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Oestrus, ovulation and peri-ovulatory hormone profiles in tethered and loose-housed sows
ANIMAL REPRODUCTION SCIENCE ELSEVIER
Animal Reproduction Science 46 ( 1997) 133- 148
Oestrus, ovulation and perk-ovulatory hormone profiles in tethered and loose-housed sows Nicoline M. Soede Wqeningen
Institute
oj’Animu1
*,
Frans A. Helmond, Willem G.P. Schouten, Bas Kemp
Sciences (WIASI,
Wqeninpn
AH Wugeningen,
A~riculturul
Uniuersity,
P.O. Box 338, 6700
The Netherlunds
Accepted 8 August 1996
Abstract
Multiparous sows that had heen tethered during lactation were put in two different housing conditions after weaning (Day 0); the sows were either tethered by neck chain, or individually housed in a pen of approximately 6 m*. After two months, ten tethered and eleven loose housed sows were used to assess stress and reproductive parameters. Stereotypic behaviour after the afternoon feeding was assessed from Day 18 onwards; at Day 53 stereotypic behaviour tended to occur during a higher percentage of time in the tethered sows (P = 0.11) and at Day 66, the differences were significant (tethered, 78 f 5 vs. loose-housed, 40 + 10% of time (mean + sem); P = 0.03). At Day 35 and 55, cortisol profiles after afternoon feeding were similar for the two groups of sows (P > 0.10). Around oestrus (approximately Day 641, the profiles of oestradiol-17P, luteinizing hormone and progesterone were measured and proved to be similar for both treatment groups (P > 0.10). The duration of oestrus was shorter in the tethered sows (42 k 4 vs. 63 + 2 h; P < 0.001) and, consequently, the timing of ovulation during oestrus (h after onset of oestrus) was advanced in the tethered sows (28 + 2 vs. 41 + 2; P < 0.001). The duration of ovulation did not differ (tethered, 2.9 f 0.5 vs. loose-housed, 2.1 k 0.2 h; P = 0.16). The sows were sacrificed at Day 5 after ovulation; ovulation rate, fertilization rate, embryo development and embryo diversity were similar for the two groups, as were adrenal weight and size of adrenal cortex. Duration of oestrus and the levels of stereotypies at Day 60 tended to be negatively related in the tethered sows (P = 0.071, but not in the loose-housed sows (P = 0.65). In conclusion, sows that had been tethered during pregnancy and lactation, and were housed loose or were tethered again at weaning,
* Corresponding author. Department of Animal Husbandry, P.O. Box 338, 6700 AH Wageningen, The Netherlands. Tel.: + 3 I 3I7 4839’73; fax: + 31 3 17 485006; e-mail: [email protected]. 0378.4320/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01609-O
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within two months differed
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both in stereotypic
behaviour
and in duration of oestrus, without
apparent effects on reproductive hormones. Keywords: Stress; Reproduction;Oestnts; Sows
1. Introduction Stressful conditions may negatively influence reproductive performance of animals (reviewed by Moberg, 1987; Rivier and Rivest, 1991; Varley and Stedman, 1994). Hormones of the HPA-axis (hypothalamic-pituitary-adrenal axis) that are released during stress, may interact with hormones of the HPG-axis (hypothalamic-pituitarygonadal axis) at many levels. Reproductive processes that are especially susceptible to stress include processes that depend on delicate hormonal changes, such as follicular growth and ovulation (Hennessy and Williamson, 1983; Dalin et al., 1988) and oestrous behaviour (Barnett et al., 1986; Hemsworth et al., 1986; Hein and Alhich, 1992). Tethering of sows is considered a stressful situation (Cronin, 1982; Schouten and Rushen, 1992; Janssens et al., 1994), resulting in increased levels of cortisol (Becker et al., 1985; Janssens et al., 1994) and increased levels of stereotypic behaviour (Cronin, 1982; Wiepkema and Schouten, 1992). Stress-related hormones are known to influence sexual functions at all three levels of the HPG-axis (Rivier and Rivest, 1991) and, consequently, effects of tethering on reproductive performance have been much debated. However, not much evidence of effects has been presented. In the present experiment, reproductive parameters (hormone patterns, oestrus, ovulation and early embryo development) were studied in sows that were either tethered or housed loose in a pen. Furthermore, stress parameters (cortisol, stereotypies, adrenal weight) were measured.
2. Materials and methods 2.1. General experimental schedule At the day of weaning, 26 sows arrived at the experimental farm and were housed either in large individual pens of 6 m* or were tethered by neck chain. To investigate effects of these housing conditions on reproductive performance, the sows were given an indwelling jugular vein catheter between 11 and 15 days after arrival. From Day 16 to 36 after arrival, all sows were put under progesterone dominance to synchronize oestrus. Around the second oestrus after imposed progesterone dominance (at approximately 65 days after arrival), the following reproductive parameters were studied: onset and duration of oestrus, onset and duration of ovulation, and profiles of oestrogen, luteinizing hormone (LH) and progesterone. At slaughter, at approximately 120 h after ovulation, Day 0
ovulation rate and embryo development and diversity were assessed. weaning, arrival at experimental farm, start of experiment
Day 4-6 Day 11-15
oestrus cannulation of the vena jugularis
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Day Day Day Day Day Day
18 16-36 40-45 55 62-66 70-74
onwards assessment of stereotypic behaviour altrenogest treatment (Day 35: cortisol profiles) oestrus cortisol profiles oestrus, ovulation, oestradiol, LH and progesterone ovarian, embryonic and adrenal parameters
135
profiles
2.2. Animals and housing In total, 26 multiparous commercial crossbred (Great Yorkshire X Dutch Landrace) sows [parity 4.2 + 0.3 (range 2 to 711 arrived at the experimental unit at the day of weaning. All sows came from one farm and had been tethered during pregnancy and lactation. At the experimental unit, sows were tethered by neck chain or placed individually in pens of 6 m2. The tethered sows were all housed in one compartment, all in one row. The penned sows were divided over two compartments. In the three compartments, no other animals were present during the experiment. The sows were fed at approximately 8 am and 4 pm, a total of 2.5 kg of a commercial sow ration and water was available ad libitum. Bedding in the pens consisted of saw dust. In the compartments, the lights were on from 7 am till 7 pm. 2.3. Jugular vein cannulation The sows were surgically fitted with a permanent jugular vein catheter between 12 and 16 days after arrival to allow repeated blood sampling without disturbing the animals. The animals were fasted overnight before surgery. Surgery was carried out under sterile conditions and general anaesthesia. The sows were premeditated by injecting 6 mg azaperone kg- ’ body mass i.m. (Stresnil; Janssen, Tilburg, The Netherlands). After 30 min, general anaesthesia was induced and maintained using O,-N,O and 2% halothane (Halothane; Trofield, Zug, Swiss). An indwelling catheter (Silastic 1.02 mm I.D., 2.16 mm O.D.; Dow Coming, Midland, MI, USA) was implanted into the external jugular vein towards the superior vena cava. The other end of the cannula was passed S.C. to the back of the animal, where it was externalized between the scapulae. A one-way luer-lock stopcock (Vygon, Veenendaal, The Netherlands) was secured to the end of the cannula to which a (10 ml) syringe could be attached easily. To protect the cannula from damage, the animals were harnessed. The harness (23 X 20 cm, PVC with nylon; Bizon Chemie, Goes, The Netherlands) was fixed at the back of the animals with belts tied around the chest. From three days before surgery until three days after surgery, all animals were given 12 ml of the antibiotic T.S. Sol (Dopharma, Raamsdonksveer, The Netherlands) orally once a day. The animals were given at least ten days to recover from surgery and anaesthesia. The patency of the cannula was maintained by flushing it three times a week and filling it with sterile heparinized physiological saline (25 IU heparin ml - ’, 0.9% NaCl; Leo, Weesp, The Netherlands) when not in use. 2.4. Detection of oestrus Detection of oestrus was performed by a back-pressure test in the presence of one of two vasectomized boars in front of the sows. Detection of first oestrus after weaning and
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the oestrus after altrenogest treatment were performed once daily. Detection of third oestrus was performed at intervals of 4 h, immediately after blood collection. The time of onset of third oestrus was defined as 2 h before the first time a sow showed a standing response to the back pressure test in the presence of the boar. The time of the end of third oestrus was defined as 2 h after the last time the sow showed a standing response to the back-pressure test in the presence of a boar. 2.5. Stereotypic behaviour From 15 min after feeding onwards, sow behaviour was scored every min for 60 min. Each observer walked quietly along the row of sows or pens and noted at 1 min intervals the behaviour of all individual sows. The observer noted the posture of the sow (standing, sitting, lying and in free sows also walking) and the occurrence of stereotypies (vacuum chewing, bar biting, chain manipulation and snout rubbing). 2.6. Cycle synchronization From Day 17 after arrival onwards, 20 ml altrenogest (Regumate, Hoechst, Amsterdam), a progesterone analogue, was spread over the morning feed for 20 days. 2.7. Blood sampling Prior to the experimental period, all animals were often handled and were adapted to the blood collection procedure. At Day 20 of the altrenogest treatment (Day 35 after weaning) and at Day 19 after altrenogest treatment (luteal phase, Day 55 after weaning), blood samples were taken every 12 min between 72 min before afternoon feeding till 72 min after afternoon feeding and were analysed for cortisol levels. To analyse reproductive hormones around the second oestrus after altrenogest treatment, blood samples were drawn from the cannula every 4 h, at 7:30, 1 I :30, 15:30, 19:30, 23:30 and 3:30 h from Day 17 of the cycle onwards. From 24 h after ovulation onwards, blood samples were drawn at 12 h intervals, at 8:00 and 20:00 h, up to five days after ovulation. Before blood samples were drawn, the luer-lock stopcock was disinfected with ethyl alcohol (70%) and the cannula was flushed with 4 ml of sterile physiological saline (NPBI, The Netherlands) to remove possible blood clots. Approximately 8 ml of blood was collected with a sterile syringe (Beckton Dickinson, Ireland). Then the cannula was filled with heparinized saline (to keep the cannula patent). Blood samples were collected in ice-cooled polypropylene tubes, containing 100 l.~l EDTA saline; Titriplex III, Merck, The Netherlands), immediately solution (144 mgml-’ placed on ice and then centrifuged at 2000 g for 10 min at 4°C. Plasma was collected and stored at -20°C until hormone analysis. Additionally, to make an estimate of the time of ovulation in advance (and thereby reduce the h of scanning of the ovaries), the 4 h blood samples were daily analysed for LH, using a rapid LH assay. Two 200 ~1 aliquots of the samples taken between 12:00 and 8:00 h were stored at 4°C till 8:30 h, after which the rapid LH assay was performed to detect a first rise in LH levels.
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2.8. Hormone analyses 2.8.1. Cortisol Plasma concentrations of cortisol were measured in duplicate in unextracted 50 p_l samples according to a single-antibody radioimmunoassay (RIA) technique previously described by Janssens et al. (1994). The sensitivity of the assay was 0.5 ngmll’ at the 90% B/B, level. The intra-assay coefficient of variation was 8.2% and the inter-assay coefficient of variation was 14.7%. 2.8.2. Oestradiol Oestradiol-17B was measured after extraction and column chromatography by RIA using a modification of the method described by Helmond et al. (1980). Samples of 100 p-1 were diluted in 900 pl phosphate buffer BSA with 0.1% BSA and mixed with 1 ml acetate buffer (0.15 M, pH 4.1). The samples were extracted with methanol over SPE columns Cl8 (Baker, The Netherlands) after addition of 500 cpm of [2,4,6,7-jH]E2 (NEN Chemicals, The Netherlands) for estimation of procedural losses. The residues of the extracts were evaporated under a stream of nitrogen, redissolved in 250 (~-1toluenemethanol (9: 1, v/v> and applied to chromatography columns (8.0 X 0.7 cm) packed with Sephadex LH-20 (Pharmacia, Uppsala, Sweden, eluting solvent: toluene-methanol 9: 1, v/v). The first 5 ml fraction was discarded and E2 was eluted in a final 3 ml fraction. The E2 fraction was dried under a stream of nitrogen and redissolved in 500 ~1 ethanol. An aliquot of 150 IJ-I was taken to determine the recovery of [3H]E2 (62%). E2 concentrations were measured in duplicate (2 aliquots of 150 pl) by RIA using a specific rabbit antiserum against 6-keto-estrone (6-CMO-BSA). The main cross-reacting steroids were oestrone (1.49%) and oestriol (0.21%). The antiserum was used in a final dilution of 1: 125 000. Oestradiol- 17p (code El 132; Sigma, Zwyndrecht) was used as a standard and [2,4,6,7-‘H]E2 (code NET 3 17; specific activity 95.4 Ci mmol- ‘, NEN Chemicals) as a tracer. The sensitivity of the assay was 20 pg ml-’ at the 80% B/B, level. The minimal detectable dose at the 90% B/B, level was 8 pgml-‘. The intra-assay coefficient of variation was 12.4% and the inter-assay coefficient of variation was 17.4%. The amount of E2 was expressed in pg ml-’ after correction for procedural losses. The moment after which E2 levels show a consistent decrease is defined as the moment of peak E2, the level of E2 at that time is defined as the peak level of E2. 2.8.3. LH Plasma concentrations of LH were measured by a double-antibody RIA as previously described by Niswender et al. (19701, using porcine LH (pLH iodination grade batch 004/3 obtained from UCB, Brussels, Belgium, potency 0.85 X NIH LHS19) as a standard and for radioiodination (specific activity 35 pCi kg-’ >. Anti-porcine LH batch 004 (also obtained from UCB> was used in a I:384000 final dilution which gave an initial binding of the labelled hormone of approximately 40%. Sac-ccl was used as second antibody (donkey anti-rabbit, Welcome, Beckenham, England). The sensitivity of the assay was 0.5 ng ml- ’ at the 80% B/B, level. The minimal detectable dose at the 90% B/B, level was 0.13 ngml- ‘. The inter-assay coefficient of variation was 13.8% and the intra-assay coefficient of variation was 10.6%. Basal LH level is the mean LH
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level in the samples taken from 44 h up to 20 h before peak LH levels. For the rapid LH assay, incubation was 2 h at 37°C instead of 16 h at 4°C. 2.8.4. Progesterone Plasma concentrations of progesterone were measured by RIA essentially as previously described by Helmond et al. (1986). A specific rabbit antiserum raised against a 4-pregnene-6P-ol-3,2O-dione-hemisuccinate-BSA-conjugate was used in a final solution of 1:75 000. Progesterone (code P-9776; Sigma) was used as a standard and [1,2,6,7‘HIprogesterone as a tracer (code NET 381, specific activity 93.5 Ci mmol- ’; NEN Chemicals). The sensitivity of the assay was 0.2 ng ml- ’ at the 80% B/B,, level. The minimum detectable dose at the 90% B/B, level was 0.15 ngml- ‘. The intra-assay coefficient of variation was 7.6% and the inter-assay coefficient of variation was 14%. The main cross-reacting steroids were pregnenolone (98%), corticosterone (2.7%), 17o-hydroxyprogesterone (15%) and 20o-hydroxy-4-pregnen-3-one, cortisol, oestrone, oestradiol-17B, oestradiol-17a, oestradiol, androstenedione, dehydroandrosterone and testosterone (all < 0.2%). 2.9. Insemination In the absence of a boar, the sows were inseminated with a commercial dose of 3 X IO9 mixed semen from 3 GY boars at 28 h after first rise in LH levels as detected with the rapid LH assay. If the sow had not ovulated within 20-22 h after first insemination, she was inseminated again. 2.10. Detection of ovulation
The procedure of transrectal ultrasonography to study the process of ovulation was described in detail by Soede et al. (1992). An annular array sector scanner (type 15OV, Pie Medical, Maastricht, The Netherlands) with a 5 and 7.5 MHz multiple scan angle transducer was used. Before scanning, faeces were removed from the rectum manually with an abundant use of a lubricant. During subsequent scanning, the number of pre-ovulatory follicles (diameter of antrum 2 5 mm) was determined. Sows were scanned at intervals of 1 h from 30 h after first rise in LH levels onwards since sows have been found to ovulate at 44 + 3 (mean &-sd; 37 to 49) h after first rise in LH levels (Soede et al., 1994). Onset of ovulation was defined based on a consistent decrease in the number of follicles on the ovary and end of ovulation for each ovary was determined based on the presence of a constant number of few or no follicles on the ovary at two consecutive scannings. 2.1 I. Parameters at slaughter
Sows were slaughtered at 122 f. 1 (range 111 to 133) h after onset of ovulation. The stomach, adrenal glands and reproductive tract were removed.
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2.11 .I. Embryo development Each oviduct was flushed with 15 ml Dulbecco’s PBS (DPBS) from the infundibulum into the uterus. Thereafter, the oviduct was separated from the uterus and each uterine horn was flushed twice with 30 ml DPBS to collect the embryos + oocytes. Recovery rate was determined as the percentage of embryos + oocytes recovered based on the corpora lutea count. The embryos + oocytes were subjected to hypotonic treatment (0.6% KC1 solution, 0°C 10 min) and subsequently placed on a fat free glass slide. Small droplets of methanol-acetic acid (3:1, v/v> were added until disruption and spreading of the embryo (generally 1 cm2), which enables counting of the nuclei and sperm cells using a microscope (magnification 200 X ) after drying and staining with 10% Giemsa in PBS solution. A nuclei count of zero or one was considered an unfertilized oocyte. Embryos with a degenerated morphological appearance together with a low number of nuclei were considered “degenerated”, others were considered “normal” embryos. Fertilization rate and rate of normal and degenerating embryos was determined based on the total number of recovered embryos + oocytes. 2.1 I .2. Reproductive organs The number of corpora lutea was counted on both ovaries. 2. 11.3. Adrenal glands The adrenal glands were weighed. At 25%, 50% and 75% of the length, the diameter of the cortex was measured at the dorsal, ventral and lateral sides. 2.11.4. Stomach The condition of the stomach?wall? was scored from 0 to 5: 0 = intact; 1 slight hyper/parakeratoses; 2 = obvious hyper/parakeratoses; 3 = hyper/parakeratoses and few, small, lesions; 4 = hyper/parakeratoses and more, bigger, lesions and 5 = hyper/parakeratoses and many, big lesions and/or active or chronic ulcers. 2.12. Statistical analyses Data were analysed using SAS (1990). Data are presented as mean & sem. To test differences in reproductive and stress parameters between the housing conditions, the GLM procedure of SAS was used. Correlations between reproductive and stress parameters were tested with the CORR procedure, both for all data and for the treatment groups separately. The GLM procedure was subsequently used to make equations of the significant correlations.
3. Results Nine out of thirteen tethered and eleven out of thirteen loose housed sows came in oestrus at 4.8 f 0.1 (4 to 6) d after weaning (P > 0.10). The duration of oestrus was 1.5 f 0.2 (1 to 3) days (P > 0.10). At Day 12 after weaning, five out of the six sows that had not shown oestrus had corpora lutea, the sixth sow (a loose housed sow) had
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inactive ovaries. After the Day 20 altrenogest treatment (given to all 26 sows), nine out of thirteen tethered sows came in oestrus after 4.3 + 0.2 (4 to 5) days and twelve out of thirteen loose housed sows came in oestrus after 5.7 &-0.3 (4 to 9) days (P < 0.01). The duration of oestrus was 2.0 & 0.2 (1 to 3) days (P > 0.10). One additional tethered sows came in oestrus after 5 days, oestrus lasted 6 days and the sow developed cystic ovaries. Of the remaining three tethered sows that did not show oestrus, two sows had a silent oestrus and one sow developed cystic ovaries; the two sows with a silent oestrus after altrenogest treatment also had a silent first oestrus after weaning. The one loose housed sow that did not show oestrns developed cystic ovaries. The three sows with cystic ovaries were taken from the experiment. In both housing conditions, a sow became ill and was culled at approximately 50 days after arrival (between second and third oestrus). As a consequence, ten tethered sows and eleven loose-housed sows were used to assess reproductive parameters around (third) oestrus at approximately 65 days after arrival. From Day 18 after weaning, the levels of stereotypic behaviour after afternoon feeding were assessed. Between Days 18 and 40 after weaning, sows showed stereotypic behaviour during on average 50-55% of the time in the ten loose-housed sows and increasing from 50 to 65% of the time in the eleven tethered sows. These differences between treatments were not significant (Fig. 1). At Day 52-53, stereotypies took place during 48 + 9% of the time in the loose-housed sows and during 68 + 8% of the time in the tethered sows (P = 0.11) and at Day 67-68, the time spent on stereotypies was 40 * 10% and 78 _t 5%, respectively (P < 0.05). When all sows (including the sows with cystic ovaries and the sows that were culled during the experiment) are taken into account, the results are similar; at Day 52-53, stereotypic behaviour took place during 46 ? 8% (n = 13) of the time in the loose-housed sows and during 67 * 8% (n = 13) of
100
90 60 i
3 .E
70
z e ; .-
60-
s : z z
40-
t
50-
30-
30 Days after
40
weaning
50
60
(d)
Fig. I. Levels of stereotypic behaviour (% of time, mean f se) during the first hour after afternoon feeding in loose-housed (--, n = 11) and tethered (---, n = 10) sows between Days 15 and 65 after weaning.
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55 50 45 = 40 E -, 35 E30
-
{
25-
0
20 15 IO L 50_’ -96
-72
-40
-24 Time relative
Fig. 2. Cortisol levels &ml-‘)
0
24
to feeding
40
72
96
(min)
around afternoon feeding in loose-housed (-,
n = 11) and tethered (-- -,
n = 10) sows at Day 45 after weaning.
time in the tethered sows (P < 0.10) and at Day 67-68, the time spent on stereotypies was 34 + 9% (n = 12) and 69 k 10% (n = 121, respectively (P < 0.05). Cortisol levels around afternoon feeding at Day 36 and at Day 56 after weaning were similar in the loose-housed and tethered sows (see Fig. 2 for data of Day 56). Cortisol levels were not different between Day 36 and Day 56, in either of the two treatment groups (P > 0.10). Peak levels of cortisol in these two hours were also similar in the eleven loose-housed and ten tethered sows, at Day 56: 58 + 15 (19-174; median 42) ngml-’ and 54 f 13 (12-141; median 47) ngml-‘, respectively. After two months, tethered sows had a significantly shorter duration of oestrus than the loose-housed sows, on average 42 f 4 compared to 63 + 2 h (P < 0.001). In both therefore, groups, ovulation took place at approximately 70% of oestrus (P > 0.101, tethered sows also had a shorter interval between onset of oestrus and ovulation than the loose-housed sows (P < 0.001; Table 1). The duration of ovulation was not influenced by treatment: tethered, 2.9 vs. loose-housed, 2.1 h (P = 0.16). The duration of ovulation was 1, 2, 3, 4 and 5 h in 0, 4, 1, 1 and 1 tethered sows and in 2, 5, 2, 0 and 0 loose-housed sows, respectively. Ovulation rate varied from 17 to 25 and was similar in both groups of sows ( P > 0.10). In Fig. 2, hormone profiles of oestradiol, LH and progesterone around third oestrus at approximately Day 65 after weaning are presented. Peak levels of oestradiol-17p were reached between 4 h and 28 h (on average 13 f 2 h; P > 0.10)) before peak levels of LH. The peak levels of oestradiol-17p varied from 23 to 105 pgml-’ and were similar for the two treatment groups (tethered, 60 + 8 pg ml-’ vs. loose-housed, 60 _+9 pgml-‘; P > 0.10). Also the total profiles of oestradiol- 17p were similar for the two treatment groups (Fig. 3a). Basal levels of LH varied considerably between sows from 1.1 to 10.8 ngml-‘, and were not significantly affected by treatment (tethered, 4.0 &-1.1
the
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Table 1 Parameters of oestrus and ovulation around Day 60 after weaning of sows that were either tethered or housed loose from weani:.g onwards Tethered n
Cycle length (d)
’
Duration of oestrus (h) Ovulation * Timing of ovulation Hours after onset of oestrus % Of oestrus Duration of ovulation (h) Ovulation rate (n)
Mean f sem
8
Loose-housed Range
n
Mean f sem
Range
21.OkO.2
20-22
11
20.5 i 0.4
17-23
IO
42&4a
24-60
11
63k2
52-76
9 9
28+2a 71*4
16-34 50-83
9 9
41f2b 67&3
28-49 50-81
7
2.9 + 0.5 21.2kO.8
2-5 17-24
8 11
2.1 kO.2 20.8 + 0.8
l-3 17-25
IO
b
a.bP <
0.01 ’ In two tethered sows, the previous oestrus was a silent oestrus. * In pne of the tethered sows ovulation was already complete at first scanning, therefore time and duration of ovulation are unknown and in two tethered sows, ovulation had just started at first scafming, therefore, duration of ovulation is unknown. Two of the loose-housed sows ovulated later than expected, therefore, time and duration of ovulation are unknown and in one loose-housed sow ovulation had just started at first scanning, therefore, duration of ovulation is unknown.
loo$e-housed, 2.9 & 0.8 ngr@- ’; P > 0.10). Peak iH levels were 8.9 f 1,O and 8.5 f 110 ngml-,’ for thk tethered and loose-housed sows, respectively (P > 0.10; Fig. 3b). Sows ovulated betwien 28 and 35 h (on average 31 k 1 h) after peak levels of LH, which was not affected by.;$zatment (P > 0.10). Progesterone pro$les up, to 132 h after p&k LH levels were similar fbr the ttio treatment groups (Fig. 3~). Of the eighteen sows
vs.
Fig. 3: Profiles: (A) oestradiol-17p (loose-housed -, n = 8 and tethered -- -, n = 8); (B) LH (loose-housed --; n = I I andt&bed - - -, n = IO) rind (C) pkogesterone (loose-housed -, n = 11 and tethkred - - 1, n = IO) around oestrus at approximately Day 60 aftei wkaning in loose-housed and tethered SOWS.
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60 !(a)
01 -48
I
-36
-24
I
1
-12
0
~/_
12
24
’ 12
24
i i
36
48
36
46
12 (b) 10 I
Q
oL.L’ -46
-36
-24
Time
-12
relative
0
to peak
LH levels
I
’
(h)
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Table 2 Embryo development at Day 5 after ovulation in tethered and loose-housed sows that were inseminated at 11k 1 (ranae 2 to 22) h before ovulation Tethered ( n = 9)
Embryo age (h)
Loose-housed (n = 9)
Mean 5 sem
Range
Mean f sem
Range
122*2
Ill-133
120+4
1P-129
Embryo quality Normal (%) Degenerating (%) Unfertilized (%)
98+1 0*0 2rtl
92-100 o-o O-8
86-1-33 1*4 13+33
O-100 O-11 O-100
Embryo development Cell cycles (n) sd cell cycles (n) Accessory sperm count(n)
6.0 f 0.2 0.4kO.03 86+ 19
4.7-7.4 0.3-0.7 22- 169
5.7 + 0.2 0.6*0.1 64*20
4.8-6.9 0.2-0.9 I-153
was not influenced by treatment (P > 0.05), but was related to the interval between insemination and ovulation (I-O) which varied from 2 to 22 h: sperm count (n) = 174 - 7.8 X I-O (h) [ R2 = 0.55, P = 0.003, n = 13 (excluding the three sows that were inseminated twice)]. Average weight of the adrenal glands was 8.7 + 0.7 g in the tethered sows and 7.4 k 0.5 g in the loose-housed sows (P > 0.10). The diameter of the adrenal cortex was 4.0 f. 0.2 mm and 3.5 k 0.2 mm, respectively (P > O.lO), and the diameter of the adrenal medulla was 3.8 f 0.3 mm and 4.3 z!-0.2 mm, respectively (P > 0.10). The condition of the stomach wall at slaughter varied from 0 (intact) to 4 (hyper/parakeratoses and lesions). An intact stomach wall was present in three out of ten tethered sows and five out of elenen loose-housed sows. Lesions occurred in five out of ten tethered sows and three out of eleven loose-housed sows (P > 0.10). The remaining sows had hyper/parakeratoses, but no lesions. In the tethered sows, the average level of stereotypies before third oestrus (measured between 12 and 17 days before oestrus) was linearly related to the duration of third oestrus: [duration of third oestrus (h)] = 63 f 0.32 X [level of stereotypies (% of time)] (P = 0.07). In the loose housed sows no such relation existed (P = 0.65). Basal LH levels before the LH surge were positively related to adrenal weight 5 days after subsequent ovulation (r = 0.65, P = 0.005) in both housing conditions. Even without the three sows with extreme high basal LH levels (more than 10 ngml-’ in two tethered and one loose housed sow, the remaining sows had basal LH levels of less than 5 ng ml- ’), the relation remained significant (P = 0.045). 4. Discussion Multiparous sows, 26 in total, that had been tethered during lactation were put in two different housing conditions after weaning; the sows were either tethered by neck chain, or put in a pen of approximately 6 m2. During a period of 60 days, five sows were taken
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from the experiment. In both housing conditions one sow became ill and was culled and, furthermore, two tethered and one loose-housed sow developed cystic ovaries. The cystic ovaries in the three sows developed during treatment with altrenogest, a progesterone analogue. Not much is known about causes or incidence of cystic ovaries in sows. However, a possible cause of cystic ovaries is a disturbed relation between progesterone and oestrogens in the follicular phase, possibly related with the use of gonadotropins (Wrathall, 1980; Ryan and Raeside, 1991a,b). At Day 18 after weaning, a first assessment was made of the level of stereotypies during one hour after the afternoon feeding. The average level of stereotypies was approximately 50% in both housing conditions. Normally, large individual differences are found in the level of stereotypies within a group of animals (Appleby and Lawrence, 1987); that was also the case in the present experiment as is apparent from the large standard error of the mean. The average level of stereotypies after afternoon feeding at Day 18 is relatively high, presumably as a consequence of the fact that the sows had been tethered during lactation. Stereotypic behaviour is considered a coping strategy of sows that are in a chronic stress condition (Cronin, 1982; Schouten and Wiepkema, 1991); during stress, low stereotyping sows have been found to show the strongest signs of uneasiness (Schouten and Wiepkema, 1991). After two months, the level of stereotypic behaviour increased in the tethered sows and decreased in the loose-housed sows, to an average level of stereotypic behaviour on Day 67 after weaning of 80% (tethered sows) vs. 40% (loose-housed sows) (P = 0.03). It seems that in the loose-housed sows, stereotypic behaviour decreased because of the less stressful housing conditions. In the ‘tethered sows, the level of stereotypies increased because of the chronic stress induced by tethering. One would expect that the difference in stereotypic behaviour between the loose-housed and tethered sows would also be reflected in other stress-related parameters. However, the levels of cortisol at Day 36 and at Day 56 did not differ between the treatment groups, nor did the weight of the adrenal glands, the size of the adrenal cortex, and the pathology of the stomach wall at approximately Day 70 after weaning. Furthermore, no relations existed between stereotypic behaviour and any of the stress parameters within the two groups of sows. Apparently, in the present experiment, stereotypic behaviour is a more sensitive measure for the difference in stress conditions, than the other stress-related parameters that were measured. During the first and second oestrus after weaning, more tethered sows had a silent oestrus (two sows once and two sows twice) than loose-housed sows (one sow once). The duration of oestrus of the sows that came in heat was not influenced by the housing conditions. During the third oestrus, the duration of oestrous of the tethered sows was significantly shorter than that of the loose-housed sows, on average 42 f 12 compared to 63 + 8 h. These differences in duration of oestrus were not reflected in the profiles of reproductive hormones (oestradiol-17l3, LH and progesterone). These profiles were not affected by the housing conditions, nor were relations found between reproductive hormones and the duration of oestrus within the two groups of sows. The higher number of SOWS with a silent first and second oestrus in the tethered sows and also the shorter duration of the third oestrus in the tethered sows may be well be related to the stressful housing conditions of tethering. In a number of experiments, a reduction in the number of animals showing oestrus or the duration of oestrus was found after an injection with
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adrenocorticotropic hormone (ACTH) or synthetic corticosteroids (pigs: Liptrap, 1970; Esbenshade and Day, 1980; ovariectomized gilts: Esbenshade et al., 1983; sheep: Ehnert and Moberg, 1991; ovariectomized cattle: Hein and Allrich, 1992). Unexpectedly, in our sows, the effects of the housing conditions on oestrous behaviour were not related with effects of housing on cortisol levels, neither did a relation exist between oestrous behaviour and cortisol within the experimental groups. Apparently, differences in oestrous behaviour between the two groups were not directly mediated through effects on cortisol. Besides cortisol, however, other stress-related hormones may be of importance. It is generally known that stress-related hormones such as endorphins, CRH and ACTH may influence sexual functions at all three levels of the HPG-axis (Rivier and Rivest, 1991). Oestrous behaviour was significantly related with the levels of stereotypic behaviour in the tethered sows; sows with a high level of stereotypic behaviour had a significantly shorter duration of oestrus. During stereotypic behaviour, endogenous opioid peptides (EOP), endorphines are released (Wiepkema and Schouten, 1992). A number of EOPs are known to suppress LH release (Malven, 1986) and may consequently affect reproductive processes. For example, Armstrong et al. (1988) showed that chronic administration of morphine (an EOP-agonist) delayed the onset of oestrus after weaning. A direct relation between LH release (LH suppression) and the duration of oestrus, however, has not been described. In the present experiment, LH levels were not influenced by the housing conditions of the sows and no relation existed between basal or peak LH levels and stereotypic behaviour. The duration of oestrus is an important parameter to predict the time of ovulation; in quite a few experiments, is has been assessed that ovulation takes place at approximately two-thirds of the duration of oestrus, independent of the (quite variable) duration of oestrus (Weitze et al., 1994; Soede et al., 1995). Even under the extreme conditions of the present experiment, sows ovulated at on average 67-71% of the duration of oestrus in both housing conditions. The duration of oestrus, therefore, is of utmost importance for the timing of insemination in sows (Soede and Kemp, 1996). Differences in ovulation time of individual oocytes have been related to differences in embryo development and, consequently, of embryo survival within sows (Pope et al., 1990). Therefore, differences in the duration of ovulation between sows may be related to differences in embryo diversity between sows and, consequently, with differences in embryo survival between sows. Differences in the duration of ovulation between sows of 1 to 4 h were not related to differences in embryo diversity at Day 5 after ovulation (Soede et al., 1992; Soede and Kemp, 1993). It was suggested that stressful conditions may influence the duration of ovulation, since in one experiment with tethered sows, durations of ovulation were found between 2 and 7 h (Soede et al., 1992). Since the sows were not inseminated, embryo diversity was not assessed. In the present experiment, the duration of ovulation was not significantly different between the tethered and loose-housed sows; the duration of ovulation varied between 1 and 3 h in the loose housed sows and between 2 and 5 h in the tethered sows. No relations were found with embryo diversity at Day 5. So apparently the longer durations of ovulation that were found in an earlier experiment with tethered sows (Soede et al., 1992), were not (solely)
due to the tethering.
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In conclusion, sows that had been tethered during lactation, and were housed loose or were tethered again at weaning, within two months differed both in stereotypic behaviour and in oestrous behaviour. These effects were not related to differences in profiles of cortisol or oestradiol-17l3, LH or progesterone. Furthermore, within the tethered sows, a short duration of oestrus was related with high levels of stereotypic behaviour. No such relation existed in the loose-housed sows. All the sows in this experiment had been tethered before. It might be that especially in these high parity sows, set points and/or regulation of the reproductive hormones are irreversibly changed and, therefore, not influenced by housing conditions any more.
Acknowledgements
The authors gratefully acknowledge the great help of Lucy d’Hoore, Joanne Maaskant, Frits Rietveld and Wouter Hazeleger during the experiment.
References Armstrong, J.D., Kraeling, R.R. and Britt, J.H., 1988. Morphine suppresses luteinizing hormone concentrations in transiently weaned sows and delays onset of es&us after weaning. J. Anim. Sci., 66: 2216-2223. Appleby, MC. and Lawrence, A.B., 1987. Food restriction as a cause of stereotyped behaviour in tethered gilts. Anim. Prod., 45: 103-l 10. Bamett, J.L., Hemsworth, P.H., Winfield, C.G. and Hansen, C., 1986. Effects of social environment on welfare status and sexual behaviour of female pigs. 1. Effects of group size. Appl. Anim. Behav. Sci., 16: 249-257. Becker, B.A., Ford, J.J., Christenson, R.K., Manak, R.C., Hahn, G.L. and DeShazer, J.A., 1985. Cortisol response of gilts in tether stalls. J. Anim. Sci., 60: 264-270. Cronin, G.M., 1982. The development and significance of abnormal stereotyped behaviours in tethered sows. Ph.D. Thesis, Agricultural University, Wageningen, The Netherlands, 146 p. Dalin, A.M., Nyberg, L. and Eliasson, L., 1988. The effect of transportation/relocation on cortisol, CBG and induction of puberty in gilts with delayed puberty. Acta Vet. Stand., 29: 207-218. Ehnert, K. and Moberg, G.P., 1991. Disruption of oestrous behaviour in ewes by dexamethasone or management-related stress. J. Anim. Sci.. 69: 2988-2994. Esbenshade, K.L. and Day, B.N., 1980. The effect of triamcinolone acetonide on plasma glucocorticoid levels and occurrence of estrus in gilts. J. Anim. Sci., 51: 153-157. Esbenshade, K.L., Paterson, A.M. and Day, B.N., 1983. Glucocorticoids and estrus in swine. I The relationship between triamcinolone acetonide and estrogen in the expression of estrus in gilts. J. Anim. Sci., 56: 460-465. Hein, K.G. and Alhich, R.D., 1992. Influence of exogenous adrenocorticotropic hormones on estrous behaviour in cattle. J. Anim. Sci., 70: 243-247. Helmond, F.A., Simons, P.A. and Hein, P.R., 1980. The effects of progesterone on estrogen-induced luteinizing hormone and follicle-stimulating hormone in the female rhesus monkey. Endocrinology, 107: 478-485. Helmond, F.A., Aamink, A. and Oudenaarden, C., 1986. Periovulatory hormone profiles in relation to embryonic development and mortality in pigs. In: J.M. Sreenan and M.G. Diskin (Editors), Embryonic mortality in farm animals, Martinus Nijhoff, Dordrecht, pp. 1 19- 125. Hemsworth, P.H., Bamett, J.L., Hansen, C. and Wintield, C.G., 1986. Effects of social environment on welfare status and sexual behaviour of female pigs. II. Effects of space allowance. Appl. Anim. Behav. Sci.. 16: 259-267.
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Hennessy, D.P. and Williamson, P., 1983. The effects of stress and of ACTH administration in hormone protiles, oestnrs and ovulation in pigs. Theriogenology, 20: 13-26. Janssens, C.J.J.G., Helmond, F.A. and Wiegant, V.M., 1994. Increased cortisol response to exogenous adrenocorticotropic hormone in chronically stressed pigs: influence of housing conditions. J. Anim. Sci., 12: 1771-1777. Liptrap, R.M.. 1970. Effect of corticotrophin and corticosteroids on oestrus, ovulation and oestrogen excretion in the sow. J. Endocrinol. 47: 197-205. Niswender, G.D., Reichert, L.E. and Zimmerman, D.R., 1970. Radioimmunoassay of serum concentrations of luteinizing hormone throughout the estrous cycle in pigs. Endocrinology, 87: 576-580. Malven, P.V., 1986. Inhibition of pituitary LH release resulting from endogenous opioid peptides. Dom. Anim. Endocrinol., 3: I35- 144. Moberg, G.P., 1987. Influence of adrenal axis upon the gonads. Oxf. Rev. Reprod. Biol., 9: 456-496. Pope, W.F., Xie, S., Broermann, D.M. and Nephew, K.P., 1990. Causes and consequences of early embryonic diversity in pigs. J. Reprod. Fert. Suppl., 40: 251-260. Rivier, C. and Rives& S., 1991. Effect of stress on the activity of the hypothalamic-pituitary-gonadal axis: peripheral and central mechanisms. Biol. Reprod., 45: 523-532. Ryan, P.L. and Raeside, J.I., 1991a. Cystic ovarian degeneration in pigs (first of two parts). A review. Irish Vet. J., 44: 22-25. Ryan, P.L. and Raeside, J.I., 1991b. Cystic ovarian degeneration in pigs (second of two parts). A review. Irish Vet. J., 44: 27-36. SAS/STAT, 1990. User’s guide, SAS Institute, Gary, NC. Schouten, W. and Rushen, J., 1992. Effects of naloxone on stereotypic behaviour and normal behaviour of tethered and loose-housed sows. Appl. Anim. Behav. Sci., 33: 17-26. Schouten. W.G.P. and Wiepkema, P.R., 1991. Coping styles of tethered sows. Behav. Process., 25: 125- 132. Soede, N.M. and Kemp, B., 19%. Timing of AI and ovulation in sows. Reprod. Dom. Anim., 31: 201-207. Soede, N.M., Noordhuizen, J.P.T.M. and Kemp, B., 1992. The duration of ovulation in pigs, studied by transrectal ultrasonography, is not related to early embryonic diversity. Theriogenology, 38: 653-666. Soede, N.M. and Kemp, B., 1993. In synchronized pigs, the duration of ovulation is not affected by insemination and is not a determinant for early embryonic diversity. Theriogenology, 39: 1043-1053. Soede, N.M., Helmond, F.A. and Kemp, B., 1994. Petiovulatory profiles of cestradiol, LH and progesterone in relation to oestrus and embryonic mortality in multiparous sows using transrectal ultrasonography to detect ovulation. J. Reprod. Fert., 101: 633-641. Soede, N.M., Wetzels, C.C.H., Zondag, W., de Koning. M.A.I. and Kemp, B., 1995. Effects of time of insemination relative to ovulation, as ‘determined by ultrasonography, on fertilization rate and accessory sperm count in sows. J. Reprod. Fert., 104: 99-106. Varley, M. and Stedman, R., 1994. Stress and reproduction. In: D.J.A. Cole, J. Wiseman and M.A. Varley (Editors), Principles of pig science. Nottingham University Press, UK, pp. 277-296. Weitze, K.F., Wagner-Rietschel, H., Waberski, D., Richter, L. and Krieter, J., 1994. The onset of heat after weaning, heat duration, and ovulation as major factors in AI timing in sows. Reprod. Dom. Anim., 29: 433-443. Wiepkema, P.R. and Schouten, W.G.P., 1992. Stereotypies in sows during chronic stress. Psychother. Psychosom., 57: 194- 199. Wrathall, A.E., 1980. Ovarian disorders in the sow. Vet. Bull., 50: 427-435.
Animal Reproduction Science 46 ( 1997) 133- 148
Oestrus, ovulation and perk-ovulatory hormone profiles in tethered and loose-housed sows Nicoline M. Soede Wqeningen
Institute
oj’Animu1
*,
Frans A. Helmond, Willem G.P. Schouten, Bas Kemp
Sciences (WIASI,
Wqeninpn
AH Wugeningen,
A~riculturul
Uniuersity,
P.O. Box 338, 6700
The Netherlunds
Accepted 8 August 1996
Abstract
Multiparous sows that had heen tethered during lactation were put in two different housing conditions after weaning (Day 0); the sows were either tethered by neck chain, or individually housed in a pen of approximately 6 m*. After two months, ten tethered and eleven loose housed sows were used to assess stress and reproductive parameters. Stereotypic behaviour after the afternoon feeding was assessed from Day 18 onwards; at Day 53 stereotypic behaviour tended to occur during a higher percentage of time in the tethered sows (P = 0.11) and at Day 66, the differences were significant (tethered, 78 f 5 vs. loose-housed, 40 + 10% of time (mean + sem); P = 0.03). At Day 35 and 55, cortisol profiles after afternoon feeding were similar for the two groups of sows (P > 0.10). Around oestrus (approximately Day 641, the profiles of oestradiol-17P, luteinizing hormone and progesterone were measured and proved to be similar for both treatment groups (P > 0.10). The duration of oestrus was shorter in the tethered sows (42 k 4 vs. 63 + 2 h; P < 0.001) and, consequently, the timing of ovulation during oestrus (h after onset of oestrus) was advanced in the tethered sows (28 + 2 vs. 41 + 2; P < 0.001). The duration of ovulation did not differ (tethered, 2.9 f 0.5 vs. loose-housed, 2.1 k 0.2 h; P = 0.16). The sows were sacrificed at Day 5 after ovulation; ovulation rate, fertilization rate, embryo development and embryo diversity were similar for the two groups, as were adrenal weight and size of adrenal cortex. Duration of oestrus and the levels of stereotypies at Day 60 tended to be negatively related in the tethered sows (P = 0.071, but not in the loose-housed sows (P = 0.65). In conclusion, sows that had been tethered during pregnancy and lactation, and were housed loose or were tethered again at weaning,
* Corresponding author. Department of Animal Husbandry, P.O. Box 338, 6700 AH Wageningen, The Netherlands. Tel.: + 3 I 3I7 4839’73; fax: + 31 3 17 485006; e-mail: [email protected]. 0378.4320/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-4320(96)01609-O
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within two months differed
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both in stereotypic
behaviour
and in duration of oestrus, without
apparent effects on reproductive hormones. Keywords: Stress; Reproduction;Oestnts; Sows
1. Introduction Stressful conditions may negatively influence reproductive performance of animals (reviewed by Moberg, 1987; Rivier and Rivest, 1991; Varley and Stedman, 1994). Hormones of the HPA-axis (hypothalamic-pituitary-adrenal axis) that are released during stress, may interact with hormones of the HPG-axis (hypothalamic-pituitarygonadal axis) at many levels. Reproductive processes that are especially susceptible to stress include processes that depend on delicate hormonal changes, such as follicular growth and ovulation (Hennessy and Williamson, 1983; Dalin et al., 1988) and oestrous behaviour (Barnett et al., 1986; Hemsworth et al., 1986; Hein and Alhich, 1992). Tethering of sows is considered a stressful situation (Cronin, 1982; Schouten and Rushen, 1992; Janssens et al., 1994), resulting in increased levels of cortisol (Becker et al., 1985; Janssens et al., 1994) and increased levels of stereotypic behaviour (Cronin, 1982; Wiepkema and Schouten, 1992). Stress-related hormones are known to influence sexual functions at all three levels of the HPG-axis (Rivier and Rivest, 1991) and, consequently, effects of tethering on reproductive performance have been much debated. However, not much evidence of effects has been presented. In the present experiment, reproductive parameters (hormone patterns, oestrus, ovulation and early embryo development) were studied in sows that were either tethered or housed loose in a pen. Furthermore, stress parameters (cortisol, stereotypies, adrenal weight) were measured.
2. Materials and methods 2.1. General experimental schedule At the day of weaning, 26 sows arrived at the experimental farm and were housed either in large individual pens of 6 m* or were tethered by neck chain. To investigate effects of these housing conditions on reproductive performance, the sows were given an indwelling jugular vein catheter between 11 and 15 days after arrival. From Day 16 to 36 after arrival, all sows were put under progesterone dominance to synchronize oestrus. Around the second oestrus after imposed progesterone dominance (at approximately 65 days after arrival), the following reproductive parameters were studied: onset and duration of oestrus, onset and duration of ovulation, and profiles of oestrogen, luteinizing hormone (LH) and progesterone. At slaughter, at approximately 120 h after ovulation, Day 0
ovulation rate and embryo development and diversity were assessed. weaning, arrival at experimental farm, start of experiment
Day 4-6 Day 11-15
oestrus cannulation of the vena jugularis
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Day Day Day Day Day Day
18 16-36 40-45 55 62-66 70-74
onwards assessment of stereotypic behaviour altrenogest treatment (Day 35: cortisol profiles) oestrus cortisol profiles oestrus, ovulation, oestradiol, LH and progesterone ovarian, embryonic and adrenal parameters
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profiles
2.2. Animals and housing In total, 26 multiparous commercial crossbred (Great Yorkshire X Dutch Landrace) sows [parity 4.2 + 0.3 (range 2 to 711 arrived at the experimental unit at the day of weaning. All sows came from one farm and had been tethered during pregnancy and lactation. At the experimental unit, sows were tethered by neck chain or placed individually in pens of 6 m2. The tethered sows were all housed in one compartment, all in one row. The penned sows were divided over two compartments. In the three compartments, no other animals were present during the experiment. The sows were fed at approximately 8 am and 4 pm, a total of 2.5 kg of a commercial sow ration and water was available ad libitum. Bedding in the pens consisted of saw dust. In the compartments, the lights were on from 7 am till 7 pm. 2.3. Jugular vein cannulation The sows were surgically fitted with a permanent jugular vein catheter between 12 and 16 days after arrival to allow repeated blood sampling without disturbing the animals. The animals were fasted overnight before surgery. Surgery was carried out under sterile conditions and general anaesthesia. The sows were premeditated by injecting 6 mg azaperone kg- ’ body mass i.m. (Stresnil; Janssen, Tilburg, The Netherlands). After 30 min, general anaesthesia was induced and maintained using O,-N,O and 2% halothane (Halothane; Trofield, Zug, Swiss). An indwelling catheter (Silastic 1.02 mm I.D., 2.16 mm O.D.; Dow Coming, Midland, MI, USA) was implanted into the external jugular vein towards the superior vena cava. The other end of the cannula was passed S.C. to the back of the animal, where it was externalized between the scapulae. A one-way luer-lock stopcock (Vygon, Veenendaal, The Netherlands) was secured to the end of the cannula to which a (10 ml) syringe could be attached easily. To protect the cannula from damage, the animals were harnessed. The harness (23 X 20 cm, PVC with nylon; Bizon Chemie, Goes, The Netherlands) was fixed at the back of the animals with belts tied around the chest. From three days before surgery until three days after surgery, all animals were given 12 ml of the antibiotic T.S. Sol (Dopharma, Raamsdonksveer, The Netherlands) orally once a day. The animals were given at least ten days to recover from surgery and anaesthesia. The patency of the cannula was maintained by flushing it three times a week and filling it with sterile heparinized physiological saline (25 IU heparin ml - ’, 0.9% NaCl; Leo, Weesp, The Netherlands) when not in use. 2.4. Detection of oestrus Detection of oestrus was performed by a back-pressure test in the presence of one of two vasectomized boars in front of the sows. Detection of first oestrus after weaning and
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the oestrus after altrenogest treatment were performed once daily. Detection of third oestrus was performed at intervals of 4 h, immediately after blood collection. The time of onset of third oestrus was defined as 2 h before the first time a sow showed a standing response to the back pressure test in the presence of the boar. The time of the end of third oestrus was defined as 2 h after the last time the sow showed a standing response to the back-pressure test in the presence of a boar. 2.5. Stereotypic behaviour From 15 min after feeding onwards, sow behaviour was scored every min for 60 min. Each observer walked quietly along the row of sows or pens and noted at 1 min intervals the behaviour of all individual sows. The observer noted the posture of the sow (standing, sitting, lying and in free sows also walking) and the occurrence of stereotypies (vacuum chewing, bar biting, chain manipulation and snout rubbing). 2.6. Cycle synchronization From Day 17 after arrival onwards, 20 ml altrenogest (Regumate, Hoechst, Amsterdam), a progesterone analogue, was spread over the morning feed for 20 days. 2.7. Blood sampling Prior to the experimental period, all animals were often handled and were adapted to the blood collection procedure. At Day 20 of the altrenogest treatment (Day 35 after weaning) and at Day 19 after altrenogest treatment (luteal phase, Day 55 after weaning), blood samples were taken every 12 min between 72 min before afternoon feeding till 72 min after afternoon feeding and were analysed for cortisol levels. To analyse reproductive hormones around the second oestrus after altrenogest treatment, blood samples were drawn from the cannula every 4 h, at 7:30, 1 I :30, 15:30, 19:30, 23:30 and 3:30 h from Day 17 of the cycle onwards. From 24 h after ovulation onwards, blood samples were drawn at 12 h intervals, at 8:00 and 20:00 h, up to five days after ovulation. Before blood samples were drawn, the luer-lock stopcock was disinfected with ethyl alcohol (70%) and the cannula was flushed with 4 ml of sterile physiological saline (NPBI, The Netherlands) to remove possible blood clots. Approximately 8 ml of blood was collected with a sterile syringe (Beckton Dickinson, Ireland). Then the cannula was filled with heparinized saline (to keep the cannula patent). Blood samples were collected in ice-cooled polypropylene tubes, containing 100 l.~l EDTA saline; Titriplex III, Merck, The Netherlands), immediately solution (144 mgml-’ placed on ice and then centrifuged at 2000 g for 10 min at 4°C. Plasma was collected and stored at -20°C until hormone analysis. Additionally, to make an estimate of the time of ovulation in advance (and thereby reduce the h of scanning of the ovaries), the 4 h blood samples were daily analysed for LH, using a rapid LH assay. Two 200 ~1 aliquots of the samples taken between 12:00 and 8:00 h were stored at 4°C till 8:30 h, after which the rapid LH assay was performed to detect a first rise in LH levels.
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2.8. Hormone analyses 2.8.1. Cortisol Plasma concentrations of cortisol were measured in duplicate in unextracted 50 p_l samples according to a single-antibody radioimmunoassay (RIA) technique previously described by Janssens et al. (1994). The sensitivity of the assay was 0.5 ngmll’ at the 90% B/B, level. The intra-assay coefficient of variation was 8.2% and the inter-assay coefficient of variation was 14.7%. 2.8.2. Oestradiol Oestradiol-17B was measured after extraction and column chromatography by RIA using a modification of the method described by Helmond et al. (1980). Samples of 100 p-1 were diluted in 900 pl phosphate buffer BSA with 0.1% BSA and mixed with 1 ml acetate buffer (0.15 M, pH 4.1). The samples were extracted with methanol over SPE columns Cl8 (Baker, The Netherlands) after addition of 500 cpm of [2,4,6,7-jH]E2 (NEN Chemicals, The Netherlands) for estimation of procedural losses. The residues of the extracts were evaporated under a stream of nitrogen, redissolved in 250 (~-1toluenemethanol (9: 1, v/v> and applied to chromatography columns (8.0 X 0.7 cm) packed with Sephadex LH-20 (Pharmacia, Uppsala, Sweden, eluting solvent: toluene-methanol 9: 1, v/v). The first 5 ml fraction was discarded and E2 was eluted in a final 3 ml fraction. The E2 fraction was dried under a stream of nitrogen and redissolved in 500 ~1 ethanol. An aliquot of 150 IJ-I was taken to determine the recovery of [3H]E2 (62%). E2 concentrations were measured in duplicate (2 aliquots of 150 pl) by RIA using a specific rabbit antiserum against 6-keto-estrone (6-CMO-BSA). The main cross-reacting steroids were oestrone (1.49%) and oestriol (0.21%). The antiserum was used in a final dilution of 1: 125 000. Oestradiol- 17p (code El 132; Sigma, Zwyndrecht) was used as a standard and [2,4,6,7-‘H]E2 (code NET 3 17; specific activity 95.4 Ci mmol- ‘, NEN Chemicals) as a tracer. The sensitivity of the assay was 20 pg ml-’ at the 80% B/B, level. The minimal detectable dose at the 90% B/B, level was 8 pgml-‘. The intra-assay coefficient of variation was 12.4% and the inter-assay coefficient of variation was 17.4%. The amount of E2 was expressed in pg ml-’ after correction for procedural losses. The moment after which E2 levels show a consistent decrease is defined as the moment of peak E2, the level of E2 at that time is defined as the peak level of E2. 2.8.3. LH Plasma concentrations of LH were measured by a double-antibody RIA as previously described by Niswender et al. (19701, using porcine LH (pLH iodination grade batch 004/3 obtained from UCB, Brussels, Belgium, potency 0.85 X NIH LHS19) as a standard and for radioiodination (specific activity 35 pCi kg-’ >. Anti-porcine LH batch 004 (also obtained from UCB> was used in a I:384000 final dilution which gave an initial binding of the labelled hormone of approximately 40%. Sac-ccl was used as second antibody (donkey anti-rabbit, Welcome, Beckenham, England). The sensitivity of the assay was 0.5 ng ml- ’ at the 80% B/B, level. The minimal detectable dose at the 90% B/B, level was 0.13 ngml- ‘. The inter-assay coefficient of variation was 13.8% and the intra-assay coefficient of variation was 10.6%. Basal LH level is the mean LH
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level in the samples taken from 44 h up to 20 h before peak LH levels. For the rapid LH assay, incubation was 2 h at 37°C instead of 16 h at 4°C. 2.8.4. Progesterone Plasma concentrations of progesterone were measured by RIA essentially as previously described by Helmond et al. (1986). A specific rabbit antiserum raised against a 4-pregnene-6P-ol-3,2O-dione-hemisuccinate-BSA-conjugate was used in a final solution of 1:75 000. Progesterone (code P-9776; Sigma) was used as a standard and [1,2,6,7‘HIprogesterone as a tracer (code NET 381, specific activity 93.5 Ci mmol- ’; NEN Chemicals). The sensitivity of the assay was 0.2 ng ml- ’ at the 80% B/B,, level. The minimum detectable dose at the 90% B/B, level was 0.15 ngml- ‘. The intra-assay coefficient of variation was 7.6% and the inter-assay coefficient of variation was 14%. The main cross-reacting steroids were pregnenolone (98%), corticosterone (2.7%), 17o-hydroxyprogesterone (15%) and 20o-hydroxy-4-pregnen-3-one, cortisol, oestrone, oestradiol-17B, oestradiol-17a, oestradiol, androstenedione, dehydroandrosterone and testosterone (all < 0.2%). 2.9. Insemination In the absence of a boar, the sows were inseminated with a commercial dose of 3 X IO9 mixed semen from 3 GY boars at 28 h after first rise in LH levels as detected with the rapid LH assay. If the sow had not ovulated within 20-22 h after first insemination, she was inseminated again. 2.10. Detection of ovulation
The procedure of transrectal ultrasonography to study the process of ovulation was described in detail by Soede et al. (1992). An annular array sector scanner (type 15OV, Pie Medical, Maastricht, The Netherlands) with a 5 and 7.5 MHz multiple scan angle transducer was used. Before scanning, faeces were removed from the rectum manually with an abundant use of a lubricant. During subsequent scanning, the number of pre-ovulatory follicles (diameter of antrum 2 5 mm) was determined. Sows were scanned at intervals of 1 h from 30 h after first rise in LH levels onwards since sows have been found to ovulate at 44 + 3 (mean &-sd; 37 to 49) h after first rise in LH levels (Soede et al., 1994). Onset of ovulation was defined based on a consistent decrease in the number of follicles on the ovary and end of ovulation for each ovary was determined based on the presence of a constant number of few or no follicles on the ovary at two consecutive scannings. 2.1 I. Parameters at slaughter
Sows were slaughtered at 122 f. 1 (range 111 to 133) h after onset of ovulation. The stomach, adrenal glands and reproductive tract were removed.
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2.11 .I. Embryo development Each oviduct was flushed with 15 ml Dulbecco’s PBS (DPBS) from the infundibulum into the uterus. Thereafter, the oviduct was separated from the uterus and each uterine horn was flushed twice with 30 ml DPBS to collect the embryos + oocytes. Recovery rate was determined as the percentage of embryos + oocytes recovered based on the corpora lutea count. The embryos + oocytes were subjected to hypotonic treatment (0.6% KC1 solution, 0°C 10 min) and subsequently placed on a fat free glass slide. Small droplets of methanol-acetic acid (3:1, v/v> were added until disruption and spreading of the embryo (generally 1 cm2), which enables counting of the nuclei and sperm cells using a microscope (magnification 200 X ) after drying and staining with 10% Giemsa in PBS solution. A nuclei count of zero or one was considered an unfertilized oocyte. Embryos with a degenerated morphological appearance together with a low number of nuclei were considered “degenerated”, others were considered “normal” embryos. Fertilization rate and rate of normal and degenerating embryos was determined based on the total number of recovered embryos + oocytes. 2.1 I .2. Reproductive organs The number of corpora lutea was counted on both ovaries. 2. 11.3. Adrenal glands The adrenal glands were weighed. At 25%, 50% and 75% of the length, the diameter of the cortex was measured at the dorsal, ventral and lateral sides. 2.11.4. Stomach The condition of the stomach?wall? was scored from 0 to 5: 0 = intact; 1 slight hyper/parakeratoses; 2 = obvious hyper/parakeratoses; 3 = hyper/parakeratoses and few, small, lesions; 4 = hyper/parakeratoses and more, bigger, lesions and 5 = hyper/parakeratoses and many, big lesions and/or active or chronic ulcers. 2.12. Statistical analyses Data were analysed using SAS (1990). Data are presented as mean & sem. To test differences in reproductive and stress parameters between the housing conditions, the GLM procedure of SAS was used. Correlations between reproductive and stress parameters were tested with the CORR procedure, both for all data and for the treatment groups separately. The GLM procedure was subsequently used to make equations of the significant correlations.
3. Results Nine out of thirteen tethered and eleven out of thirteen loose housed sows came in oestrus at 4.8 f 0.1 (4 to 6) d after weaning (P > 0.10). The duration of oestrus was 1.5 f 0.2 (1 to 3) days (P > 0.10). At Day 12 after weaning, five out of the six sows that had not shown oestrus had corpora lutea, the sixth sow (a loose housed sow) had
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inactive ovaries. After the Day 20 altrenogest treatment (given to all 26 sows), nine out of thirteen tethered sows came in oestrus after 4.3 + 0.2 (4 to 5) days and twelve out of thirteen loose housed sows came in oestrus after 5.7 &-0.3 (4 to 9) days (P < 0.01). The duration of oestrus was 2.0 & 0.2 (1 to 3) days (P > 0.10). One additional tethered sows came in oestrus after 5 days, oestrus lasted 6 days and the sow developed cystic ovaries. Of the remaining three tethered sows that did not show oestrus, two sows had a silent oestrus and one sow developed cystic ovaries; the two sows with a silent oestrus after altrenogest treatment also had a silent first oestrus after weaning. The one loose housed sow that did not show oestrns developed cystic ovaries. The three sows with cystic ovaries were taken from the experiment. In both housing conditions, a sow became ill and was culled at approximately 50 days after arrival (between second and third oestrus). As a consequence, ten tethered sows and eleven loose-housed sows were used to assess reproductive parameters around (third) oestrus at approximately 65 days after arrival. From Day 18 after weaning, the levels of stereotypic behaviour after afternoon feeding were assessed. Between Days 18 and 40 after weaning, sows showed stereotypic behaviour during on average 50-55% of the time in the ten loose-housed sows and increasing from 50 to 65% of the time in the eleven tethered sows. These differences between treatments were not significant (Fig. 1). At Day 52-53, stereotypies took place during 48 + 9% of the time in the loose-housed sows and during 68 + 8% of the time in the tethered sows (P = 0.11) and at Day 67-68, the time spent on stereotypies was 40 * 10% and 78 _t 5%, respectively (P < 0.05). When all sows (including the sows with cystic ovaries and the sows that were culled during the experiment) are taken into account, the results are similar; at Day 52-53, stereotypic behaviour took place during 46 ? 8% (n = 13) of the time in the loose-housed sows and during 67 * 8% (n = 13) of
100
90 60 i
3 .E
70
z e ; .-
60-
s : z z
40-
t
50-
30-
30 Days after
40
weaning
50
60
(d)
Fig. I. Levels of stereotypic behaviour (% of time, mean f se) during the first hour after afternoon feeding in loose-housed (--, n = 11) and tethered (---, n = 10) sows between Days 15 and 65 after weaning.
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55 50 45 = 40 E -, 35 E30
-
{
25-
0
20 15 IO L 50_’ -96
-72
-40
-24 Time relative
Fig. 2. Cortisol levels &ml-‘)
0
24
to feeding
40
72
96
(min)
around afternoon feeding in loose-housed (-,
n = 11) and tethered (-- -,
n = 10) sows at Day 45 after weaning.
time in the tethered sows (P < 0.10) and at Day 67-68, the time spent on stereotypies was 34 + 9% (n = 12) and 69 k 10% (n = 121, respectively (P < 0.05). Cortisol levels around afternoon feeding at Day 36 and at Day 56 after weaning were similar in the loose-housed and tethered sows (see Fig. 2 for data of Day 56). Cortisol levels were not different between Day 36 and Day 56, in either of the two treatment groups (P > 0.10). Peak levels of cortisol in these two hours were also similar in the eleven loose-housed and ten tethered sows, at Day 56: 58 + 15 (19-174; median 42) ngml-’ and 54 f 13 (12-141; median 47) ngml-‘, respectively. After two months, tethered sows had a significantly shorter duration of oestrus than the loose-housed sows, on average 42 f 4 compared to 63 + 2 h (P < 0.001). In both therefore, groups, ovulation took place at approximately 70% of oestrus (P > 0.101, tethered sows also had a shorter interval between onset of oestrus and ovulation than the loose-housed sows (P < 0.001; Table 1). The duration of ovulation was not influenced by treatment: tethered, 2.9 vs. loose-housed, 2.1 h (P = 0.16). The duration of ovulation was 1, 2, 3, 4 and 5 h in 0, 4, 1, 1 and 1 tethered sows and in 2, 5, 2, 0 and 0 loose-housed sows, respectively. Ovulation rate varied from 17 to 25 and was similar in both groups of sows ( P > 0.10). In Fig. 2, hormone profiles of oestradiol, LH and progesterone around third oestrus at approximately Day 65 after weaning are presented. Peak levels of oestradiol-17p were reached between 4 h and 28 h (on average 13 f 2 h; P > 0.10)) before peak levels of LH. The peak levels of oestradiol-17p varied from 23 to 105 pgml-’ and were similar for the two treatment groups (tethered, 60 + 8 pg ml-’ vs. loose-housed, 60 _+9 pgml-‘; P > 0.10). Also the total profiles of oestradiol- 17p were similar for the two treatment groups (Fig. 3a). Basal levels of LH varied considerably between sows from 1.1 to 10.8 ngml-‘, and were not significantly affected by treatment (tethered, 4.0 &-1.1
the
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Table 1 Parameters of oestrus and ovulation around Day 60 after weaning of sows that were either tethered or housed loose from weani:.g onwards Tethered n
Cycle length (d)
’
Duration of oestrus (h) Ovulation * Timing of ovulation Hours after onset of oestrus % Of oestrus Duration of ovulation (h) Ovulation rate (n)
Mean f sem
8
Loose-housed Range
n
Mean f sem
Range
21.OkO.2
20-22
11
20.5 i 0.4
17-23
IO
42&4a
24-60
11
63k2
52-76
9 9
28+2a 71*4
16-34 50-83
9 9
41f2b 67&3
28-49 50-81
7
2.9 + 0.5 21.2kO.8
2-5 17-24
8 11
2.1 kO.2 20.8 + 0.8
l-3 17-25
IO
b
a.bP <
0.01 ’ In two tethered sows, the previous oestrus was a silent oestrus. * In pne of the tethered sows ovulation was already complete at first scanning, therefore time and duration of ovulation are unknown and in two tethered sows, ovulation had just started at first scafming, therefore, duration of ovulation is unknown. Two of the loose-housed sows ovulated later than expected, therefore, time and duration of ovulation are unknown and in one loose-housed sow ovulation had just started at first scanning, therefore, duration of ovulation is unknown.
loo$e-housed, 2.9 & 0.8 ngr@- ’; P > 0.10). Peak iH levels were 8.9 f 1,O and 8.5 f 110 ngml-,’ for thk tethered and loose-housed sows, respectively (P > 0.10; Fig. 3b). Sows ovulated betwien 28 and 35 h (on average 31 k 1 h) after peak levels of LH, which was not affected by.;$zatment (P > 0.10). Progesterone pro$les up, to 132 h after p&k LH levels were similar fbr the ttio treatment groups (Fig. 3~). Of the eighteen sows
vs.
Fig. 3: Profiles: (A) oestradiol-17p (loose-housed -, n = 8 and tethered -- -, n = 8); (B) LH (loose-housed --; n = I I andt&bed - - -, n = IO) rind (C) pkogesterone (loose-housed -, n = 11 and tethkred - - 1, n = IO) around oestrus at approximately Day 60 aftei wkaning in loose-housed and tethered SOWS.
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60 !(a)
01 -48
I
-36
-24
I
1
-12
0
~/_
12
24
’ 12
24
i i
36
48
36
46
12 (b) 10 I
Q
oL.L’ -46
-36
-24
Time
-12
relative
0
to peak
LH levels
I
’
(h)
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Table 2 Embryo development at Day 5 after ovulation in tethered and loose-housed sows that were inseminated at 11k 1 (ranae 2 to 22) h before ovulation Tethered ( n = 9)
Embryo age (h)
Loose-housed (n = 9)
Mean 5 sem
Range
Mean f sem
Range
122*2
Ill-133
120+4
1P-129
Embryo quality Normal (%) Degenerating (%) Unfertilized (%)
98+1 0*0 2rtl
92-100 o-o O-8
86-1-33 1*4 13+33
O-100 O-11 O-100
Embryo development Cell cycles (n) sd cell cycles (n) Accessory sperm count(n)
6.0 f 0.2 0.4kO.03 86+ 19
4.7-7.4 0.3-0.7 22- 169
5.7 + 0.2 0.6*0.1 64*20
4.8-6.9 0.2-0.9 I-153
was not influenced by treatment (P > 0.05), but was related to the interval between insemination and ovulation (I-O) which varied from 2 to 22 h: sperm count (n) = 174 - 7.8 X I-O (h) [ R2 = 0.55, P = 0.003, n = 13 (excluding the three sows that were inseminated twice)]. Average weight of the adrenal glands was 8.7 + 0.7 g in the tethered sows and 7.4 k 0.5 g in the loose-housed sows (P > 0.10). The diameter of the adrenal cortex was 4.0 f. 0.2 mm and 3.5 k 0.2 mm, respectively (P > O.lO), and the diameter of the adrenal medulla was 3.8 f 0.3 mm and 4.3 z!-0.2 mm, respectively (P > 0.10). The condition of the stomach wall at slaughter varied from 0 (intact) to 4 (hyper/parakeratoses and lesions). An intact stomach wall was present in three out of ten tethered sows and five out of elenen loose-housed sows. Lesions occurred in five out of ten tethered sows and three out of eleven loose-housed sows (P > 0.10). The remaining sows had hyper/parakeratoses, but no lesions. In the tethered sows, the average level of stereotypies before third oestrus (measured between 12 and 17 days before oestrus) was linearly related to the duration of third oestrus: [duration of third oestrus (h)] = 63 f 0.32 X [level of stereotypies (% of time)] (P = 0.07). In the loose housed sows no such relation existed (P = 0.65). Basal LH levels before the LH surge were positively related to adrenal weight 5 days after subsequent ovulation (r = 0.65, P = 0.005) in both housing conditions. Even without the three sows with extreme high basal LH levels (more than 10 ngml-’ in two tethered and one loose housed sow, the remaining sows had basal LH levels of less than 5 ng ml- ’), the relation remained significant (P = 0.045). 4. Discussion Multiparous sows, 26 in total, that had been tethered during lactation were put in two different housing conditions after weaning; the sows were either tethered by neck chain, or put in a pen of approximately 6 m2. During a period of 60 days, five sows were taken
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from the experiment. In both housing conditions one sow became ill and was culled and, furthermore, two tethered and one loose-housed sow developed cystic ovaries. The cystic ovaries in the three sows developed during treatment with altrenogest, a progesterone analogue. Not much is known about causes or incidence of cystic ovaries in sows. However, a possible cause of cystic ovaries is a disturbed relation between progesterone and oestrogens in the follicular phase, possibly related with the use of gonadotropins (Wrathall, 1980; Ryan and Raeside, 1991a,b). At Day 18 after weaning, a first assessment was made of the level of stereotypies during one hour after the afternoon feeding. The average level of stereotypies was approximately 50% in both housing conditions. Normally, large individual differences are found in the level of stereotypies within a group of animals (Appleby and Lawrence, 1987); that was also the case in the present experiment as is apparent from the large standard error of the mean. The average level of stereotypies after afternoon feeding at Day 18 is relatively high, presumably as a consequence of the fact that the sows had been tethered during lactation. Stereotypic behaviour is considered a coping strategy of sows that are in a chronic stress condition (Cronin, 1982; Schouten and Wiepkema, 1991); during stress, low stereotyping sows have been found to show the strongest signs of uneasiness (Schouten and Wiepkema, 1991). After two months, the level of stereotypic behaviour increased in the tethered sows and decreased in the loose-housed sows, to an average level of stereotypic behaviour on Day 67 after weaning of 80% (tethered sows) vs. 40% (loose-housed sows) (P = 0.03). It seems that in the loose-housed sows, stereotypic behaviour decreased because of the less stressful housing conditions. In the ‘tethered sows, the level of stereotypies increased because of the chronic stress induced by tethering. One would expect that the difference in stereotypic behaviour between the loose-housed and tethered sows would also be reflected in other stress-related parameters. However, the levels of cortisol at Day 36 and at Day 56 did not differ between the treatment groups, nor did the weight of the adrenal glands, the size of the adrenal cortex, and the pathology of the stomach wall at approximately Day 70 after weaning. Furthermore, no relations existed between stereotypic behaviour and any of the stress parameters within the two groups of sows. Apparently, in the present experiment, stereotypic behaviour is a more sensitive measure for the difference in stress conditions, than the other stress-related parameters that were measured. During the first and second oestrus after weaning, more tethered sows had a silent oestrus (two sows once and two sows twice) than loose-housed sows (one sow once). The duration of oestrus of the sows that came in heat was not influenced by the housing conditions. During the third oestrus, the duration of oestrous of the tethered sows was significantly shorter than that of the loose-housed sows, on average 42 f 12 compared to 63 + 8 h. These differences in duration of oestrus were not reflected in the profiles of reproductive hormones (oestradiol-17l3, LH and progesterone). These profiles were not affected by the housing conditions, nor were relations found between reproductive hormones and the duration of oestrus within the two groups of sows. The higher number of SOWS with a silent first and second oestrus in the tethered sows and also the shorter duration of the third oestrus in the tethered sows may be well be related to the stressful housing conditions of tethering. In a number of experiments, a reduction in the number of animals showing oestrus or the duration of oestrus was found after an injection with
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adrenocorticotropic hormone (ACTH) or synthetic corticosteroids (pigs: Liptrap, 1970; Esbenshade and Day, 1980; ovariectomized gilts: Esbenshade et al., 1983; sheep: Ehnert and Moberg, 1991; ovariectomized cattle: Hein and Allrich, 1992). Unexpectedly, in our sows, the effects of the housing conditions on oestrous behaviour were not related with effects of housing on cortisol levels, neither did a relation exist between oestrous behaviour and cortisol within the experimental groups. Apparently, differences in oestrous behaviour between the two groups were not directly mediated through effects on cortisol. Besides cortisol, however, other stress-related hormones may be of importance. It is generally known that stress-related hormones such as endorphins, CRH and ACTH may influence sexual functions at all three levels of the HPG-axis (Rivier and Rivest, 1991). Oestrous behaviour was significantly related with the levels of stereotypic behaviour in the tethered sows; sows with a high level of stereotypic behaviour had a significantly shorter duration of oestrus. During stereotypic behaviour, endogenous opioid peptides (EOP), endorphines are released (Wiepkema and Schouten, 1992). A number of EOPs are known to suppress LH release (Malven, 1986) and may consequently affect reproductive processes. For example, Armstrong et al. (1988) showed that chronic administration of morphine (an EOP-agonist) delayed the onset of oestrus after weaning. A direct relation between LH release (LH suppression) and the duration of oestrus, however, has not been described. In the present experiment, LH levels were not influenced by the housing conditions of the sows and no relation existed between basal or peak LH levels and stereotypic behaviour. The duration of oestrus is an important parameter to predict the time of ovulation; in quite a few experiments, is has been assessed that ovulation takes place at approximately two-thirds of the duration of oestrus, independent of the (quite variable) duration of oestrus (Weitze et al., 1994; Soede et al., 1995). Even under the extreme conditions of the present experiment, sows ovulated at on average 67-71% of the duration of oestrus in both housing conditions. The duration of oestrus, therefore, is of utmost importance for the timing of insemination in sows (Soede and Kemp, 1996). Differences in ovulation time of individual oocytes have been related to differences in embryo development and, consequently, of embryo survival within sows (Pope et al., 1990). Therefore, differences in the duration of ovulation between sows may be related to differences in embryo diversity between sows and, consequently, with differences in embryo survival between sows. Differences in the duration of ovulation between sows of 1 to 4 h were not related to differences in embryo diversity at Day 5 after ovulation (Soede et al., 1992; Soede and Kemp, 1993). It was suggested that stressful conditions may influence the duration of ovulation, since in one experiment with tethered sows, durations of ovulation were found between 2 and 7 h (Soede et al., 1992). Since the sows were not inseminated, embryo diversity was not assessed. In the present experiment, the duration of ovulation was not significantly different between the tethered and loose-housed sows; the duration of ovulation varied between 1 and 3 h in the loose housed sows and between 2 and 5 h in the tethered sows. No relations were found with embryo diversity at Day 5. So apparently the longer durations of ovulation that were found in an earlier experiment with tethered sows (Soede et al., 1992), were not (solely)
due to the tethering.
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In conclusion, sows that had been tethered during lactation, and were housed loose or were tethered again at weaning, within two months differed both in stereotypic behaviour and in oestrous behaviour. These effects were not related to differences in profiles of cortisol or oestradiol-17l3, LH or progesterone. Furthermore, within the tethered sows, a short duration of oestrus was related with high levels of stereotypic behaviour. No such relation existed in the loose-housed sows. All the sows in this experiment had been tethered before. It might be that especially in these high parity sows, set points and/or regulation of the reproductive hormones are irreversibly changed and, therefore, not influenced by housing conditions any more.
Acknowledgements
The authors gratefully acknowledge the great help of Lucy d’Hoore, Joanne Maaskant, Frits Rietveld and Wouter Hazeleger during the experiment.
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