Effect of parity order and litter weaning age on the performance and body energy balance of rabbit does

Livestock Production Science 85 (2004) 239 – 251 www.elsevier.com/locate/livprodsci

Effect of parity order and litter weaning age on the performance and body energy balance of rabbit does G. Xiccato *, Angela Trocino, A. Sartori, P.I. Queaque Dipartimento di Scienze Zootecniche, Universita` degli Studi di Padova, Agripolis, via Romea 16, I-35020 Legnaro (PD), Italy Received 18 September 2002; received in revised form 2 April 2003; accepted 22 April 2003

Abstract This study aimed to evaluate the effects of doe parity order and litter weaning age on reproductive performance and lactation and body energy balance of rabbit does from one kindling to the following. To this aim, 138 lactating does of three parity orders (first, second and third kindling: K1, K2 and K3) were remated 11 days after kindling. Their litters were weaned at 21, 26 and 32 days of age in a 3  3 factorial arrangement (three parity orders by three weaning ages). Thirty does at initial kindling and 69 pregnant does at final kindling were slaughtered to determine body tissue and energy balance. When increasing parity order, milk production, feed and digestible energy intake during lactation increased linearly while body energy deficit decreased (from 20.5% of the initial content in K1 does to 9.2% in K3 does). When weaning age was decreased from 32 to 21 days, body energy deficit decreased ( 19.4% to 8.0%). Significant interactions between parity order and weaning age were recorded in energy balance and reproductive performance. According to these results, multiparous does showed a lower but still relevant energy deficit than primiparous does. Early weaning permitted us to reduce body energy deficit, especially at lower parity orders. D 2003 Elsevier B.V. All rights reserved. Keywords: Rabbit; Parity order; Weaning age; Reproductive performance; Energy balance

1. Introduction Rabbit does are susceptible to an intense body energy deficit during lactation, especially highly-productive hybrid lines whose voluntary feed intake is often insufficient to cover nutrient requirements for lactation and concurrent pregnancy (Partridge et al., 1986a,b; Maertens, 1992; Xiccato, 1996). The relationship between feed and energy intake and body

* Corresponding author. Tel.: +39-049-8272639; fax: +39-0498272669. E-mail address: [email protected] (G. Xiccato). 0301-6226/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0301-6226(03)00125-8

energy deficit has been widely demonstrated in lactating and pregnant does from their first to second kindling (Parigi Bini et al., 1992; Xiccato et al., 1995; Fortun-Lamothe and Lebas, 1996). The higher feed intake measured in multiparous rabbits (Battaglini and Grandi, 1991; Castellini and Battaglini, 1991; Pascual et al., 1998) suggests that body energy deficit could decrease with higher parity order, even if the limited research conducted demonstrates the occurrence of body energy losses in multiparous does as well (Partridge et al., 1983, 1986a; Pascual et al., 2000). Previous studies on nutritional strategies in prepubertal and reproducing females to stimulate energy intake and improve body condition during reproduc-

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tive activity have not yielded appreciable results (Fortun-Lamothe, 1997, 1998; Xiccato et al., 1999; Pascual et al., 2002) whereas the application of less intensive reproductive rhythms has been shown more effective (Parigi Bini et al., 1996). Early weaning of litters has been proposed as a way to both reduce doe body energy output by decreasing the lactation period and ensure better coverage of kit nutritional requirements by separate kit and dam feeding (De Blas et al., 1999; Xiccato et al., 2000; Nicodemus et al., 2002). This study was carried out to establish how doe parity order and litter weaning age can (1) affect voluntary energy intake and reproductive and lactation performance; (2) change doe body composition and energy reserves; and (3) decrease energy and tissue losses between kindling.

2. Materials and methods 2.1. Rearing building and equipment The rabbits were kept in a brick shed maintained at a minimum temperature of 18 jC by a forced heating system and submitted to a constant photoperiod of 16h light, 8-h darkness. The animals were kept in individual cages for reproducing does (400  600  350 mm) made of galvanized wire net equipped with an automatic drinker and a manual feeder. The nest (400  220  300 mm) was formed by galvanized sheet walls and a double wire floor, and presented a manual closure to permit programmed lactation. The nest was prepared with wood shavings and attached to the front side of the maternal cage 2 –3 days before kindling. 2.2. Animals and experimental groups Twelve days before kindling, 138 pregnant does of a hybrid maternal line (Grimaud Fre`res, France) were moved from a commercial breeding unit to the experimental facilities. The rabbits were grouped according to three parity orders: 46 does were at their first kindling (K1), 46 does at their second kindling (K2), and 46 does at their third kindling (K3). The K1 does had been mated at 130 days of age and K2 and K3 does had been previously submitted to a 42-

day reproductive rhythm, i.e. they had been successfully mated 11 days after kindling. The experimental period lasted 42 days, from the initial kindling to the final kindling. At the initial kindling, 30 does (10 per parity) were slaughtered to estimate the initial body composition of the remaining does according to the comparative slaughter technique (Parigi Bini et al., 1992). The remaining 108 does (36 per parity order) were further divided within parity into three groups, with the same average weight and variability, whose litters were weaned at 21 (W21), 26 (W26) or 32 days of age (W32), in a 3  3 factorial arrangement (three parity orders by three weaning ages). 2.3. Experimental diets From initial kindling to 21 days of lactation, all does were given ad libitum access to a lactation diet, and from 21 days to final kindling, a weaning diet. Litters were given ad libitum access to the weaning diet from 18 until 32 days of age. The ingredients and chemical composition of the experimental diets are listed in Table 1. The weaning diet contained almost the same ingredients as the lactation diet but at different inclusion rates. The chemical composition and nutritive value of the diets followed the recommendations of De Blas and Mateos (1998). The lactation diet was higher in starch and lower in fibre than the weaning diet due to the higher proportion of barley meal and the lower inclusion of dehydrated alfalfa meal, and was also higher in ether extract and gross energy concentration due to the addition of fat. Digestibility coefficients of dry matter (DM) and almost all nutrients were significantly higher in the lactation diet than in the weaning diet, with the exception of crude fibre (Table 2). Energy digestibility was also higher in the lactation diet, which had higher digestible energy (DE) concentration (11.77 vs. 10.50 MJ/kg DM). Digestible protein to DE ratio was similar in the two diets. 2.4. Data recording Doe live weight was recorded immediately after initial kindling. The litters were standardized to nine kits by cross-fostering within parity order, weighed and maintained in the closed nest. The does were allowed to enter the nest and suckle their litters once

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Table 1 Ingredients and chemical composition of the experimental diets Diet Lactation

Weaning

Ingredients (%) Dehydrated alfalfa meal, 17% CP Wheat middlings Barley (six rows) Sugar beet pulp Soybean meal, 44% CP Sunflower meal, 30% CP Animal fat Molasses (50% cane + 50% sugar beet) Dibasic calcium phosphate Limestone Salt DL-Methionine HCl – lysine Premixa

24.00 22.00 24.00 5.00 9.50 9.50 1.50 2.00 0.35 1.30 0.35 0.10 0.00 0.40

35.00 25.00 16.00 8.00 4.00 8.00 0.00 2.00 0.42 0.55 0.45 0.08 0.10 0.40

Chemical composition Dry matter (%) Crude protein (% DM) Ether extract (% DM) Crude fiber (% DM) Ash (% DM) NDF (% DM) ADF (% DM) ADL (% DM) Starch (% DM) Gross energy (MJ/kg DM)

90.2 18.1 3.7 15.2 8.3 34.2 17.3 3.9 18.0 18.5

90.4 16.9 2.1 17.9 8.8 37.3 19.6 4.7 14.4 18.1

a

Premix provided per kilogram of complete diet: vitamin A, 12,000 IU; vitamin D3, 1000 IU; vitamin E acetate, 50 mg; vitamin K3, 2 mg; biotin, 0.1 mg; thiamin, 2 mg; riboflavin, 4 mg; vitamin B6, 2 mg; vitamin B12, 0.1 mg; niacin, 40 mg; pantothenic acid, 12 mg; folic acid, 1 mg; choline chloride, 300 mg; Fe, 100 mg; Cu, 20 mg; Mn, 50 mg; Co, 2 mg; I, 1 mg; Zn, 100 mg; Se, 0.1 mg; Robenidine, 66 mg.

daily (09:00 h) for a maximum 10 min (programmed lactation). Milk yield was measured daily by weighing the doe immediately before and after suckling (Partridge and Allan, 1983; Parigi Bini et al., 1992). Feed intake and live weight of does were recorded daily during lactation and from weaning to kindling. Live weight of litters from 0 to 32 days of age and their solid feed intake from 18 to 32 days of age were measured daily. During lactation, the litters were weighed before suckling. At initial kindling and during the first 2 weeks of lactation, eight does (one doe each from groups K1W21, K1-W26, K1-W32, K2-W21, K2-W26 and K3-W21; two does from group K2-W32) were discarded due to insufficient milk production or health problems.

Eleven days after kindling, the remaining 100 does were artificially inseminated using a fresh semen pool from Grimaud ‘parent’ bucks. Ovulation was induced by GnRH injection (10 Ag of Receptal, Ste´ Distrivet, Igoville, France). Fourteen days after mating, pregnancy status was tested by abdominal palpation and 31 non-pregnant does (three of group K1-W21, four of group K1-W26, four of group K1-W32, five of group K2-W21, four of group K2-W26, three of group K2-W32, three of group K3-W21, three of group K3W26, and two of group K3-W32) were excluded from the study. At 18 days of age, the litters were moved from their nests to cages (400  600  350 mm) opposite those of their mothers and given ad libitum access to the weaning diet. Until weaning, the mothers were

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Table 2 Apparent digestibility and nutritive value of the experimental diets Diet Lactation No. of rabbits Digestibility coefficients (%) DM Organic matter CP Ether extract Crude fiber NDF ADF Gross energy Nutritive valueb Digestible energy (DE) (MJ/kg DM) Digestible protein (DP) (g/kg DM) DP/DE (g/MJ) a b

P

R.S.D.a

*** *** *** *** n.s. *** *** ***

0.9 0.9 0.6 1.0 2.0 1.7 1.3 0.9

Weaning

12

12

65.2 65.6 76.4 70.7 17.6 30.0 14.2 63.6

60.1 60.5 71.7 42.8 16.1 25.5 6.8 58.0

11.77 138 11.72

10.50 121 11.52

Residual standard deviation. Not analyzed statistically.

moved to the litter cages to suckle once a day. The day of weaning, the does were given a limited feed amount (200 g) to favor the interruption of milk production. The 69 pregnant does gave birth at 30 –31 days after insemination. Fertility rate was calculated as: no. of kindling/no. of insemination  100. 2.5. Comparative slaughter The comparative slaughter technique was used to estimate the chemical and energy balance of the does from initial to final kindling (Parigi Bini et al., 1992). Thirty does (10 per parity order) were slaughtered immediately after initial kindling and 69 does were slaughtered after final kindling. The does were weighed immediately before slaughter and killed by intracardiac injection of a commercial product for animal euthanasia (Tanax, Hoechst AG, Frankfurt am Main, Germany). The gut and bladder were removed and emptied. Then the fresh empty organs were joined to the whole body and weighed to determine the empty body (EB) weight. 2.6. Digestibility trial The apparent digestibility of nutrients and the DE concentration of the experimental diets were measured in a digestibility assay carried out at the same time on

24 growing rabbits (12 animals of both sexes per diet) according to the European standardized method (Perez et al., 1995). The animals were selected from the weaned litters (four rabbits per diet per weaning age) and were moved to individual digestibility cages at 35 days of age (live weight 751 F 112 g). The digestibility trial started at 38 days of age with a 7-day adaptation period followed by a 4-day collection period. 2.7. Chemical analysis Diets and feces were analyzed by AOAC (1990) methods. Ether extract was determined after acidhydrolysis treatment. Fibre fractions were analyzed by the Goering and Van Soest (1970) method as modified by Robertson and Van Soest (1981) using the sequential procedure. The NDF determination was carried out using a thermo-resistant amylase (Thermamyl L120, Novo Nordisk, Denmark). Starch content was measured by an enzymatic kit (Boehringer Mannheim, Gmbh, Mannheim, Germany). The frozen doe empty bodies were cut in small pieces by a belt saw and ground by a mincer. The samples were freeze-dried and analyzed by AOAC (1990) methods. Gross energy concentration of diets, feces and empty bodies was measured by adiabatic bomb calorimeter (Martillotti et al., 1987).

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parity order during the lactation period (linear component of variance, L < 0.001). In the dry period (from weaning to final kindling), feed and energy intake were not affected by parity order. As weaning age increased from 21 to 32 days of age, doe live weight at final kindling decreased linearly (L < 0.05). The different lactation length obviously influenced both total milk production and total feed intake during lactation and dry period. Daily DE intake per kg of metabolic weight was not affected by weaning age during lactation but was lower in W21 does than in W32 does (655 vs. 794 kJ/day kg LW 0.75; L < 0.001) during the dry period. Litter performance from birth to weaning is shown in Table 4. Litter size at weaning and at 32 days of age were not affected by the experimental treatments, while litter weight at the same days (L < 0.001), weight gain from birth to 32 days (L < 0.001) and total feed intake (L < 0.05) from 18 to 32 days of age increased with parity order. Litter weight at weaning obviously differed among the weaning groups and increased with age (L < 0.001; quadratic component of variance Q < 0.001). At 32 days of age, litter weight was higher in groups weaned later (from 4737 g in W21 litters to 5258 g in W32 litters; L < 0.01). Total feed intake from 18 to 32 days decreased with increasing weaning age (L < 0.001). No significant interaction between doe parity order and litter weaning age was recorded for the performance of does and litters.

2.8. Data treatment and statistical analysis The energy and chemical balances of the 69 pregnant does were calculated on the basis of the difference between their EB composition at final kindling and their initial EB composition estimated on the initial slaughter group composition within parity (Parigi Bini et al., 1992). The GLM procedure of the Statistical Analysis System (SASI, 1991) was used for two-way analysis of variance (three parity orders by three weaning ages with interaction) of data concerning performance and for one-way analysis of variance of nutrient digestibility coefficients of the two diets. The Chi-square procedure was used to analyze fertility rate. Orthogonal polynomial contrasts were used to compare means by group of parity order and weaning age in order to estimate the linear (L) and quadratic ( Q) component of variance.

3. Results 3.1. Doe and litter performance Performance of lactating and concurrently pregnant rabbits between initial and final kindling is reported in Table 3. Initial and final live body weight were not influenced by parity order. On the other hand, total milk production, feed and DE intake increased linearly with

Table 3 Performance of lactating and concurrently pregnant does between initial and final kindling Parity order (K) K1 No. of does Live body weight (LW) at: Initial kindling (g) Final kindling (g) Total milk production (g) Food intake (g) During lactation From weaning to kindling DE intake (kJ/day kg LW 0.75) During lactation From weaning to kindling a b

K2

P K3

L

a

W26

W32

22

23

24

n.s. n.s. n.s.

3624 3769 4242

3603 3650 4964

*** n.s.

n.s. n.s.

6291 3651

*** n.s.

n.s. n.s.

1195 655

20

27

3547 3652 4548

3620 3638 5023

3646 3745 5410

n.s. n.s. ***

7276 2721

7993 2945

8313 2888

1099 685

1203 757

1237 726

Q

P

W21

22

L, linear component of variance. Q, quadratic component of variance.

Weaning age (W) b

a

R.S.D. b

L

Q

3586 3617 5774

n.s. * ***

n.s. n.s. n.s.

235 238 625

7657 2963

9634 1941

*** ***

n.s. n.s.

813 404

1166 719

1178 794

n.s. ***

n.s. n.s.

123 105

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Table 4 Performance of weaning litters Parity order (K) K1 No. of litters Litter size at weaninga Litter size at 32 days Litter weight (g) Initiala At weaning At 32 days Litter weight gain (0 – 32 days) (g) Litter feed intake (18 – 32 days) (g) a

K2

22 8.5 8.3 431 3428 4650 4219 2321

20 8.5 8.5 467 3824 4988 4521 2455

P K3 27 8.7 8.7 480 3963 5384 4904 2848

Weaning age (W)

L

Q

n.s. n.s.

n.s. n.s.

** *** *** *** *

n.s. n.s. n.s. n.s. n.s.

W21 22 8.5 8.4 464 2619 4737 4273 3050

W26 23 8.6 8.6 454 3337 5027 4573 2622

P W32 24 8.5 8.5 459 5258 5258 4799 1951

R.S.D.

L

Q

n.s. n.s.

n.s. n.s.

n.s. *** ** * ***

n.s. *** n.s. n.s. n.s.

0.6 0.7 55 489 645 617 915

Initial litter size was standardized to nine kits.

Fertility rate was not significantly influenced by either parity order or weaning age.

Some of the reproductive variables controlled for rabbit does at their final kindling were affected by parity order (Table 5). The litter size increased with parity order (L < 0.05), while the average weight of kits born alive decreased (L < 0.05). Weaning age did not affect reproductive performance at final kindling, but important interactions ( P < 0.01) between parity order and weaning age were recorded in litter size. In particular, in K1 does, the number of kits born per litter was lower in W32 does (7.9, 8.7 and 6.2 in W21, W26 and W32, respectively), while in K3 does, it was lower in W21 does (6.7, 9.9 and 11.2). The number of kits alive and the weight of kits born per litter changed according to the number of kits born per litter.

3.2. Body composition and energy balance between kindling Data obtained from the 30 does initially slaughtered (Table 6) were used to calculate the chemical and energy balance of the 69 lactating and concurrently pregnant rabbits. At initial slaughter, doe live weight and EB weight were not significantly affected by parity order, while gut content increased (from 177 g in K1 does to 292 g in K3 does, L < 0.01). Empty body fat decreased markedly (from 19.1% in K1 to 9.3% in K3 does; L < 0.001) and was mostly substi-

Table 5 Reproductive performance of pregnant does at the final kindling Parity order (K)

No. of does Pregnancy length (days) No. of kits born per litterab No. of kits born alive per litterc Weight of kits born per litterad (g) Weight of kits born alive per litter (g) Average weight of newborn kitsa (g) Average weight of kits born alive (g) Fertility rate (%)e a

P

K1

K2

K3

22 30.8 7.8 7.0 382 355 49.6 51.1 66.7

20 30.8 8.2 8.1 407 405 50.4 50.9 62.5

27 31.0 9.3 8.8 426 417 47.0 46.9 77.1

L n.s. * * n.s. * n.s. *

Weaning age (W)

P

R.S.D.

Q

W21

W26

W32

L

Q

n.s. n.s. n.s. n.s. n.s. n.s. n.s.

22 30.9 7.6 7.5 375 369 50.2 50.6 66.7

23 30.9 9.0 8.1 433 409 49.0 49.3 67.6

24 30.9 8.7 8.4 407 399 47.7 49.0 72.7

n.s. n.s. n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s. n.s. n.s.

0.3 2.3 2.7 100 111 6.3 6.0

Kits born dead included. Parity order  weaning age interaction (K  W), P < 0.01 (K1 does: 7.9, 8.7, 6.2; K2 does: 8.2, 8.2, 8.2; K3 does: 6.7, 9.9, 11.2 in W21, W26 and W32, respectively). c K  W, P < 0.05 (K1 does: 7.6, 7.5, 6.0; K2 does: 8.2, 8.2, 8.0; K3 does: 6.6, 8.6, 11.1 in W21, W26 and W32, respectively). d K  W, P < 0.05 (K1 does: 384, 429, 332 g; K2 does: 410, 412, 399 g; K3 does: 329, 459, 488 g in W21, W26 and W32, respectively). e Test m2, P>0.05. b

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Table 6 Empty body weight and composition of does slaughtered at the initial kindling Parity order (K)

No. of does Live body weight (g) Gut content (g) Empty body weight (g) Composition of empty body Water (%) Protein (%) Fat (%) Energy (MJ/kg)

P

R.S.D.

K1

K2

K3

L

Q

10 3530 177 3353

10 3622 223 3399

10 3668 292 3376

n.s. ** n.s.

n.s. n.s. n.s.

*** ** *** ***

n.s. n.s. n.s. n.s.

58.6 19.3 19.1 11.81

64.1 19.6 13.0 9.53

tuted by water (from 58.6% to 66.9%; L < 0.001) and by protein in a lower amount (L < 0.01). In consequence, body energy content decreased with parity order. At final kindling, parity order also affected doe gut content and body composition (Table 7). Gut content was lower in K1 does than in K3 does (L < 0.05). Empty body composition showed a further substitution of fat (from 12.7 to 7.3%) by water (from 64.8 to 69.0%) and a significant increase in protein content from does at their second kindling (K1 group) to does at their fourth kindling (K3 group) (L < 0.001). As a result, energy content decreased with parity order also at final kindling (L < 0.001). When weaning age increased from 21 to 32 days, live weight and EB weight of does at final kindling

66.9 20.5 9.3 8.30

242 81 224

2.2 0.9 2.6 0.87

decreased significantly (L < 0.05) without modification in gut content. Moreover, EB water content increased (from 65.6% in W21 does to 67.8% in W32 does; L < 0.01) while fat and energy decreased (L < 0.01). The interaction between parity order and weaning age was significant ( P < 0.05) for EB fat and energy. In K1 does, these concentrations were higher in W21 than in W26 and W32 does; in K2 does, they were lower in W21 and W26 than in W32 does; in K3 does, they were similar in all weaning groups. The doe EB gain composition and the chemical and energy balance from initial to final kindling are provided in Table 8. Gut content increased more in K1 than in K2 and K3 does (L < 0.01). The EB gain composition differed significantly by parity, with

Table 7 Doe empty body composition at final kindling Parity order (K)

No. of does Live body weight (g) Gut content (g) Empty body weight (g)

P

Weaning age (W)

P

R.S.D.

K1

K2

K3

L

Q

W21

W26

W32

L

Q

22 3661 305 3356

20 3638 295 3343

27 3745 359 3386

n.s. * n.s.

n.s. n.s. n.s.

22 3768 314 3454

23 3650 307 3343

24 3626 339 3287

* n.s. *

n.s. n.s. n.s.

*** *** *** ***

n.s. * n.s. n.s.

** n.s. ** **

n.s. n.s. n.s. n.s.

Composition of empty body Water (%) 64.8 Protein (%) 19.2 Fat (%)a 12.7 Energy (MJ/kg)b 9.37

66.4 20.2 10.0 8.53

69.0 20.2 7.3 7.48

65.6 19.8 11.3 8.89

66.7 19.8 10.0 8.48

67.8 20.0 8.8 8.01

238 80 217

2.2 0.7 2.4 0.88

a Parity order  weaning age interaction (K  W), P < 0.05 (K1 does: 15.3, 11.4, 11.6%; K2 does: 11.3, 11.2, 7.6%; K3 does: 7.3, 7.5, 7.2% in W21, W26 and W32, respectively). b K  W, P < 0.05 (K1 does: 10.3, 8.9, 8.9 MJ/kg; K2 does: 8.9, 8.9, 7.7 MJ/kg; K3 does: 7.5, 7.6, 7.4 MJ/kg in W21, W26 and W32, respectively).

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Table 8 Composition of empty body gain and energy balance of lactating and pregnant does between initial and final kindling Parity order (K) K1 No. of does Live body weight gain (g) Gut content gain (g) Empty body gain (g)

K2

22 122 123 1

P K3

Weaning age (W)

L

Q

W21

W26

R.S.D.

L

Q

24 52 107 55

* n.s. **

n.s. n.s. n.s.

154 78 140

20 40 71 31

27 83 65 18

n.s. ** n.s.

n.s. n.s. n.s.

22 144 79 65

Composition of empty body gain Water (g) 211 Protein (g) 2 Fat (g)a 217 Energy (MJ)b 8.36

51 11 94 3.30

84 9 64 2.60

*** n.s. *** ***

** n.s. * *

125 10 75 2.68

104 4 129 4.94

118 6 170 6.65

n.s. n.s. *** ***

n.s. n.s. n.s. n.s.

107 37 86 3.42

Chemical and energy balancec Water Protein Fatd Energye

2.6 1.8 23.3 11.2

3.8 1.1 20.2 9.2

*** n.s. * ***

** n.s. n.s. n.s.

6.1 1.8 16.9 8.0

5.0 0.5 24.5 13.4

5.7 0.8 35.3 19.4

n.s. n.s. ** ***

n.s. n.s. n.s. n.s.

5.1 5.4 20.1 10.4

10.5 0.2 33.0 20.5

23 48 73 25

P W32

a Parity order  weaning age interaction (K  W), P < 0.01 (K1 does: 112, 274, 264 g; K2 does: 49, 55, 179 g; K3 does: 65, 58, 67 g in W21, W26 and W32, respectively). b K  W, P < 0.05 (K1 does: 3.9, 10.8, 10.4 MJ; K2 does: 1.9, 1.7, 6.7 MJ; K3 does: 2.6, 2.3, 2.9 MJ in W21, W26 and W32, respectively). c Percentage variation on the empty body composition at initial kindling. d K  W, P < 0.05 (K1 does: 16.9, 41.9, 40.2%; K2 does: 12.8, 13.3, 43.9%; K3 does: 20.9, 18.1, 21.7% in W21, W26 and W32, respectively). e K  W, P < 0.05 (K1 does: 9.4, 26.7, 25.5%; K2 does: 5.4, 5.7, 22.4%; K3 does: 9.4, 7.8, 10.3 in W21, W26 and W32, respectively).

progressively lower body fat and energy losses from K1 to K3 does (L < 0.001; Q < 0.05). Body water gain was higher in K1 than K2 and K3 does (L < 0.001 and Q < 0.01). The same pattern was observed when the changes in EB composition between kindling were expressed as percentages of initial content. Fat and energy balances were negative in all parity groups and significantly decreased with increasing parity order. When weaning age was reduced from 32 to 21 days, doe live body and EB weight gain increased significantly; EB fat and energy losses decreased (L < 0.001) and their balance became less negative ( 35.3 to 16.9% for fat and 19.4 to 8.0% for energy; L < 0.01). Significant interactions between the two experimental factors were also recorded for fat and energy balance. In particular, fat and energy losses in K1 does (in absolute or percentage values) were lower in W21 than in W26 and W32 does; in K2 does, they were

lower in W21 and W26 than in W32 does; in K3 does, they were similar in all weaning groups. Protein balance was near zero in all groups and unaffected by experimental factors.

4. Discussion 4.1. Effect of the parity order Studies on doe reproductive performance usually show a high fertility rate in nulliparous does, a lower fertility in primiparous does and intermediate values in multiparous does (Barge and Masoero, 1986; Rebollar et al., 1992; Castellini, 1995). The body energy deficit of primiparous does and the negative interactions between lactation and fertility have been considered the main reasons for these results (TheauCle´ment and Roustan, 1992; Fortun-Lamothe and Bolet, 1995; Parigi Bini and Xiccato, 1998; Fortun-

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Lamothe et al., 1999). In our study, fertility was numerically higher in K3 does, but differences among parity orders were not significant. As regards the relationships between parity order and reproductive traits, our results agreed with existing literature describing greater litter size and weight at birth, and consequently lower average kit weight, with increasing parity order (Parigi Bini et al., 1989; Pascual et al., 1998; Szendro¨, 2000). We observed that kit survival was unaffected by parity order but litter weight at weaning and 32 days of age were stimulated by the higher milk production of does in their second and third lactation than of primiparous does, as also noted by Szendro¨ (2000). Despite the negative correlation with milk intake usually observed (Szendro¨, 2000), solid feed intake from 18 to 32 days of age was higher in the heavier litters of multiparous does. Differences in weight and probably also in digestive physiology development among litters from different parities may account for this result. During the first lactation, chemical and energy changes confirmed previous results on primiparous lactating and concurrently pregnant does (Parigi Bini et al., 1992; Xiccato et al., 1992, 1995), where similar or even higher fat mobilisation (from 37% to 59% of the initial content) and energy losses (from 24% to 32% of the initial content) were recorded. The insufficient feed intake of primiparous does and the consequent inability to cover lactation and pregnancy requirements are widely recognized as the main reasons for their body energy deficit (Maertens, 1992; Xiccato, 1996; Parigi Bini and Xiccato, 1998; Pascual et al., 1998). This is especially true in highly productive hybrid rabbits, for which selection and crossbreeding strategies have been aimed at increasing prolificacy and milk production rather than voluntary feed intake and/or career length. Unlike primiparous rabbits, multiparous does are usually considered capable of ingesting the higher amounts of feed required to achieve body energy and protein equilibrium. Partridge et al. (1983, 1986a) and Pascual et al. (2000), however, reported substantial body fat and energy mobilisation in multiparous lactating does. Various authors described significant increases (of 5 – 15%) in feed intake from the first to the second, and from the second to the third kindling, followed by lower but not significant increases for

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successive parities (Parigi Bini et al., 1989; Battaglini and Grandi, 1991; Castellini and Battaglini, 1991). In the present study as well, voluntary intake during lactation significantly increased with parity order. DE intake rose by 9% from K1 to K2 does, and only by 3% from K2 to K3 does. Milk production increased by 10 and 8%, respectively. The unchanged substantial gap between dietary energy intake and milk energy output accounted for the body deficit also maintained at higher parities. Pascual et al. (2000) found that non-pregnant does lost 16% of their initial fat content during second lactation. A total energy loss of 12.7 MJ was also calculated over 32 days of lactation in multiparous non-pregnant does fed ad libitum (Partridge et al., 1983). On the basis of the energy requirement for maintenance and the coefficients of energy utilization for milk production and foetal growth reported by Parigi Bini and Xiccato (1998), the energy deficit for K2 and especially K3 rabbits should have been even higher than our findings. Other metabolic factors might have contributed to limiting the body energy deficit during the second and third lactation. According to Partridge et al. (1986a), fatter does (like our primiparous does) tended to mobilise body energy reserves for milk synthesis, while leaner does (like our multiparous does) tended to save body energy by either partitioning energy differently (e.g. decreasing milk energy output) or using dietary energy more efficiently. In this context, a lower milk energy concentration was found in leaner than fatter does by Partridge et al. (1986a) but no literature is available on the effect of parity order on milk composition. In our study, protein seemed to play a secondary role in the energy balance of reproducing does, given that the experimental diets had an adequate digestible protein to DE ratio, and the digestible protein intake appeared sufficient to cover the protein requirement (Maertens, 1992; De Blas and Mateos, 1998). Previous studies showed the protein requirement to be higher during the last 10 days of pregnancy than during lactation and found limited body protein losses ( 5 to 10%) only in concurrently pregnant and lactating does subjected to intensive reproductive rhythm (Parigi Bini et al., 1992; Xiccato et al., 1995). Other authors have also reported a negative protein balance in non-pregnant multiparous does (Partridge et al., 1986b; Pascual et al., 2000).

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The large variation in gut content that we recorded at initial and final kindling confirmed that live weight is a poor predictor of body tissue and energy changes, as stated by Partridge et al. (1983, 1986a). Gut content increased from the first to the second parity, as also reported in other studies (Parigi Bini et al., 1992; Xiccato et al., 1992, 1995), due to the marked reduction in feed intake a few days prior to first kindling, while prior to second kindling, feed intake decreased less, probably as a result of the lactation energy deficit (Lebas, 1972; Partridge et al., 1986b). 4.2. Effect of the weaning age The feasibility of early weaning has been demonstrated in studies describing the good condition and survival rate of kits and the evolution of digestive physiology in kits and young rabbits (De Blas et al., 1981; Xiccato et al., 2000, 2001; Gutie´rrez et al., 2002). Our results confirmed a very low mortality of early weaned rabbits that had reached a suitable live weight (>550 g at 32 days of age). Moreover, the studies above report good performance during the growing period and similar slaughter weight in both early and traditionally weaned rabbits. The main goal of early weaning is the reduction of doe body energy deficit, through the decrease of body energy utilization for milk synthesis by shortening the lactation length (period of energy deficit), and the increase of body energy restoration by prolonging the dry period (period of energy surplus). Decreasing the lactation length undoubtedly reduces body energy output. Moreover, during the third decade of lactation (from 20 days onward), the simultaneous increase of milk dry matter and fat concentration (Lebas, 1971, 1972; Pascual et al., 1999) maintains the energy partitioned for milk production at a high level. In this period, however, daily milk production decreases while feed intake remains at maximum level or decreases only slightly for nearly a week, thereby making possible the achievement of the daily energy equilibrium or even a positive balance (Maertens and De Groote, 1988; Parigi Bini and Xiccato, 1998). Prolonging the dry period increases the energy recovery time, as occurs when extensive remating rhythms are applied (Partridge et al., 1984; Cervera et al., 1993; Parigi Bini et al., 1996). However, the lower intake in the dry period compared to the

lactation period reduces the daily energy gain and delays the complete restoration of body reserves. In this study, voluntary feed intake decreased sharply from around 350 g/day prior to weaning to around 180 – 200 g/day within 4– 5 days after weaning. In the first week after weaning, moreover, the W21 does showed a lower consumption (about 20 g/day) than the W26 and W32 does. The metabolic stress created by the sudden interruption of lactation in W21 does at top milk yield might have accounted for this feeding behavior. On the basis of the measured milk production and feed intake of the does weaned at 32 days (average of the three parity groups) and the maintenance requirements and coefficients of energy utilization reported by Parigi Bini and Xiccato (1998), we estimated that the does were in positive balance for a few days after kindling before showing increasing energy deficits up to 0.8 MJ/day at 20 days of lactation. Due to the decreased milk production and stable feed intake from 20 days onward, the lactating does reached daily energy equilibrium at about 26 days. At 32 days, daily energy gain reached + 0.3 MJ/day and remained stable until the last days before kindling when it returned negative due to the increasing pregnancy requirement and decreasing feed intake. Partridge et al. (1986a) reported a similar change in body energy balance over a 32-day lactation period. On the basis of the variation in daily balance described above, we calculated that W32 rabbits lost around 7 MJ from initial kindling to 21 days of lactation and a further 2 MJ from 21 to 26 days. Thereafter they gained around 1 MJ in the period from 26 to 32 days and another 1.5 MJ until 37 days, while the energy equilibrium was reached in the last 5 days before final kindling on average. From initial to final kindling, W32 does therefore lost around 6.5 MJ of body energy, primarily in the form of fat. For W21 and W26 does, the energy gain was estimated to be + 0.5 MJ/day in the first 5 days immediately after weaning and + 0.3 MJ/day onwards. Therefore, from initial to final kindling, W26 does lost around 5 MJ ( = 7 2 + 2.5 + 1.5 + 0) and W21 does lost 1.5 MJ ( = 7 + 2.5 + 1.5 + 1.5 + 0). The underestimation of the W21 doe energy deficit compared to the value measured by comparative slaughter ( 2.68 MJ) can be easily corrected by considering the lower feed intake of these does after weaning.

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These calculations showed that increasing dry period length by early weaning permitted only a slow recovery of body energy reserves, due to the substantial decrease in post-weaning feed intake, and only partially prevented the occurrence of body deficit. The significant interaction observed between parity order and weaning age showed only weaning at 21 days of age to be effective in reducing body deficits in does during the first-to-second kindling interval, but weaning at both 21 and 26 days of age improved the body condition of does during their second-to-third kindling interval. Early weaning did not efficiently reduce the doe body energy deficits during the thirdto-fourth kindling interval. This interaction might be explained by the different evolution of the daily energy balance from one kindling to the next. In particular, on the basis of the model above and the different feed intake and milk yield curves, primiparous does seem to reach daily energy equilibrium at the end of lactation, and therefore only weaning at 21 days would reduce the energy deficit that remains high even during the entire third decade of lactation. On the other hand, does in their third lactation appear likely to reach daily energy equilibrium earlier (i.e. at 20 days of lactation) and achieve slightly positive balance during the third lactation decade. In such a case, early weaning would not substantially improve the total energy balance of multiparous does. With no corroborating experimental reference, these initial results on the effects of early weaning on the doe body balance require further investigation. Reference data on the reproductive performance of does submitted to early weaning is also scarce. Nicodemus et al. (2002) compared an intensive reproductive system associated with early weaning (mating 4 days post partum and weaning at 25 days of age) with a traditional reproductive and weaning system (mating 11 days post partum and weaning at 35 days of age). Despite the fact that the effects of weaning age are closely linked to those of the remating interval, the authors observed significantly higher prolificacy and litter size at weaning in does mated 4 days post partum whose litters were weaned at 25 days than in does subjected to traditional rhythm. In light of our results, weaning age did not affect doe reproductive performance at final kindling, while the significant interactions measured with parity order are not easily explainable. The lower number of kits born and born

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alive per litter recorded in does at their fourth kindling whose litters were weaned at 21 days of age might be ascribed to the marked interference in the metabolic and hormonal pattern at the time of foetus implantation from 7 to 11 days of pregnancy (Fortun-Lamothe and Bolet, 1995), due to the sudden interruption of lactation in multiparous does with the highest milk production. In any case, the number of does in this study was insufficient to definitely evaluate these highly-variable reproductive traits.

5. Conclusions This study confirmed the occurrence of body energy deficits in lactating and concurrently pregnant primiparous does and also measured substantial energy deficits in multiparous highly productive does. The dietary energy intake increased with parity order but was not sufficient to permit the complete recovery of body reserves lost during lactation even at the fourth kindling. The feasibility of early weaning at 21 days of age was confirmed as indicated by the low mortality and suitable kit weight. Early weaning reduced doe body energy deficit by decreasing milk energy output but the sharp decrease in feed intake immediately after weaning delayed and limited the recovery of body tissue and energy. Moreover, early weaning at 21 days probably caused the does metabolic stress, as demonstrated by an even further decrease in feed intake and reduced reproductive performance, especially in multiparous rabbits. In perspective, earliest weaning (21 days) should be limited to primiparous does, which are more susceptible to body deficit and less likely to suffer from metabolic stress caused by sudden interruption in lactation. Intermediate weaning (26 days) and less intensive reproductive rhythms might be used with multiparous does to overcome body energy deficits and avoid adverse effects on reproductive performance.

Acknowledgements This study was funded by Ministero dell’Universita` e della Ricerca Scientifica (year 2000; Contr. MM07193821). The Authors wish to thank Dr Andrea

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