10. Fig. 8 presents the numerical relation between the PCA-scores and D-index. PCA-scores are shaded according to the value of the D-index.

Testing of the D-index Uterine involution in extreme groups

There was a significantly greater intercept for high-index cows when compared with low-index cows in the regression of pregnant horn diameter on DFC, but no significant effect of index group on the slope of the regression, i.e. the rate of uterine involution. When both slope and intercept effects of index group were included in the regression of cervix diameter on DFC, neither was significant.

When the regression model was simplified by removing the effect of index group on slope, a significant difference in intercept was found, where the high-index group had a greater intercept. Overall, high-index cows had a delayed uterine involution since at any given time from calving both cervix and pregnant horn diameter was greater in high-index cows (Table 4).

Uterine involution in all animals

The next test of uterine involution was performed using all animals. The maximum value of the D-index within 21 days after calving (maxD-index) was used as a constant value of uterine inflammation. Values of D-index after 21 days postpartum would not significantly influence the rate of uterine involution, because most of the process is already completed [START_REF] Morrow | Postpartum ovarian activity and uterine involution in dairy cattle[END_REF][START_REF] Gier | Uterus of the cow after parturition:involutional changes[END_REF].

There was a significantly greater intercept for increasing values of maxD-index in the regression of uterine cervix and pregnant horn on DFC (Table 5). There was also a significant effect of maxD-index on the slope (i.e. involution rate) in the regression of pregnant horn, but not in the regression of uterine cervix.

Overall, at any given time, cows with a higher maxDindex had a significantly delayed involution of uterine cervix and pregnant horn and significantly slowed down the rate of involution of pregnant horn.

Discussion

In this study, clinical findings of dairy cows during the postpartum period were described and related, using a multivariate statistical model. The overall purpose was to identify the key combinations of uterine discharge symptoms and their severity, which possible reflect degrees of uterine inflammation, since it has been shown that uterine discharge reflects well the bacterial uterine infection and immune response [START_REF] Williams | Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle[END_REF]). To achieve this, a PCA model was designed and used. This is the first time a multivariate PCA approach has been applied to uterine discharge symptoms.

Using this approach, and deconstructing symptoms into classes to avoid making assumptions about the symptom's scales, the PCA analysis provided the basis for building a simple score. The constructed discharge index (D-index) gives a continuous value between 0 and 10. Calculated D-coefficients are ready for use under field conditions. To obtain the D-index value, the vaginal discharge needs to be scored as indicated in the methods section (Table 1). Then the D-index value is created by summing the D-coefficients for each discharge feature, rectal temperature and offset (Table 3). Notice that rectal temperature, as a non-class variable, Table 3. Coefficients p1, p2 used for calculating of PC1 and PC#2, coefficients p1* (which are p1 coefficients adjusted for the autoscaling of the data) used for creating an index of severity of discharge symptoms (D-index) and D-coefficients used for calculating this index after the linear transformation into a 0-10 range needs to be first multiplied with D-coefficient. Notice also that after the simplification of the model, the discharge colours red, brown and black have the same D-coefficient ('brownish'). For example, a cow recorded as: pus3, amount3, consistency-semi-viscous, smell-yes, colour-pink and temperature 39.5°C will have a D-index value = 9.89 (see Table 6).

At first glance calculating of the value of D-index may seem to be complicated, but it mainly consists of adding the coefficients. In the light of easy access to on-farm hand held recorders ⁄ calculators, etc., the added complexity is not seen as being a serious drawback given the advantages of the additional information provided by the D-index. The D-index can be used without any adjustment at any time from calving (up to 6 weeks post-calving), because time from calving (DFC) did not influence the model, it remained stable across time. Time is correlated with uterine infection but is not a cause of the disease. It is a condition of the animal associated with suppressed immunity after calving, which predisposes to a higher risk of uterine infection, especially in the first 3 weeks postpartum [START_REF] Mallard | Alteration in immune responsiveness during the peripartum period and its ramification on dairy cow and calf health[END_REF][START_REF] Burton | Gene expression signatures in neutrophils exposed to glucocorticoids: A new paradigm to help explain ''neutrophil dysfunction'' in parturient dairy cows[END_REF].

Using PCA analysis, it became clear how uterine discharge features relate to each other and how they cluster together, possibly representing different degrees of uterine inflammation. The lowest D-index values are associated with pus0, amount1, no-blood and mucus like consistency, which are strongly correlated. Discharges with flecks of pus (pus1) give higher D-index values between 3 and 4. This feature is not correlated with others, but is localized on the border between Pregnant horn involution 1.57 )0.0122 0.0263 )0.000792 0.43 0.2081 p = 0.000 p = 0.000 p = 0.003 p = 0.017 Cervix Involution 1.82 )0.0144 0.0102 )0.000171 0.60 0.1431 p = 0.000 p = 0000 p = 0.042 p = 0.385 'clean' and 'not clean' uterus indicating an increasing process of inflammation. The next two clusters of features have a similar D-index value between 4 and 6, and these are clusters: pus2 correlated with amount2 and watery discharge correlated with high temperature. This second group, when associated with brownish colour, is defined in the literature as puerperal metritis. The highest D-index values have discharges with a strong correlation between pus3 and fetid smell, which is associated with amount3, semi-viscous consistency and sometimes pink colour (haemorrhagic discharge). The brownish colour feature is not strongly correlated with other features, but its localization between other clusters means that it may be associated with high temperature and watery discharge (puerperal metritis), pus2 correlated with amount2, semi-viscous consistency, or with the group with amount3, pus3 and fetid smell. All these different combinations occur especially often in the two first weeks after calving.

In the current study, only the first principal component was used to create the D-index as it contrasts the symptoms of 'clean uterus' with 'not clean uterus'. However, further PCs may bring additional, more detailed information about uterine inflammation. They account for lower percent of total variance and describe less important and smaller combinations of uterine discharge symptoms. PC#2 in this model contrasts cluster pus2 and amount2 with amount3 and watery consistency.

The combination of discharge symptoms in the PCA model may be related to different types of uterine infection, which have been described before in the literature [START_REF] Sheldon | Defining postpartum uterine disease in cattle[END_REF]. In order to establish if one scoring scale could be made across time, no attempt was made in this study to differentiate between the types of uterine infection, only severity of discharge symptoms was assessed. This permitted us to obtain one number, the D-index, on a continuous scale, which possibly reflects the uterine inflammation process.

In this paper, the D-index was tested by using the data of the uterine involution process. It is generally known that uterine infection may negatively affect involution [START_REF] Morrow | A review of postpartum ovarian activity and involution of the uterus and cervix in cattle[END_REF][START_REF] Hajurka | Influence of health status of reproductive organs on uterine involution in dairy cows[END_REF]. Most of this process is completed within 3 weeks after calving [START_REF] Morrow | Postpartum ovarian activity and uterine involution in dairy cattle[END_REF][START_REF] Gier | Uterus of the cow after parturition:involutional changes[END_REF], therefore, a maximum value of the discharge index within 21 days was used as a potential factor affecting the involution. Uterine involution was significantly delayed in the high-D-index group of cows indicating that the D-index correctly described the known effect of uterine inflammation on involution. Similarly, in the test with all cows, involution was progressively delayed and the rate of involution of pregnant horn was slowed down with the increase of the D-index values.

Postpartum contamination of the uterus is ubiquitous in dairy cows [START_REF] Sheldon | Uterine diseases in cattle after parturition[END_REF]), but it does not mean that any type of discharge within the first 3 weeks after calving is acceptable. In this PCA model, only about half of the observations had 'not clean' uterus with a high D-index score for the first 3 weeks after calving. These cows had a delayed involution process, compared with low-D-index animals. It may be that, among other factors, breed and hygienic conditions influence the prevalence of contaminated animals. The incidence of positive bacterial cultures varies in normal calving cows, e.g. [START_REF] Griffin | Non-specific uterine infection and bovine fertility : I. Infection patterns and endometritis during the first seven weeks post-partum[END_REF] 96% of normal cows, [START_REF] Bekana | Intrauterine bacterial findings and hormonal profiles in post-partum cows with normal puerperium[END_REF] 22% of normal cows, [START_REF] Kask | Uterine bacteriology, histology, resumption of ovarian activity and granulocyte function of the postpartum cows in different milking frequencies[END_REF] 33% of normal cows. Unhygienic conditions may increase the bacterial contamination of the vestibulum and vagina, and the bacteria can than easily migrate to the uterus after parturition [START_REF] Bretzlaff | Incidence and treatments of postpartum reproductive problems in a dairy herd[END_REF].

In the examined herd, there were high hygienic standards, which may explain the better than average uterine health. Also, Danish Jersey cows were more resistant to both contamination in the early period and infection in the late period than Danish Holstein cows.

In future investigations the authors hope to use the D-index to assess the extent of the relationship between the clinical symptoms of uterine inflammation and ovarian activity.

Conclusions

This study provided for the first time a continuous scale for severity of uterine inflammation clinical symptoms. This scale is universal for any type of uterine discharge, any type of uterine infection associated with the presence of the discharge in vagina (defined in the literature as puerperal metritis, metritis, clinical metritis, clinical endometritis) and for any time up to 6 weeks after calving. The scale was built without any assumptions about the correlations between discharge features and thus reflects degree of uterine inflammation based on the natural, biological pattern. drafted paper. Carsten Ridder has analyzed data, drafted paper. Henrik Callesen has designed study, drafted paper.

Fig. 1 .

 1 Fig. 1. Plot of PCA loadings for principal components 1 and 2, showing the relations between all the new class-variables. The close correlation between colour-variables 'red', 'brown' and 'black' is indicated

Fig. 2 .

 2 Fig. 2. PCA model with transformed 'brownish' variable, which did not change the relations between the other variables

Fig. 4 .Fig. 5 .

 45 Fig. 4. Scores plot shaded according to days from calving (DFC). The shading scale is shown below the main plot

Fig. 6 .Fig. 7 .Fig. 8 .

 678 Fig. 6. Scores plot dividing observations into two time intervals: weeks 1-3 and 4-6

Table 1 .

 1 Scoring of uterine discharge

				Score
	Discharge features	0	1	2	3	4
	Proportion of pus	No	Flecks	£ 50%	>50%	-
	Amount	No <1 handful	1 handful	>1 handful	-
	Consistency	No	Watery	Semi-viscous Mucus-like Dense
	Smell	No	Yes	-	-	-
	Colour	No	Pink	Red	Brown	Black

  was used to estimate the influence of the value of the D-index on the rate of uterine involution.

Table 2 .

 2 Loadings and percent of explained variance for PC1 to PC#12

						PCA scores and loadings [model 2]
	Scores PC#2 (12.405%)	-4 -2 0 2 4
				-3	-2	-1		0	1	2	3	4
								Scores PC#1 (30.136%)
	Loadings PC#2 (12.405%)	-0.5 0 0.5	-0.5 -0.4 -0.3 No blood -0.2 -0.1 Amount1 Pus0 Mucus-li	0 Pus1	0.1 Amount2 0.2 Brownish 0.3 Amount3 Pus3 0.4 Semi-vis Smel Pink Temp. Pus2 Watery	Fig. 3. PCA model of scores and loadings for uterine discharge symptoms. The position of the objects (each examination) in scores plot (upper panel) was calculated using coefficients (p1, p2) for each variable. The loadings plot (lower panel) shows
						"Clean uterus"	Loadings PC#1 (30.136%)	"Not clean uterus"			how the variables are correlated with each other. PC1 differentiates symptoms into 'clean' and 'not clean' uterus
	Loadings		PC#1	PC#2	PC#3	PC#4	PC#5	PC#6	PC#7	PC#8	PC#9	PC#10	PC#11	PC#12
	Temp.		0.06	0.12	0.05	0.32	)0.20	0.02	)0.17	)0.89	)0.10	0.02	0.03	0.00
	Smell			0.33	0.22	0.14	)0.06	)0.08	)0.03	)0.02	0.02	0.06	)0.84	)0.32	0.00
	No blood		)0.31	)0.04	0.21	)0.48	0.02	0.35	)0.07	)0.17	)0.06	)0.07	)0.03	0.00
	Pink			0.21	0.14	0.15	0.01	)0.49	)0.61	)0.16	0.18	)0.03	0.22	0.00	0.00
	Brownish		0.20	)0.06	)0.35	0.55	0.35	0.06	0.21	0.06	0.09	)0.09	0.04	0.00
	Watery		0.05	0.34	)0.42	)0.19	)0.09	0.02	0.25	0.02	)0.69	)0.05	0.16	0.31
	Semi-visc		0.38	)0.21	0.24	0.01	0.18	0.14	)0.14	0.01	)0.10	0.26	)0.40	0.67
	Mucus-like		)0.40	0.05	)0.05	0.08	)0.14	)0.16	0.03	)0.02	0.42	)0.23	0.31	0.68
	pus0			)0.33	0.20	0.20	0.20	)0.09	)0.03	0.50	0.01	)0.02	0.12	)0.45	)0.01
	pus1			)0.07	0.12	)0.52	)0.27	0.28	)0.26	)0.45	)0.12	0.16	0.04	)0.28	0.00
	pus2			0.06	)0.47	)0.19	0.15	)0.49	0.33	)0.23	0.16	)0.12	)0.15	0.12	0.00
	pus3			0.33	0.13	0.36	)0.13	0.33	)0.07	0.05	)0.06	0.01	)0.02	0.56	0.01
	amount1		)0.32	0.07	0.22	0.30	0.21	)0.08	)0.39	0.17	)0.36	)0.12	0.01	0.00
	amount2		0.09	)0.53	)0.10	)0.26	0.01	)0.34	0.40	)0.25	0.01	)0.09	)0.03	0.00
	amount3		0.25	0.41	)0.14	)0.08	)0.23	0.39	0.05	0.05	0.38	0.22	0.01	0.00
	% expl. variance	30.1	42.5		53.2	62.5	70.4	77	83.4	89.3	94.3	97.6	99.9	100

Ó 2010 Blackwell Verlag GmbH

Acknowledgements

The paper is a part of a PhD project funded by the Danish Research Council, Aarhus University, the Faculty of Agricultural Sciences and Lattec I ⁄ S. The authors would like to thank Kurt Myrup Pedersen for helpful comments and general support.

Conflict of interests

The authors report a financial relationship with Lattec I ⁄ S. We declare that Lattec I ⁄ S had no involvement in the conception, implementation or reporting of this work.

Author contributions

Justyna Gorzecka has designed study, collected data, analyzed data, drafted paper. Nicolas Friggens has designed study, analyzed data,