# Animal performance and stress at weaning when dairy cows suckle their calves for short versus long daily durations

A. Nicolao, Isabelle Veissier, Matthieu Bouchon, E. Sturaro, Bruno Martin, Dominique Pomiès

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A. Nicolao, Isabelle Veissier, Matthieu Bouchon, E. Sturaro, Bruno Martin, et al.. Animal performance and stress at weaning when dairy cows suckle their calves for short versus long daily durations. Animal, 2022, 16 (6), pp.100536. 10.1016/j.animal.2022.100536. hal-03682979

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1 Animal performance and stress at weaning when dairy cows suckle their calves for short versus long daily durations

3 A. Nicolao¹,², I. Veissier¹, M. Bouchon³, E. Sturaro², B. Martin¹ and D. Pomiès¹

4 ¹Université Clermont Auvergne, INRAE, VetAgro Sup, UMR Herbivores, F-63122

5 Saint-Genès-Champanelle, France

6 ²DAFNAE, University of Padova, Viale dell'Università 16, 35020 Legnaro, Italy

7 ³Herbipôle, INRAE, F-63122, Saint-Genès-Champanelle, France

8

9 Corresponding author: Dominique Pomiès. Email: dominique.pomies@inrae.fr

10

11 Abstract

12 Calves in most dairy farms are separated from their dams either immediately or within a few hours after birth, prompting increasing concern of the society for reasons 13 of animal welfare. The aim of this study was to identify systems to maintain cow-calf 14 contact (CCC) that balance the benefits for calf growth and health against the 15 negative impacts on sellable milk and stress at weaning. We tested reuniting cows 16 and calves for 20 min before (Before-group) or 2.5 h after (After-group) morning 17 milking (in Trial 1) or for a 9 h period between the morning and evening milkings 18 (Half-day-group, in Trial 2). In Control-groups, calves were separated from their dam 19 at birth and fed with artificial nipple with tank milk provided daily at 13% (Trial 1) and 20 14% (Trial 2) of their BW. In both trials, each practice was applied on a group of 14 21 dam-calf pairs (7 Holstein [Ho] and 7 Montbéliarde [Mo]). All calves were weaned at 22 a BW of at least 100 kg. In Trial 1, the After-group was prematurely stopped when the 23
24 calves were 8 weeks of age as calf growth became limited (340 g/d) due to low milk intakes (2.97 kg/d). During the first 8 weeks of lactation, milk yield at the parlour was 25 29%, 51% and 42% lower in After-, Before- and Half-day-cows respectively 26 compared to Controls. From Week 14 to 16 when all calves were separated from 27 their dam, Before-cows still produced 25% less milk than Control-cows while Half- 28 day-cows reached the milk yield of Control-cows within a week. There were no 29 significant differences in milk somatic cell count and in frequency of health disorders 30 (cows and calves) between suckling and Control- groups. Compared to Control- 31 calves, calf growth until weaning was higher in the suckling calves in Trial 1 (861 vs. 32 699 g/d) and similar in Trial 2 (943 vs. 929 g/d). At weaning, Before- and Half-day- 33 calves started to vocalize earlier and continued to vocalize longer than Controls. In 34 conclusion, the best compromise between cow milk yield and calf growth is a long 35 period of CCC (9 h) between the morning and evening milkings. Still abrupt weaning 36 stresses both cows and calves even if CCC has been restricted before separation. 37

38 **Keywords:** cow-calf contact, milk feeding, milk yield, growth, weaning

39 **Implications**

40 Consumers are increasingly questioning the practice of separating calves from their
41 dams at birth. Compared to short periods of suckling immediately before or after
42 milking, a half-day contact during the day between milkings provides a good
43 compromise between sellable milk and calf growth. This practice could conciliate
44 consumers and farmers views. Solutions should be explored to reduce calves and
45 cows stress due to abrupt weaning.
46 **Introduction**

47 Calves in most dairy farms are separated from their dams either immediately or
48 within a few hours after birth (Busch et al., 2017). They are fed milk replacers or non-
49 marketable milk, distributed by an automatic milk feeder or in buckets, and receive
50 increasing amounts of solid feed until weaning (Le Cozler et al., 2012). Female
51 calves, which are the future replacement heifers, are generally weaned off from milk
52 at 8–10 weeks of life, or 12 weeks of age or more in case of organic farming. Male
53 calves are generally sold at a few weeks of age to be fattened in specialized farms.

54 Dairy calves therefore very rarely suckle their dam (Pardon et al., 2012). Early
55 separation between cows and calves enables farmers to get the most milk from the
56 cows and control the amount of colostrum and milk ingested by the calves. This
57 practice however creates animal welfare issues for consumers (Placzek et al., 2021)
58 who associate the welfare of an animal with possibilities to express its natural
59 behaviours (Lund et al., 2006). In dairy farming, this includes calves suckling their
60 dam. Some farmers already let calves suckle their dam or other cows at least for
61 short periods after birth, either to promote calf health or to reduce workload and/or
62 production costs (Michaud et al., 2018).

63 Several experiments were carried out to assess how cow-calf contact (CCC) affects
64 milk production and composition. Considering the total milk produced by cows (either
65 milked or taken by the calves), a combination of milking and suckling can make cows
66 produce more milk due to teat stimulation by the calves and better udder emptying
67 (Sandoval-Castro et al., 2000). This increase in total milk production — and even
68 sellable milk output — was reported in dual purpose breeds, such as Salers or
69 Brahman x Holstein cows, in which calf stimulation before milking is necessary to
70 activate milk ejection (Cozma et al., 2013; Cozma et al., 2016; Tesorero et al., 2001).
71 However, in dairy cows selected for high milk production, total milk production is
72 sometimes reported to be unaffected by nursing (Mendoza et al, 2010) in particular
73 when sucking occurs just before and after milking (Cozma et al., 2013). Most of the
74 time, whole-day cow-calf contact is found to significantly reduce the milk yield by 10
75 to 12 kg/d (Pomiès et al., 2010; Zipp et al., 2018). Krohn (2001) suggested that
76 reducing the duration of CCC could limit the suppressive effects of suckling on milk
77 ejection.

78 Suckling may also affect milk fat content, because milk fat content changes over the
79 course of milking (Rico et al. 2014) and the proportion of the milk produced by the
80 cow that is collected during milking varies with suckling. Consequences of suckling
81 on milk protein content are not clear so far (Johnsen et al., 2016). Boden and Leaver
82 (1994) and Barth (2020) found an increase of milk protein content in suckled cows
83 while Cozma et al. (2013) found a decrease, and Froberg et al. (2007) and Sandoval-
84 Casto et al. (1999) found no differences.

85 Calves reared with their dam can grow faster compared to artificially-reared calves, in
86 particular when the latter are fed a restricted milk diet (Flower & Weary, 2001). The
87 faster growth may be due to higher milk consumption as well as to stimulation of
88 anabolism by the higher release of oxytocin in suckling calves than bucket-fed calves
89 (discussed by Uvnäs-Moberg et al., 2001). Therefore, suckling may have positive
90 effects on calves' growth that are not solely related to the higher ingestion of milk.

91 A common belief is that separating the calf at birth limits the risk of transmission of
92 diseases and is better for both calf and cow health. Beaver et al. (2019) reviewed the
93 available literature and found no evidence that suckling has negative effects on
94 health. For instance, when latency to first suckling and quality of colostrum are
95 controlled, leaving the calf with its dam at birth has no effect on risk for diarrhoea or
96 on mortality (Meagher et al., 2019). In addition, suckling can benefit cows by reducing
97 the incidence of mastitis while others could not find an effect (reviewed by Johnsen et
98 al., 2016). Pomiès et al. (2010) and Cozma et al. (2013) reported lower milk somatic
99 cell count (SCC) in nursing cows.

100 Under natural conditions, weaning occurs gradually at 8–10 months of age
101 (Reinhardt & Reinhardt, 1981), and the cow–calf bond persists after weaning
102 (Veissier et al., 1990). In dairy farming promoting CCC over long duration, weaning is
103 provoked by an abrupt separation of dam and calf that causes stress to both cows
104 and calves (Flower & Weary, 2001; Hudson & Mullord, 1977; Lidfors, 1996; Weary &
105 Chua, 2000). Cow–calf bonding is viewed as positive for animal welfare, but the
106 stress induced by the separation is a welfare problem (Weary et al., 2008).

107 Habituating calves to be separated from their dam has been shown to reduce stress
108 at weaning in beef calves (Price et al., 2003).

109 Restricted suckling, i.e. suckling for only some time during the day, is likely to be
110 beneficial to calf growth and health while limiting losses in total and sellable milk
111 production and limiting stress at weaning. The aim of this study was to propose
112 suckling practices that achieve these balanced effects. We tested reuniting cows and
113 calves for short periods before or after milking or for a half-day contact during the day
114 between the morning and evening milkings. Reuniting cows and calves was generally
115 performed only once a day with a view to limit farmers’ labour while still allowing
116 adequate calf growth (Ackerman et al., 1969; Saldana et al., 2019).

117 **Material and methods**

118 We conducted two trials at the Herbipôle experimental farm of the French National
119 Institute for Agriculture, Food and Environment (INRAE)
120 (https://doi.org/10.15454/1.5572318050509348E12) located in Marcenat, France

121 (45.30°N, 2.84°E, 1 080 m a.s.l.). All procedures were carried out in accordance with

122 French Ministry of Agriculture guidelines on animal research and with all other

123 applicable national and European regulations governing experiments with animals.

124 All researchers responsible for the study (D. Pomiès, B. Martin, M. Bouchon, and I.

125 Veissier) and all animal caretakers had adequate appropriate training, and the

126 experimental farm is accredited for running experiments (C15-114-01).

127 Trial 1 was carried out between February and July 2017, and Trial 2 was carried out

128 between February and July 2018. In Trial 1, two practices where calves had access

129 to short-time CCC every day (**Before** and **After** milking) were compared to milk

130 feeding with teat buckets and automatic milk feeder (**Control**). In Trial 2, a suckling

131 practice where calves had access to day-time CCC every day (**Half-day**) was

132 compared to artificial suckling practice (**Control**). Each practice was applied on a

133 group of 14 dam-calf pairs (7 Holstein [Ho] and 7 Montbéliarde [Mo]). Within each

134 trial, the 14 dam-calf pairs were balanced for lactation number (2.55 ± 1.68 in Trial 1,

135 2.71 ± 1.56 in Trial 2), date of calving (28 March ± 22 d in Trial 1, 17 March ± 14 d in

136 Trial 2) and milk yield genetic index (84 ± 294 in Trial 1, 106 ± 205 in Trial 2). The

137 groups were decided before calving; the sex of calves was therefore not balanced. In

138 Trial 1, 78%, 29% and 50% of the calves were female in Before-, After- and Control-

139 groups respectively and in Trial 2, 36% and 29% of the calves were female in the

140 Half-day- and the Control-group respectively. Cows were milked in a 2 x 14

141 herringbone milking parlour (Delaval, France) twice a day at 07:00 am and 04:30 pm.

142 **Animal management**

143 *Before weaning in Trial 1*
The Control-calves were separated from their dams within a few hours (up to 6 h) after birth. They received at least 2.0 L of fresh colostrum from a feeding bottle. If there was no good-quality fresh colostrum available (< 24% Brix, measured by refractometer), then good-quality thawed and reheated colostrum was provided. The calves were housed in individual pens for 7-9 days and fed bulk milk twice a day using teat buckets. After this period calves were group housed in a collective straw-bedded pen of 60 m² with access to water and a hay rack until weaning. They were fed bulk milk by an automatic feeder equipped with teats (Förster Technik Engen, Germany), specific starter-age concentrate (from Centraliment factory, Aurillac, France) distributed in a bowl within the same automatic feeder as for milk, and hay (permanent grassland, first cut). Amount of milk provided to the calves was about 13% of their BW (12% to 15% depending on the age) during the first seven weeks of life, and then the amount of milk was gradually reduced until weaning at about the 13th week (Table 1). In the two remaining groups, the calves spent the first three days after birth with their dams in a 20 m² individual calving pen or a 40 m² collective pen for three cows and their calves, depending on pen availability. All pens were equipped with water troughs. During these days, animal caretakers checked at least twice a day if the calves suckled properly their dam. Then calves were separated and accommodated in a collective pen until weaning and cows returned to the herd.

Before-calves had access to their dam in a specific collective pen facing the milking parlour (90 m²) for 20 min before the morning milking and 10 min before the evening milking for the first two weeks, then only for 20 min before the morning milking. After-calves had access to their dam in a cow pen for 2.5 h after the morning and evening milkings for the first two weeks, then only for 2.5 h after the morning milking. The
168 calves were also fed concentrate by an automatic feeder. The calf feeding plan is
detailed in Table 1.

170 Before weaning in Trial 2

171 Control-calves were reared in the same way as those in Trial 1, except that the
172 amount of milk available in the automatic milk dispenser was 14% of calf BW during
173 the first seven weeks then decreased from 8% BW in Week 8 to 3% BW from week
174 10 (Table 1). Because there were fewer calves to be suckled by their dam in Trial 2
175 than in Trial 1, Half-day-calves spent the first five days after birth (and not only the
176 first three days as in Trial 1) alone with their dam in a straw-bedded 20 m² individual
177 calving pen (and never in a collective calving pen as sometimes done in Trial 1). The
178 calves received colostrum directly from their dam. The calves were then housed in a
179 collective straw-bedded pen next to the cowshed (50 m²), from which they could see
180 their dams. From 07:30 am when cows came back from the milking parlour to 04:30
181 p.m., the separation gate between the calves' pen and the cowshed was left open so
182 that the calves could suckle their dams. Calves had free access to water, a hay rack
183 and a bucket with concentrate. The calf feeding plan is detailed in Table 1.

184 Cow feeding in Trials 1 and 2

185 The cows were fed ad libitum with a mixed ration (82% 1st-cut hay, 18% 2nd-cut hay
186 in Trial 1; 40% 1st-cut hay, 60% 2nd-cut hay in Trial 2) plus 5 kg/d of concentrates
187 (from Centraliment factory) distributed twice a day until early May (1.5 kg/d of protein
188 concentrate and 3.5 kg/d of energy concentrate in Trial 1; 1.0 kg/d of protein
189 concentrate and 4.0 kg/d of energy concentrate in Trial 2). From early May, the cows
190 went to pasture day and night, and received 2.0 kg of a pasture-specific concentrate
191 after each milking. During the two periods, cows had free access to water. In Trial 1,
192 the three groups of cows grazed together during the daytime and all calves stayed inside the barn. In Trial 2, the two groups of cows grazed in two adjacent plots, swapping every morning to ensure the same feeding, during the daytime. Half-day-calves went to pasture with the cows, while Control-calves stayed inside the barn.

196 Calf weaning in Trials 1 and 2

197 Weaning took place in batches, every two weeks. The calves were weaned at a BW of at least 100 kg, which was reached in average on Week 13 in Trial 1 (108 ± 3.9 kg) and Week 11 in Trial 2 (114 ± 2.2 kg). On the day of weaning, the calves to be weaned were moved to two collective pens [one for each group, 10 x 70 m] for one week, and then to the same collective pen (Trial 2) or were moved directly to a collective pen (Trial 1) due to lack of pen availability. After weaning calves and cows could not see each other, but they could hear each other as they were housed in the same cowshed. Post-weaning calves were fed concentrate and hay (Table 1).

205 Measurements

206 Trial 1

207 Individual milk yield at parlour was measured at each milking using milk flow meters (MM27BC, DeLaval, Tumba, Sweden). Individual milk samples of 30 mL were taken on four consecutive milkings per week and analyzed at Agrolab's (Aurillac, France) to determine milk fat and milk protein contents by mid-infrared spectroscopy. Milk SCC were measured by epi-fluorescence on two consecutive milkings per week until 16 weeks after calving to calculate average individual milk yield at the parlour and milk composition by week of lactation.
214 Calf BW was measured at birth then every Tuesday morning until 15 weeks of age to
215 calculate individual average daily gain (ADG) (ID 300 scale, TRU-TEST; 0.5 kg
216 precision, up to 250 kg). Before- and After-calves were weighed just before and 20
217 min after joining the cows, and the measures served to estimate the individual milk
218 intake by suckled calves per week until weaning as the difference in BW after and
219 before suckling. Daily milk intake by Control-calves was recorded by the automatic
220 milk feeder until weaning.

221 Animal caretakers checked clinical signs on animals at least once a day, they applied
222 Standard Operating Procedures to cure the animal affected and recorded the
223 disorder and the treatment used in a sanitary logbook. The quality of the records was
224 checked once a week by one of the authors of the present paper (M. Bouchon).
225 Health disorders were sorted into reproductive disorders (metritis, retention of the
226 placental membrane, ovarian cysts, and vaginitis) and non-reproductive disorders
227 (mastitis, milk fever, lameness, etc.) for cows, and respiratory disorders (runny nose,
228 coughing, dyspnoea, etc.) and non-respiratory disorders (diarrhoea, umbilical
229 infection, etc.) for calves.

230 Around weaning (the day before weaning [Day 0], the day of weaning [Day 1], and
231 Day 2, Day 4 and Day 7 after weaning), the calves and their respective dams were
232 observed with continuous direct observations by two observers trained by an
233 experienced ethologist. The consistency between observers and the trainer was
234 checked during pilot observations. Observers checked that they recorded similarly
235 the behaviour. Observations started at 02.00 p.m. and lasted for 5 min to note
236 whether they vocalized *frequently* (seven vocalizations per min or more), *from time to
237 time* (from one to six vocalization per min), or not (zero vocalizations), with no
238 distinction between high or low pitched vocalizations. Calculations were thereafter
239 made of the daily percentage of animals that vocalized (frequently or from time to time), by group and by type of animal (calf or cow).

241 *Trial 2*

242 Milk yield, milk composition, health disorders and observations around weaning were measured in the same way as for Trial 1. Cow BW was measured at calving (on two consecutive days) and at weaning (IRW q/ scale, DELAVAL; 1 kg precision, up to 15 243 244 000 kg). On the same dates, cow body condition score (BCS) was estimated on a 245 scale of 0 (very thin cow) to 5 (very fat cow; Bazin, 1984). Calf BW was measured at 246 birth then every Tuesday morning until 16 weeks of age to calculate individual ADG 247 between birth and weaning and at three weeks before and after weaning (same scale 248 as in Trial 1). Daily intake of milk by calves was not controlled. 249

250 From the beginning of March to the start of the grazing period (beginning of May), 251 once a week, Half-day-cow-calf pairs were observed during the first hour after the 252 morning milking by two trained observers who recorded all the calves' successful and 253 refused suckling attempts from their dam or from other cows. To estimate the 254 acceptance by cows of calves other than their own, we calculated the ratio between 255 percentage of successful sucking attempts by their own calf and that by other calves. 256

257 **Statistical analyses**

258 The data were analyzed using the MIXED procedure of the SAS 9.4 software 259 package (SAS Institute Inc., Cary, NC). SCC were log10-transformed to achieve 260 normal distribution. Individuals - cow or calf - were considered as statistical unit and 261 used in the models as random factors. For milk yield, milk composition, cow BW and 262 BCS, the model took into account the effects of practice (Control group and two CCC
groups in Trial 1; Control group and one CCC group in Trial 2), week of lactation
(repeated factor, for milk yield and milk composition only), breed (Ho or Mo), parity
(primiparous or multiparous) and interactions group × week, group × breed and group
× parity as fixed factors. Date of calving as well as initial values at calving (milk yield
index, fat content index, protein content index, and BCS) were used as covariates.
For calf BW, ADG and daily milk intake, the model considered the effects of practice,
week of age (repeated factor, for BW and milk intake only), breed, sex, and the
interactions group × week, group × breed and group × sex as fixed factors., Date of
birth and BW at birth were used as covariates. For all data, normality of residuals was
checked using the Shapiro-Wilk test and their homogeneity was checked visually. To
obtain best-fit models for each variable, fixed factors (other than group) and
interactions with P > 0.15 were discarded using a manual stepwise backwards
selection.

Frequency of health disorders and vocalizations around weaning was compared
between groups using a Chi-square test. Significance was set at P ≤ 0.05 and
tendency at 0.05 < P ≤ 0.10. The ratio acceptance of other calves by cows was
compared to 1 (no difference between own calves and others) using a Wilcoxon rank
sum test. As the weaning took place between the 9th and the 13th week, the
statistical analyses were carried out successively on 2 periods: before weaning
(Weeks 1-8), with t-test for paired comparisons between groups in Trial 1, and after
weaning (Weeks 14-16). Results are expressed as means and Standard Errors in
tables and figures. In the text, averages of the differences between groups or breeds
are reported.

286 **Results**
287 **Trial 1**

288 The experiment was stopped for the After-group on 12 June 2017, when After-calves
289 were about 8 weeks of age (see the subsection headed *Cow and calf health* for
290 details). The results reported in Table 1 refer to Weeks 1 to 8 for all groups and to
291 Weeks 14 to 16 for Control- and Before-groups only.

292 *Milk yield at parlour*

293 During the first 8 weeks of lactation, Before-cows produced 12.5 kg/d less milk per
cow and After-cows produced 7.0 kg/d less milk per cow than Control-cows. The
difference was marked especially at morning milking in Before-cows (-10.3 kg/d;
295 Table 2). The difference in milk yield between Before- and Control-cows increased up
to 13.1 kg/d from Week 1 to Week 4, then it stabilized at 11.0 kg/d from Week 5 to
297 Week 12 (Figure 1). The difference in milk yield between After- and Control-cows
decreased by up to 6.3 kg/d from Week 1 to Week 4, then it stabilized at 4.5 kg/d
299 until the trial was stopped for After-cows on Week 8. Milk yield was higher for Ho
cows than Mo cows from Week 1 to Week 8 (+4.6 kg/d). There was no interaction
301 between breed and cow group on milk yield at parlour.
302

303 The estimated total milk yield of nursing cows – including the milk suckled by calves
(see below) – was lower than the milk produced by Control-cows, by 5.3 kg/d in
304 Before-cows and 4.0 kg/d for After-cows. The difference was especially marked on
305 Week 2, with Before- and After-cows producing 9.3 kg/d and 8.1 kg/d less than
306 Control-cows. The difference thereafter diminished and disappeared on Week 7.
307 From Week 14 to Week 16, milk yield of Before-cows increased but remained lower
308 than that of Controls (-5.2 kg/d).
309

310 *Milk composition at parlour*
During the first eight weeks of lactation, average milk fat content was lower for After-cows compared to Control-cows (-0.47 percent point [pp]) or to Before-cows (-0.67 pp; Table 2). At morning milking, milk fat content was lower in Before-cows than in the other groups, whereas at evening milking milk fat content was lower in After-cows than in the other groups. A Group × Breed interaction was observed in both morning and evening milking: in After-cows, milk fat content was lower in Mo cows than in Ho cows (-0.45 pp, on average). Milk protein content was higher in Before-cows than in the other groups (0.28 pp on average) and this difference was similar in morning and evening milk. Milk protein content was higher in Mo cows than in Ho cows in all three groups in morning and evening milk (+0.22 pp on average). Before- and After-cows had about 30 000 somatic cells/mL of milk more than Control-cows, but this difference was not significant. From Week 14 to Week 16, the differences in milk fat content between Before- and Control-cows increased up to 0.28 pp and became significant, whereas the difference in protein content decreased to 0.14 pp but stayed significant. SCC was again higher in Before-cows than Control-cows (+75 000) but the difference was still not significant.

### Milk intake by calves

During their first eight weeks of life, Before-calves ingested more milk (+1.24 kg/d) and After-calves ingested less milk (-3.03 kg/d) than Control-calves (Table 2), with no overall between-breed difference but a significant breed × group interaction. Within Before-calves, Ho ingested more milk than Mo (+1.20 kg/d) whereas within After-calves, Ho ingested less milk than Mo (-1.04 kg/d). From birth to weaning, Before-calves ingested 2.72 kg/d more milk than Control-calves; and within Before-calves, Ho again ingested more milk than Mo (+1.13 kg/d).

### Calf growth
336 From Week 1 to Week 8, After-calves had 42% less ADG and Before-calves 26%
337 more ADG than Control-calves (Table 2). The BW difference between Control-calves
338 and After-calves increased progressively from 2.5 kg to 13.9 kg (Figure 2). Because
339 of this low growth and a number of health disorders (see under Cow and calf health),
340 the experiment was stopped for the After-group. The BW difference between Before-
341 calves and Control-calves was constant up to 6 weeks of age (+3.5 kg on average)
342 and then became higher from Week 7 to Week 15 (+10.2 kg on average). From birth
343 to weaning, Before-calves had 162 g/d higher ADG than Control-calves.

344 *Cow and calf health*

345 During the 16 weeks of the trial, the frequency of health disorders was not
346 significantly different between groups of cows. Reproductive disorders occurred once
347 in Before-cows and once in After-cows, and non-reproductive disorders occurred six
348 times in Control-cows, seven times in Before-cows and four times in After-cows. We
349 observed statistical difference between groups of calves both in respiratory (P = 0.02)
350 and non-respiratory (P = 0.001) disorders. No disorders were observed in Control-
351 calves. Three Before-calves and six After-calves were diagnosed with respiratory
352 disorder due to bovine respiratory syncytial virus (BRSV). Despite medical treatment
353 (anti-inflammatory drugs, vaccine against BRSV, and antibiotics to prevent
354 complications), two Before-calves and four After-calves died. Four After-calves had
355 episodes of diarrhoea, including two affected by the respiratory disorder. We decided
356 to stop the experiment for the After-calves on 12 June due to the high calf morbidity
357 and mortality and their low BW gain.

358 *Vocalizations at weaning*
As the trial was stopped prematurely for the After-group, observations at weaning were not performed. Control-calves vocalized mainly on Day 2 after weaning (75% of the calves; Figure 3). Before-calves started to vocalize earlier (92% on Day 1) and continued to vocalize later (75% on Day 4). On Day 7, only 17% of Before-calves were still vocalizing. Before-cows started to vocalize on Day 1, but less frequently than their calves (55%), and stopped vocalizing earlier than their calves (27% on Day 4). On Day 7, no Before-cows were vocalizing.

## Trial 2

### Milk yield at parlour

During the first 8 weeks of lactation, Half-day-cows produced 11.4 kg/d less milk than Control-cows (Table 3). The milk loss was distributed between morning (-4.4 kg/d) and evening (-7.3 kg/d) milkings. From Week 1 to Week 3, the milk yield of Half-day-cows increased by 7.9 kg/d, and the difference between Control- and Half-day-cows was about 9.4 kg/d (Figure 4). The milk yield of Control-cows stabilized at 28.6 kg/d from Week 3 to Week 11, and then started to decrease from Week 12 (Figure 4). The milk yield of Half-day-cows decreased significantly from Week 3 to Week 8 (-3.0 kg/d; $P = 0.02$) and then increased steadily to reach the same milk yield as Control-cows on Week 14 (25.7 kg/d). During the first 8 weeks of lactation, the loss in milk yield due to nursing was higher for Ho tan Mo cows especially at evening milking (Ho, -8.3 kg/d i.e. 72%; Mo, -6.8 kg/d i.e. -62%) whereas only a tendency was observed for daily milk yield (Ho, -14.6 kg/d i.e. -50%; Mo, -8.33 kg/d i.e. -34%).

### Milk composition at parlour
During the first 8 weeks of lactation, milk fat content was lower in Half-day-cows than Control-cows (-0.78 pp), with a similar difference between morning and evening milk (Table 3). Conversely, milk protein content was higher in Half-day-cows than in Control-cows (+0.13 pp). No significant difference in SCC was noticed despite higher values in Before-cows (+36 000 cell/mL).

From Week 14 to Week 16, no difference was found on milk yield, milk composition and milk SCC between Half-day- and Control-cows (Table 3).

### Body condition of cows

At the time of calf weaning, Half-day- and Control-cows had similar BW (633 kg) and BCS (1.53 points; Table 3).

### Calf growth

Half-day- and Control-calves had similar BW until the end of the trial (Figure 5).
From birth to weaning, ADG was similar in Half-day- and Control-calves, and was higher in Mo than in Ho calves (+176 g/d; Table 3). However, during the three weeks before weaning ADG was higher in Half-day-calves than in Control-calves (+230 g/d).
During the 3 weeks after weaning, ADG was the same between groups and was lower in Mo than in Ho calves (-222 g/d). The difference between Mo and Ho was marked in Control-calves (-365 g/d; P < 0.001) whereas it was not observed in Half-day-calves (-81 g/d; P = 0.42).

### Cow and calf health

During the 16 weeks of Trial 2, the frequency of health disorders was not significantly different between Half-day- and Control-cows. Reproductive disorders occurred five times in Half-day-cows and two times in Control-cows, and non-reproductive
405 disorders occurred seven times in Half-day-cows and five times in Control-cows. In
406 calves, there were no significant between-group differences in frequency of
407 respiratory disorders (seven occurrences in Half-day-calves vs. five in Control-calves)
408 or non-respiratory disorders (two occurrences in Half-day-calves vs. three in Control-
409 calves). No calves died during Trial 2.

410 Vocalizations at weaning

411 Most of the Control-calves vocalized from Day 2 to Day 4 after weaning (Figure 3).
412 Half-day-calves started vocalizing earlier than Control-calves (100% on Day 1) and
413 continued vocalizing throughout the week (93% on Day 4, 43% on Day 7). All the
414 Half-day-cows started vocalizing on Day 1, but tended to stop vocalizing earlier than
415 their calves (64% vs. 93% on Day 4).

416 Acceptance of calves by cows

417 On average, the acceptance by Mo cows of calves other than their own was
418 significantly higher than 1 (1.46 ± 0.14, P < 0.05). Conversely, the acceptance of
419 another calf by Ho cows was not significantly higher than 1 (1.28 ± 0.14, P > 0.05).

420 Discussion

421 Here we tested three practices of restricted CCC that allowed suckling: two practices
422 that allowed only short contacts during the day, i.e. just before (for 20 min/d) or after
423 (for 2.5 h/d) the morning milking, and one practice that allowed a long period of free
424 cow-calf contact (for 9 h/d) between the morning and evening milkings. First, we
425 discuss the pro and cons of each practice before addressing common aspects in
426 terms of milk production and weaning.
427 **Suckling for 2.5 h immediately after milking fails to cover the calves' nutritional needs while significantly reducing the amount of sellable milk**
428

429 We expected calves to be able to ingest the milk left after milking with minimal impact
430 on milk yield at parlour. Allowing calves to suckle their dam for 2.5 h immediately
431 after milking (group referred to as After) was thus tested as a way to minimize the
432 impact of suckling on sellable milk. Under this practice, the cows yielded on average
433 29.0% less sellable milk than Controls. The calves however suckled little milk (3.0
434 kg/d). As a consequence, they had very low BW gain (half that of Control-calves) and
435 were in poor health. We had to stop this suckling practice after eight weeks to avoid
436 putting the calves at too much risk. When After-calves joined the cows, not enough
437 milk may have remained in the udder. We conclude that there is no benefit in
438 allowing calves to suckle for a limited time immediately after milking. If suckling takes
439 place after milking, it seems more beneficial to reunite cows and calves later - e.g. 2
440 h after milking - and to keep them together until the calf has suckled (De Passillé et
441 al., 2008).

442 **Suckling for 20 min before milking satisfies the calves' nutritional needs but drastically reduces the amount of sellable milk**
443

444 Allowing calves to suckle their dam for 20 min before milking (group referred to as
445 Before) was tested here as a way to give calves a short period of access to large
446 quantities of milk that should cover their nutritional needs. Indeed, during the first
447 eight weeks, Before-calves ingested 20% more milk than the Control-calves that
448 were provided milk from an automatic feeder (in quantity equivalent to 13% of their
449 BW). Not all Control-calves ingested all the milk offered to them. Being able to suckle
450 the dam seems thus to stimulate milk ingestion more than delivering milk by a feeder,
even when the feeder is equipped with teats. Control-calves had free access to a single teat for the whole group, which may have limited the intake; we however did not notice competition between calves probably due to the milk being available for the whole day. Before-calves grew faster than Control-calves (ADG: +23.2% in Before-calves), which confirms earlier findings that suckling is beneficial to calf growth (e.g. Roth et al., 2009). This benefit may come from the higher amounts of milk ingested or from the positive effect of suckling on calf metabolism (Uvnäs-Moberg et al., 2001). We did not observe any improvement in the health of Before-calves compared to Controls. Testing benefits on health however would require close clinical examination of large numbers of calves, which was not the case in our study.

Milk release by cows during milking was strongly reduced when the cows suckled their calves just before the morning milking (51.2% less milk than in Control-cows).

However, this decrease was still lower than that reported by Barth (2020) in cows suckling their calves just before both daily milkings (71% less milk). In our experiment, the reduction in milk yield was very marked at morning milking (75.5% less milk), which was expected because the calves started to empty the udder before milking, but was still visible at the evening milking (14.9% less milk).

**Allowing cow-calf contact from morning to evening milking offers a good compromise between meeting calves nutritional requirements and preserving sellable milk**

To minimize the impact of suckling on the amount of milk collected at the milking parlour while at the same time allowing calves to drink enough milk from their dam, we tested a third practice that consisted of reuniting cows and calves for 9 h a day between the morning and the evening milkings (group referred to as Half-day). In
475 parallel, we increased the amount of milk offered to Control-calves (14% BW),
476 because some of the Control-calves in Trial 1 may have not been provided enough
477 milk as some of them drank all that was offered. On average, the Half-day-cows
478 produced 42.4% less milk than Controls. One could expect no reduction at the
479 morning milking because the cows and calves were separated at night, but this was
480 not the case (reduction of 26.3%).

481 Half-day- and Control-calves had a similar growth, which suggests they ingested
482 similar amounts of nutrients.

483 *Suckling reduces milk yield and affects milk composition in all scenarios*

484 In our experiments, suckling cows always yielded less milk at the milking parlour than
485 non-suckling cows: 28.7% less when the calves had access to the cows just after the
486 morning milking (for 2.5 h), 42.4% less when the calves had access to the cows
487 between milkings (for 9 h), and 51.2% less when they had access to the cows for a
488 short period (20 min) just before the morning milking. These reduced yields are
489 consistent with those reported in the literature: 24% less when suckling takes place
490 for 2 h after milking (de Passillé et al., 2008), and 43% less when the calves stay with
491 the dam between evening and morning milkings (Barth, 2020). As already reported
492 by Fröberg et al. (2005) and Barth (2020), the reduction in milk volume collected at
493 the milking parlour cannot be explained solely by the milk ingested by calves. Indeed,
494 in Trial 1 where milk ingested by calves was measured, the reduction of sellable milk
495 exceeded the amount of milk suckled by calves, and therefore total milk production
496 was still lower than that of Control-cows, by 21.7% in Before-cows and 16.5% in
497 After-cows. The oxytocin release at milking is less marked when cows suckle their
498 calves, either when suckling occurs just before milking (Lupoli et al., 2001) or after
499 milking (de Passillé et al., 2008). This could explain partly why all suckling cows in
500 this experiment released less milk at the milking parlour even when their calves drank
501 very little milk. Nevertheless, the decreased total milk production occurred only at the
502 time of the lactation peak, which was absent in Before- and After-cows, whereas
503 interestingly, total milk production of Control- and Before-cows was similar after
504 Week 8 although calves were still suckling. This observation, never reported before,
505 deserve to be confirmed in further trials where milk ingestion by suckled calves is
506 measured.

507 In the Half-day-cows, the higher reduction in milk obtained at the parlour in Ho cows
508 compared to Mo cows - especially at evening milking - could be at least partly
509 explained by their slightly higher acceptance of calves other than their own.
510 Moreover, the higher milk yield of Ho cows may make them more attractive to calves.

511 After separation from their calves, the milk yield at parlour increased in all suckling
512 cows. Half-day-cows reached the milk yield of Control-cows within a week, whereas
513 Before-cows never managed to 'catch up' with Control-cows (-25.2% from week 14 to
514 16). Similar observations were reported by Barth (2020) in cows suckling their calves
515 before milking. Cows from some breeds (like Salers or Zebu-Holstein) can only be
516 milked if the calves first initiate milk release by a short suckling (Fröberg et al., 2007;
517 Guiadeur et al., 2011). We suspect that this same kind of process sets in when dairy
518 cows get used to suckle just before milking, making it more difficult to milk them at
519 the parlour when suckling ends (Tančin & Bruckmaier, 2001).

520 Suckling had effects on milk composition. Milk fat content decreased when cows
521 suckled after milking or between milkings and increased when they suckled just
522 before milking. When suckling occurs after milking, the calves mainly consume the
523 residual milk, which has a high fat content, whereas when suckling occurs before
524 milking, the calves suckle the cisternal milk, which has a low fat content (Rico et al., 2014). These variations in turn affect the fat content of the milked milk. Milk protein content increased in Before- and Half-day-cows, which confirms previous results from Margerison et al. (2002) and Barth (2020). Milk protein content increases when the energy balance of the cows is higher (Coulon & Rémond, 1991). Because suckling cows produced less total (suckled + milked) milk and presumably had similar feed intake, their energy balance was probably higher than that of Control-cows.

531 Suckling is thought to improve udder health (Fröberg et al., 2005; Margerison et al., 2002). However, in our studies we never found difference in SCC or frequency of mastitis between CCC practices and Controls. As already noticed by Johnsen et al. (2016) in their review on CCC, the beneficial effect of suckling on cows' udder health is not always observed.

536 **Weaning is stressful for both calves and cows**

537 All calves vocalised at weaning. Weaning has a psychological component due to the associated changes in environment: separation from the dam for calves suckling their dam, changes in accommodation (for all calves in our experiments), and changes in feeding routines (Jasper et al., 2008; Veissier et al., 1989; Weary et al., 2008).

541 Almost all the Before-calves and Half-day-calves vocalized on the day of weaning whereas Control-calves vocalized about 24 h later. Vocalizations can be at least partly due to hunger since calves vocalize less when they have access to milk after the separation from their dams (Johnsen et al., 2018). According to Thomas et al. (2001), vocalizations in the first hours after weaning are due to the separation from the dam, and vocalizations later on are due to hunger. This suggests that at weaning, Control-calves reacted to the lack of milk whereas Before- and Half-day-calves
548 reacted also to the separation from the dam and were stressed for longer as they
549 vocalised for several days.

550 The cows vocalized for two days after separation, either from time to time (Before-
551 cows) or more frequently (Half-day-cows). In our study, calves were weaned
552 according to their age and were therefore removed not all at the same time. As most
553 cows suckled other calves after their own calves had been weaned, we can rule out
554 the hypothesis of vocalizations reflecting discomfort produced by a distended udder
555 due to lack of suckling after weaning. Cows establish strong bonds with their
556 offspring, and if bonding is followed by an abrupt separation, the cows manifest
557 stress reactions such as restlessness and vocalizations (Flower & Weary, 2001;
558 Weary & Chua, 2000). Cow vocalizations at weaning are thus likely to reflect stress
559 experienced by cows due to separation from their calves.

560 In conclusion, a short cow-calf contact (2.5 h) immediately after milking does not
561 provide enough milk for the calves, whereas a short contact (20 min) immediately
562 before milking strongly decreases the amount of sellable milk. Allowing a long period
563 of CCC (9 h) between morning and evening milkings makes good compromise
564 between sellable milk and calf growth. Contrary to what was expected, weaning
565 induces a stress in cows and calves that have experienced restricted suckling. To
566 promote animal welfare by allowing cow-calf contact and suckling, it is therefore
567 necessary to reduce weaning-related stress. Restricted suckling probably needs to
568 be followed by a two-step weaning process by using nose-flaps for a few days before
569 the separation or by using a fence-line separation to enable continued visual and
570 some tactile contact after weaning before complete separation (Haley et al., 2005;
571 Johnsen et al., 2018; Loberg et al., 2008; Price et al., 2014).
The reduction in sellable milk and in its fat content due to suckling will affect the revenue of the farmer from the milk production. Suckling however may affect positively calves' growth and health, cows' career, farmer workload, etc. All these effects should be considered to assess the net impact on farmers' income. If the reduction in sellable milk is not balanced by benefits then the opportunity to generate added value for this practice should be investigated, considering the demand for certification aiming at identifying "animal welfare" practices or "husbandry systems" traceability (Beaver et al., 2020; Janssen et al., 2016).

### 580 Ethics approval

All procedures were carried out in accordance with French Ministry of Agriculture guidelines on animal research and with all other applicable national and European regulations governing experiments with animals. All researchers responsible for the study (D. Pomiès, B. Martin, M. Bouchon, and I. Veissier) and all animal caretakers had adequate appropriate training, and the experimental farm is accredited for running experiments (C15-114-01). The two trials did not require approval by an ethics committee.

### 588 Data and model availability statement

None of the data were deposited in an official repository. The data that support the study findings are available from authors upon request.

### 591 Author ORCIDs

Alessandra Nicolao, https://orcid.org/0000-0003-1316-1869

Isabelle Veissier, https://orcid.org/0000-0002-8497-5395
594 Matthieu Bouchon, https://orcid.org/0000-0002-7175-7233

595 Enrico Sturaro, https://orcid.org/0000-0001-9508-5622

596 Bruno Martin, https://orcid.org/0000-0003-2501-8306

597 Dominique Pomiès, https://orcid.org/0000-0002-4043-4423

598 **Author contributions**

599 **Alessandra Nicolao**: Methodology, Validation, Formal analysis, Investigation,

600 Writing - Original Draft, Writing - Review & Editing, Visualization. **Isabelle Veissier**:

601 Conceptualization, Methodology, Formal analysis, Writing - Original Draft, Writing -

602 Review & Editing, Supervision. **Matthieu Bouchon**: Methodology, Validation,

603 Investigation, Resources, Data Curation, Writing - Review & Editing. **Enrico Sturaro**:

604 Conceptualization, Writing - Review & Editing, Supervision, Funding acquisition.

605 **Bruno Martin**: Conceptualization, Methodology, Formal analysis, Writing - Original

606 Draft, Writing - Review & Editing, Supervision, Project administration, Funding

607 acquisition. **Dominique Pomiès**: Conceptualization, Methodology, Validation, Formal

608 analysis, Investigation, Writing - Original Draft, Writing - Review & Editing,

609 Visualization, Supervision, Funding acquisition.

610 **Declaration of interest**

611 None.

612 **Acknowledgements**

613 The authors thank the staff of the INRAE Herbipôle farm in Marcenat for animal care,

614 Nadège Aigueperse for her help in experimental design and behavioural data
615 processing, Anne de la Foye for her advice on statistical analyses, and Hélène Bec
616 for her active participation in Trial 2.

617 **Financial support statement**

618 This study was part of the EU project ProYoungStock funded by CORE organic. This
619 research was supported by French government IDEX-ISITE initiative 16-IDEX-0001
620 (CAP 20-25). Trial 1 was partly funded by the PHASE Research Division of INRAE.
621 A. Nicolao received a doctoral fellowship from the Fondazione Cariparo at University
622 of Padova (Italy).

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<caption>800 Table 1</caption>

801 Feeding plans used for the three groups of calves in Trial 1 and the two groups of calves in Trial 2 during the first 16 weeks of age.

<table><thead><tr><th rowspan="2">Trial</th><th rowspan="2">Group<sup>1</sup></th><th rowspan="2">Feed<sup>2</sup></th><th colspan="16">Weeks of age</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th><th>13</th><th>14</th><th>15</th><th>16</th></tr></thead><tbody><tr><td rowspan="16">1</td><td rowspan="3">Control</td><td>Milk<sup>3</sup> (kg/d)</td><td>6.0</td><td>6.0</td><td>7.0</td><td>8.0</td><td>8.0</td><td>8.0</td><td>8.0</td><td>7.0</td><td>6.0</td><td>5.0</td><td>4.0</td><td>3.0</td><td>Weaning</td><td></td><td></td><td></td></tr><tr><td>Concentrate<sup>4</sup> (kg/d)</td><td>0.0</td><td>0.0</td><td>0.2</td><td>0.2</td><td>0.3</td><td>0.5</td><td>0.7</td><td>0.9</td><td>1.1</td><td>1.4</td><td>1.7</td><td>2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td></tr><tr><td>Hay<sup>5</sup></td><td>0.0</td><td>0.0</td><td colspan="16">Ad libitum</td></tr><tr><td rowspan="3">Before</td><td>Milk (suckling)<sup>6</sup></td><td>Twice a day</td><td colspan="16">During 20 min before the morning milking</td></tr><tr><td>Concentrate (kg/d)</td><td>0.0</td><td>0.0</td><td>0.2</td><td>0.2</td><td>0.3</td><td>0.5</td><td>0.7</td><td>0.9</td><td>1.1</td><td>1.4</td><td>1.7</td><td>2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td></tr><tr><td>Hay</td><td>0.0</td><td>0.0</td><td colspan="16">Ad libitum</td></tr><tr><td rowspan="3">After</td><td>Milk (suckling)</td><td>Twice a day</td><td colspan="16">For 2.5 hours after the morning milking</td></tr><tr><td>Concentrate (kg/d)</td><td>0.0</td><td>0.0</td><td>0.2</td><td>0.2</td><td>0.3</td><td>0.5</td><td>0.7</td><td>0.9</td><td>1.1</td><td>1.4</td><td>1.7</td><td>2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td></tr><tr><td>Hay</td><td>0.0</td><td>0.0</td><td colspan="16">Ad libitum</td></tr><tr><td rowspan="16">2</td><td rowspan="3">Control</td><td>Milk (kg/d)</td><td>6.0</td><td>7.0</td><td>9.0</td><td>10.0</td><td>10.0</td><td>10.0</td><td>9.0</td><td>7.0</td><td>5.0</td><td>3.0</td><td>Weaning</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Concentrate (kg/d)</td><td>0.0</td><td>0.0</td><td>0.2</td><td>0.4</td><td>0.6</td><td>0.9</td><td>1.2</td><td>1.5</td><td>1.8</td><td>2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td><td>~2.0</td></tr><tr><td>Hay</td><td>0.0</td><td colspan="16">Ad libitum</td></tr><tr><td rowspan="3">Half-day</td><td>Milk (suckling)</td><td>24 h/d</td><td colspan="16">From morning to evening milking</td></tr><tr><td>Concentrate (kg/d)</td><td>0.0</td><td colspan="16">Ad libitum</td></tr><tr><td>Hay</td><td>0.0</td><td colspan="16">Ad libitum</td></tr></tbody></table>

<footnote n="802">802 <sup>1</sup>Control-calves were separated from their dam at birth; Before-calves were able to suckle their dam for 20 min before the morning milking; After-calves were able to suckle their dam for 2.5 h after the morning milking; Half-day-calves were able to suckle their dam for 9 h between the morning and evening milkings.</footnote>

<footnote n="803">803 <sup>2</sup> All quantities are expressed as fed per day.</footnote>

<footnote n="805">805 <sup>3</sup> Bulk milk distributed individually by an automatic feeder.</footnote>
806 ⁴Starter-age concentrate distributed individually by an automatic feeder (Trial 1 and Control group in Trial 2) or in a collective bucket (Half-day-group in Trial 2).

807 ⁵Permanent grassland hay (first cut) distributed in a rack.

808 ⁶Milk available by suckling dams during specific periods.
<table><thead><tr><th rowspan="2"></th><th colspan="3">Group<sup>1</sup></th><th colspan="2">Breed<sup>2</sup></th><th rowspan="2">SEM</th><th colspan="3">P-value<sup>3</sup></th></tr><tr><th>Control</th><th>Before</th><th>After</th><th>Ho</th><th>Mo</th><th>Group</th><th>Breed</th><th>Group × Breed</th></tr></thead><tbody><tr><td colspan="10"><strong>Week 1 to week 8<sup>4</sup></strong></td></tr><tr><td>Milk yield (kg/d)</td><td>24.4<sup>c</sup></td><td>11.9<sup>a</sup></td><td>17.4<sup>b</sup></td><td>20.2</td><td>15.6</td><td>0.79</td><td>&lt;0.001</td><td>&lt;0.001</td><td></td></tr><tr><td>Morning milk yield (kg/d)</td><td>14.4<sup>c</sup></td><td>4.1<sup>a</sup></td><td>11.6<sup>b</sup></td><td>11.1</td><td>8.95</td><td>0.46</td><td>&lt;0.001</td><td>&lt;0.001</td><td></td></tr><tr><td>Evening milk yield (kg/d)</td><td>9.85<sup>c</sup></td><td>8.38<sup>b</sup></td><td>5.91<sup>a</sup></td><td>9.13</td><td>6.97</td><td>0.35</td><td>&lt;0.001</td><td>&lt;0.001</td><td></td></tr><tr><td>Milk fat content (%)</td><td>3.58<sup>b</sup></td><td>3.78<sup>b</sup></td><td>3.11<sup>a</sup></td><td>3.55</td><td>3.44</td><td>0.08</td><td>&lt;0.001</td><td>0.132</td><td>0.079</td></tr><tr><td>Morning milk fat content (%)</td><td>3.12<sup>b</sup></td><td>2.64<sup>a</sup></td><td>2.92<sup>b</sup></td><td></td><td></td><td>0.08</td><td>&lt;0.001</td><td></td><td>0.055</td></tr><tr><td>Evening milk fat content (%)</td><td>4.23<sup>b</sup></td><td>4.24<sup>b</sup></td><td>3.38<sup>a</sup></td><td></td><td></td><td>0.08</td><td>&lt;0.001</td><td></td><td>0.028</td></tr><tr><td>Milk protein content (%)</td><td>2.97<sup>a</sup></td><td>3.27<sup>b</sup></td><td>3.00<sup>a</sup></td><td>2.98</td><td>3.19</td><td>0.04</td><td>&lt;0.001</td><td>&lt;0.001</td><td></td></tr><tr><td>Morning milk protein content (%)</td><td>2.98<sup>a</sup></td><td>3.32<sup>b</sup></td><td>3.01<sup>a</sup></td><td>2.94</td><td>3.21</td><td>0.03</td><td>&lt;0.001</td><td>&lt;0.001</td><td></td></tr><tr><td>Evening milk protein content (%)</td><td>2.97<sup>a</sup></td><td>3.26<sup>b</sup></td><td>2.96<sup>a</sup></td><td>2.97</td><td>3.15</td><td>0.04</td><td>0.001</td><td>0.001</td><td>0.103</td></tr><tr><td>Milk SCC (log<sub>10</sub>/mL)</td><td>4.64</td><td>4.88</td><td>4.87</td><td></td><td></td><td>0.11</td><td>0.186</td><td></td><td></td></tr><tr><td>Milk ingested by calf (kg/d)</td><td>6.00<sup>b</sup></td><td>7.24<sup>c</sup></td><td>2.97<sup>a</sup></td><td></td><td></td><td>0.23</td><td>&lt;0.001</td><td></td><td>0.004</td></tr><tr><td>ADG until 8 weeks of age (g/d)</td><td>587<sup>b</sup></td><td>740<sup>c</sup></td><td>340<sup>a</sup></td><td>493</td><td>617</td><td>40.1</td><td>&lt;0.001</td><td>0.012</td><td></td></tr><tr><td colspan="10"><strong>Week 14 to week 16<sup>5</sup></strong></td></tr><tr><td>Milk yield (kg/d)</td><td>20.4</td><td>15.3</td><td></td><td></td><td></td><td>0.85</td><td>&lt;0.001</td><td></td><td></td></tr><tr><td>Milk fat content (%)</td><td>3.47</td><td>3.75</td><td></td><td></td><td></td><td>0.06</td><td>0.004</td><td></td><td></td></tr><tr><td>Milk protein content (%)</td><td>2.84</td><td>2.98</td><td></td><td>2.84</td><td>2.97</td><td>0.04</td><td>0.035</td><td>0.049</td><td></td></tr><tr><td>Milk SCC (log<sub>10</sub>/mL)</td><td>4.71</td><td>4.91</td><td></td><td>5.05</td><td>4.57</td><td>0.14</td><td>0.351</td><td>0.026</td><td></td></tr><tr><td>Milk ingested until weaning<sup>6</sup> (kg/d)</td><td>5.63</td><td>8.35</td><td></td><td>7.31</td><td>6.84</td><td>0.20</td><td>&lt;0.001</td><td>0.063</td><td>0.018</td></tr><tr><td>Calf ADG until weaning (g/d)</td><td>699</td><td>861</td><td></td><td></td><td></td><td>35.6</td><td>0.004</td><td></td><td></td></tr></tbody></table>

Abbreviations: SCC = somatic cell count; ADG = average daily gain.
812 ¹ In Control-group, calves were separated from their dam at birth; in Before-group, calves were able to suckle their dam for 20 min before the morning milking; in

813 After-group, calves were able to suckle their dam for 2.5 h after the morning milking.

814 ² Each group consisted of 7 Holstein (Ho) and 7 Montbéliarde (Mo) cow-calf pairs.

815 ³ Adjusted values and *P*-values by group (Control, Before, After), breed (Ho, Mo), and group × breed interactions.

816 ⁴ When all Before- and After-calves could suckle their dam.

817 ⁵ When all calves were separated from their dam.

818 ⁶ Weaning took place when the calves weighed at least 100 kg.

819 a,b Values within a row with different superscripts differ significantly at *P*<0.05.
<table><thead><tr><th rowspan="2"></th><th colspan="2">Group¹</th><th colspan="2">Breed²</th><th rowspan="2">SEM</th><th colspan="3">P-value³</th></tr><tr><th>Control</th><th>Half-day</th><th>Ho</th><th>Mo</th><th>Group</th><th>Breed</th><th>Group × Breed</th></tr></thead><tbody><tr><td colspan="9"><strong>Week 1 to week 8⁴</strong></td></tr><tr><td>Milk yield (kg/d)</td><td>26.9</td><td>15.5</td><td></td><td></td><td>1.19</td><td>&lt;0.001</td><td></td><td>0.092</td></tr><tr><td>Morning milk yield (kg/d)</td><td>16.7</td><td>12.3</td><td></td><td></td><td>0.88</td><td>0.001</td><td></td><td></td></tr><tr><td>Evening milk yield (kg/d)</td><td>10.8</td><td>3.54</td><td></td><td></td><td>0.37</td><td>&lt;0.001</td><td></td><td>0.052</td></tr><tr><td>Milk fat content (%)</td><td>3.84</td><td>3.06</td><td></td><td></td><td>0.13</td><td>&lt;0.001</td><td></td><td></td></tr><tr><td>Morning milk fat content (%)</td><td>3.46</td><td>2.96</td><td></td><td></td><td>0.15</td><td>0.024</td><td></td><td></td></tr><tr><td>Evening milk fat content (%)</td><td>4.38</td><td>3.22</td><td>3.97</td><td>3.63</td><td>0.10</td><td>&lt;0.001</td><td>0.023</td><td>0.106</td></tr><tr><td>Milk protein content (%)</td><td>2.98</td><td>3.11</td><td></td><td></td><td>0.04</td><td>0.034</td><td></td><td></td></tr><tr><td>Morning milk protein content (%)</td><td>2.99</td><td>3.11</td><td></td><td></td><td>0.04</td><td>0.058</td><td></td><td></td></tr><tr><td>Evening milk protein content (%)</td><td>2.98</td><td>3.09</td><td></td><td></td><td>0.05</td><td>0.115</td><td></td><td></td></tr><tr><td>Milk SCC (log<sub>10</sub>/mL)</td><td>4.68</td><td>4.92</td><td>4.94</td><td>4.67</td><td>0.11</td><td>0.115</td><td>0.087</td><td></td></tr><tr><td colspan="9"><strong>Week 14 to week 16⁵</strong></td></tr><tr><td>Milk yield (kg/d)</td><td>23.7</td><td>23.1</td><td>24.3</td><td>22.5</td><td>0.80</td><td>0.542</td><td>0.116</td><td></td></tr><tr><td>Milk fat content (%)</td><td>3.33</td><td>3.39</td><td></td><td></td><td>0.05</td><td>0.387</td><td></td><td></td></tr><tr><td>Milk protein content (%)</td><td>2.88</td><td>2.98</td><td></td><td></td><td>0.04</td><td>0.142</td><td></td><td></td></tr><tr><td>Milk SCC (log<sub>10</sub>/mL)</td><td>4.90</td><td>4.85</td><td></td><td></td><td>0.12</td><td>0.755</td><td></td><td></td></tr><tr><td colspan="9"><strong>Week 1 to weaning⁶</strong></td></tr><tr><td>Cow BCS at weaning (0-5 scale)</td><td>1.52</td><td>1.54</td><td>1.45</td><td>1.62</td><td>0.11</td><td>0.852</td><td>0.108</td><td></td></tr><tr><td>Cow BW at weaning (kg)</td><td>633</td><td>639</td><td></td><td></td><td>8.50</td><td>0.617</td><td></td><td></td></tr><tr><td>Calf ADG until weaning (g/d)</td><td>943</td><td>929</td><td>848</td><td>1024</td><td>36.5</td><td>0.779</td><td>0.004</td><td></td></tr><tr><td>Calf ADG 3 weeks before weaning (g/d)</td><td>1176</td><td>1406</td><td>1208</td><td>1374</td><td>60.1</td><td>0.015</td><td>0.111</td><td></td></tr></tbody></table>
Calf ADG 3 weeks after weaning (g/d) 510 410 571 349 44.7 0.136 0.007 0.034

822

Abbreviations: SCC = somatic cell count; BCS = body condition score; ADG = average daily gain.

823

¹ In Control-group, calves were separated from their dam at birth; in Half-day-group, calves were able to suckle their dam for 9 h between morning and evening milkings.

824

825

² Each group consisted of 7 Holstein (Ho) and 7 Montbéliarde (Mo) cow-calf pairs.

826

³ Adjusted values and *P*-values by group (Control, Half-day), breed (Ho, Mo), and group × breed interactions.

827

⁴ When all Half-day-calves could suckle their dam.

828

⁵ When all calves were separated from their dam.

829

⁶ Weaning took place when the calves weighed at least 100 kg.
**Figure 1** Daily milk yield at the parlour for the three groups of cows in Trial 1, by week of lactation (means and SE of raw data). In Control-group, calves were separated from their dam at birth; in Before-group, calves were able to suckle their dam for 20 min before the morning milking; in After-group, calves were able to suckle their dam for 2.5 h after the morning milking. Weaning took place when the calves weighed at least 100 kg. Each group consisted of 7 Holstein (Ho) and 7 Montbéliarde (Mo) cow-calf pairs. The grey lines represent the sum of the daily milk yield at parlour of cows and the milk drunk by calves (Before and After groups).

**Figure 2** Body weight of the calves in Trial 1 by week of age (means and SE of raw data). Control-calves (n = 14) were separated from their dam at birth, Before-calves (n = 14) were able to suckle their dam for 20 min before the morning milking, and After-calves (n = 14) were able to suckle their dam for 2.5 h after the morning milking.

**Figure 3** Percentage of animals that vocalized (frequently or from time to time) during one week around weaning (on Day 1) in Trial 1 and in Trial 2. Control-calves (n = 12 in Trial 1 and 14 in Trial 2) were separated from their dam at birth, Before-calves (n = 12) were able to suckle their dam for 20 min before the morning milking, and Half-day-calves (n = 14) were able to suckle their dam for 9 h between the morning and evening milkings. Chi-square test comparing per-day data with Before-calves in Trial 1 and with Half-day-calves in Trial 2.

**Figure 4** Daily milk yield at the parlour for the two groups of cows in Trial 2, by week of lactation (means and SE of raw data). In Control-group, calves were separated from their dam at birth, and in Half-day-group calves were able to suckle their dam for 9 h between the morning and evening milkings. Weaning took place when the calves weighed at least 100 kg. Each group consisted of 7 Holstein (Ho) and 7 Montbéliarde (Mo) cow-calf pairs.

**Figure 5** Body weight of the calves in Trial 2 by week of age (means and SE of raw data). Control-calves (n = 14) were separated from their dam at birth and Half-day-calves (n = 14) were able to suckle their dam for 9 h between the morning and evening milkings.
<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>
Trial 1

<figure><img src="image_4.png" /></figure>

Trial 2

<figure><img src="image_5.png" /></figure>

<footnote>*** P<0.001; * P<0.05; + P<0.10</footnote>
<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /></figure>