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Effect of passive immunization against testosterone on reproductive hormone secretion and ovarian function in dairy cows and pubertal beef heifers
Theriogenology
39:507-526,1993
EFFECT OF PASSIVE IMMUNIZATION AGAINST TESTOSTERONE ON REPRODUCTIVE HORMONE SECRETION AND OVARIAN FUNCTION IN DAIRY COWS AND PUBERTAL BEEF HEIFERS V, Lermitel, L. Delaby2, J. Thimonierx, R. Dufour4 and M. Terquil 1 INRA Station de Physiologie de La Reproduction Centre de Recherches de Tours - 37380 Nouzilly, France 2 INRA Station de Recherches sur la vache laitiere 35590 Saint Gilles, France 3 Ecole Nationale Superieure Agronomique Chaire de Zootechnie Place Viala 36060 Montpellier CEDEX, France 4 Laboratoires IFFA, Rhone-Merieux, BP 7009 69342 Lyon CEDEX, France Received for publication: August 1, 1991 Accepted: November 14, 1992 ABSTRACT Multiparous dairy cows were divided in 3 groups from Day 5 up to Day 56 post partum: high energy level (Group H, n=lO), low energy level (Group L, n=lO) and low energy level plus anti-testosterone bovine immunoglobulins (Group LI, n= 10). Undernutrition decreased body weight, body condition score, milk yield and energy balance in Groups L and LI compared to Group H (P
Acknowledgements V. Lermite was a recipient of a RhBne-Merieux Doctoral Fellowship. The authors thank D. ANDRE and his staff for help in LH and Progesterone radioimmunoassay, M. PETIT for providing Salers heifers and J.L. TOUZE and his staff for ultrasound examinations and management of the experimental animals.
Copyright 0 1993 Butterworth-Heinemann
Theriogenology
508 INTRODUCTION
Failure to reproduce is a major factor limiting production efficiency of cattle (1). The deleterious effect of undernutrition and of negative energy balance on postpartum rebreeding performance of beef cattle (2-6) and of high producing dairy cattle (7) has been well recognized. Ovarian inactivity has been induced by limiting energy intake in post-pubertal heifers (8-10). Endocrine studies (11) suggest two main conditions resulting in ovarian inactivity in ewe and cow: normal FSH secretion and low LH secretion result in weak ovarian inactivity, while low FSH and low LH secretion result in profound ovarian inactivity. In dairy and beef cows during postpartum, weak ovarian inactivity has been described by various groups (12-15). Deep ovarian inactivity is observed in underfed suckling beef cows (5, 16, 17). In the ewe during seasonal inactivity the gonadotrophin requirement to induce ovulation have been determined by Oussaid (18): in profound ovarian inactivity, ovulation induction requires both pulsatile FSH and LH injections; when ewes are in weak inactivity only pulsatile injections of LH are needed. In the cow the sequence seems to be the same (Terqui, personal communication). Steroid negative feedback variations are possible modulators of these events, since undernutrition in cattle induces changes in sensitivity of the hypothalamo-pituitary axis to the negative feedback action of estradiol-17P (9, 10). A sex steroid-binding-protein (SBP) exists in plasma of bull and cow and binds mainly the 5a-dihydrotestosterone and testosterone and weakly estradiol-17b (19-21). Underfeeding decreased the steroid binding capacity by SBP in heifers (21); thus antibody to steroid could be a way to restore steroid binding levels and reduce the amount of steroid available for feedback effects. Our experiment was designed to test: (I) the influence of passive immunization against testosterone on LH secretory pattern and ovarian activity in postpartum underfed dairy cows;. (II) the influence of passive immunization against testosterone on LH secretory pattern, and on ovarian activity when exogenous LH stimulation is combined with immunization, in heifers that were maintained in anestrus by undernutrition and presumed to have profound ovarian inactivity. MATERIALS AND METHODS Passive immunization was performed by using anti-testosterone bovine immunoglobulins. The cows were injected subcutaneously with 100 + 9 ml and heifers with 56 f 4 ml of bovine antiserum. Further details concerning the preparation and characterization of the antiserum have been described elsewhere (22). Plasma LH concentrations were measured in duplicate by a double antibody-radioimmunoassay developed in sheep by Pelletier et al (23) and modified by Montgomery et al (24). All samples were included in the same assay to avoid problems of inter-assay variation. The limit of detection was 0.1 ng/ml and the intra-assay coefficients of variation were 9.4 and 11.5 % for 0.4 and 4 ng/ml, respectively.
Theriogenology Serial blood samples were analyzed to determine mean and basal concentrations (ng/ml) of LH, frequency (pulses / 6h) and amplitude (ng/ml) of pulses of LH, through the use of Pulsar algorithms developed by Merriam and Watchter (25). Plasma progesterone was measured in duplicate by a antibody-radioimmunoassay developed by Palmer and Jousset (26) extraction from plasma with hexan. All samples were included same assay. The limit of detection was 6.25 pg per tube and the assay coefficients of variation were 6 and 11.5 % for 1.5 and 5 respectively.
double after in the intrang/ml.
Blood samples were collected from the jugular vein into heparinized tubes and immediately centrifuged. Aliquots of plasma were stored at -20°C. Heifers and cows were weighed before feeding on two consecutive days at two weeks intervals throughout the experiment; the average weight on two consecutive days was used to determine weight changes. To describe level and variation of body reserve (fatness) the 5-points BCS system (body condition score system) was used (27). It is a combination of visual appraisal plus palpation (l=very thin, 5= very fat). Ovaries were observed in all females using ultrasound echotomography. The instrument used was an ultrasound scanner (Toshiba SAL 32B) equipped with a linear-array, 5 MHz transducer designed for intrarectal placement. Follicles > 3mm in diameter were detectable and a large follicle was defined as a follicle ;?10mm in diameter for cows and t qmm for heifers, while a cystic follicle was defined as one that exceeded 20 mm in diameter. Estrous behavior was observed twice daily. Experiment 1 This experiment involved 30 multiparous postpartum dairy cows (Friesian x Holstein), all calving in autumn. All the cows were weighed at Day 5 post partum and averaged 614 f 49 kg body weight (mean t SD). The mean milk yield of these cows was 6265 kg in 309 days during the preceding lactation. During the first week post partum, cows were fed a high plane of nutrition. From Week 2 until Week 8 post partum, cows were divided in 3 groups of 10 cows on the basis of their previous milk production, live weight, age and calving date. Ten cows remained on a high energy diet (Group H), while the 20 remaining cows were on a low energy diet (Group L). cows in Groups H and L were fed a fixed amount (13 kg/cow/day) of forage dry-matter consisting of maize silage and lucerne hay. The amount of concentrate (soya bean meal and cereal) was apportioned individually by milk production and experimental level of
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Theriogenology
nutrition (H or L). Nutritional values in net energy of the total ration per cow and per day are described in table 1. Nitrogen supply was adequate at all production levels, only an energy deficit was applied. After Week 8 post partum, all cows were fed a high plane of nutrition. All the cows received minerals and vitamins as a supplement according to recommendations of INRA (Institut National de Recherche Agronomique. 28). throughout the study. Table 1. Energy values of the high and low nutrition plane
Milk production
High nutrition
Low nutrition
Group H (n=lO)
Group L (n=20)
25-29
kg
30-37 kg
25-29
kg
30-37&z
Weeks post partum +l +2 +3 +4 +5 +6 +7
+a
24a
25
24
29 29
;z
24
28
Z?
24 26
27 27 27
;76 36
24 24
28
25
26 26 27 27 27 27 26
a Net energy (Meal) per cow per day. Between Days 11 and 15 post partum, cows of the Group H and 10 cows of the Group L received sub-cutaneously a single injection of normal bovine immunoglobulins. The remaining cows of the Group L received anti-testosterone bovine immunoglobulins (Group LI, n=lO). From Day 0 post partum, daily blood samples were collected to monitor the anti-testosterone immunoglobulin titre, plasma progesterone to determine resumption of ovarian activity designated in our study to be when plasma progesterone rose above lng/ml). Serial blood samples were collected at 20-minute intervals for 12 hours during the day before and the day after administration of immunoglobulins, to determine secretory patterns of LH. Ultrasound examination of ovarian follicles was performed once weekly until Day 56 post partum, starting 48 hours before administration of immunogloglobulin. Cows were milked twice daily and milk yield was recorded once weekly. Cows were inseminated artificialy at the first estrus period following Week 8 post partum. Three inseminations were permitted to ensure pregnancy.
51-l
Theriogenology Experiment 2
The second experiment involved 15 cyclic heifers from a rustic breed (Salers) that were 16 months old and averaged 378 i: 31 kg body weight (mean f SD) when underfeeding was initiated. Each heifer was subjected to a high energy diet (10 Meal net energy per animal per day) before the beginning of the experiment. Then heifers received a low energy diet (3 Meal net energy per animal per day) to induce anestrus. This low energy diet was maintained for the remainder of the experiment. The composition of diets is described in Table 2. Nitrogen supply was adequate and all heifers received minerals and vitamins as a supplement according to INRA recommendations (28). Anestrus was defined as an absence of ovulation. Onset of anestrus was determined by finding progesterone concentrations < lng/ml for 2 weeks. Table 2. Composition and Experiment 2
energy
values
Group H
Net energyb
diets
fed
to heifers in
Amount (DM)a
Ingredients
Straw Maize silage Pasture hay Soya bean meal
of
1.8 3.5 1.7 0.4 10
Group L
3.5 0.9 0.2 3
a DM : kg dry-matter per heifer per day. b Net energy (Meal) per heifer per day.
Blood samples were collected at 3-day intervals throughout the study to monitor plasma progesterone. Heifers in Group C (n = 7) were given normal bovine immunoglobulin 83 f 24 days (mean + SD) after ovarian activity had stopped as determined by plasma progesterone. Treated heifers (Group I, n = 8) received a single dose antitestosterone bovine immunoglobulin. Daily blood samples were collected to follow anti-testosterone immunoglobulins titre after injection. Twelve hours before and 96 hours after immunoglobulin treatment, blood samples were collected from each heifer over a 12 hours period every 20 mn for LH assay. Seven days after immunoglobulin administration, 30 of porcine luteinizing hormone (pLH) dissolved in 0.9% (w/v) w saline solution, were given intravenously to each heifer every 2 hours for 48 hours. PLH (lmg pLH CY 1300 = 0.2 mg pure pLH) was obtained from Dr. Y. Combarnous Nouzilly, France). Ultrasound (INRA,
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examination of ovarian follicles was performed 8 days before and 3 days after immunoglobulin, and for five days after first pLH injection. Data for body weight, antiserum titre, LH and progesterone concentrations, milk yield, energy balance and follicular score were subjected to an analysis of variance with split plots for repeated measurements (29). Number of LH pulses, body condition score, number of follicles and resumption of ovarian activity were assessed by a non parametric test (30). RESULTS Postpartum dairy cows Between Days 5 and 56 post partum changes of mean body weight were different (P
Body weight loss (kg) Body condition score
Day 5 Day 56
Total net energy deficit (MCal)b
Group H
Group L
29 t 24
53 f 30a
60 f 3oa
4.2 * 0.4 1.8 f o.6a
4.5 + 0.6 1.9 f o.6a
4.3 f 0.4 2.8 + 0.4 -77
-266a
Group LI
-283a
a Different from Group H (P
Milk yield (figure 1) was identical for all cows during the first 2 weeks post partum (31 + 3 kg per day, mean + SD). Cows in Group H showed an increase in milk yield up to Week 5 post partum, after that it remained stable during the remaining 3 weeks. In cows of Groups L and LI milk yield declined after Week 3 post partum (Figure 1).
513
Theriogenology +-
--.-0.-- Group L
Group H
-
Group LI
Milk yield (kg/day)
34 c 32. 30282624’ 0
1
2
3
4
Weeks
Figure 1.
5
8
I a
7
postpartum
Milk yield changes in the 3 Groups. Values are means.
Changes in mean energy balance (Figure 2) differed between the cows in Group H and those in Group L and LI (P
----D-- Group L
-+- Group H 2Net
energy
balance
-
Group LI
(M&/day)
0 -2 -4 -6 -8 0
2
4
Weeks
Figure 2. Net energy means.
balance
6
8
10
postpartum
changes in
the
3
Groups. Values
are
514
Theriogenology
Administration of bovine anti-testosterone immunoglobulin into Group LI resulted in an increase (P
-
Group LI
40nmoI [SHltestosterone
-
Groups H+L bound /
I
i 3020. IO-
0’ -5
0
I I v 5 10 15 20 25 30 days after immunoglobulin
m 35
’ 40
Figure 3. Tritiated testosterone binding in vitro by plasma from Groups H and L and from Group LI shown during a 32 days period anti-testosterone normal after or bovine immunoglobulins injection. Values are means f SD. Before immunoglobulin treatment mean and basal concentrations, median number of pulses and amplitude of pulses of LH were not different between the Group H and the Groups L and LI. Injection of normal immunoglobulins did not modify the secretory pattern of LH in Groups H and L. After injection of anti-testosterone immunoglobulins in Group LI, mean concentration of LH increased and was 57% higher (PCO.05) than in Group L. Baseline was not modified by immunization, but median number of pulses increased in Group LI (P
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Theriogenology
passive immunization against underfeeding and Table 4. Effect of testosterone on the secretory pattern LH in dairy cows, 24 hours before and 24 hours after immunoglobulin injection. Values are means f SD (ng/ml). Pulse number values are reported as medians.
Time from treatment
Group H
Group L
Group LI
-24 hours Mean concentration Baseline Pulse number Amplitude
0.7 f 0.4 0.6 f 0.2 7 0.7 + 0.4
0.8 0.7
f. 0.5 t 0.4 8
0.6 i:0.3
0.9 0.7
f 0.3 f 0.3 8
0.7 * 0.2
+24 hours Mean concentration Baseline Pulse number Amplitude
0.8 + o.4ab 0.7 t o.3a 0.7
f 0.4 8a 0.7 + o.4ab
0.6
t 0.3 7a 0.5 t o.2a
1.1 f 0.3 0.7 f 0.2 lob 1.0 f 0.4
a,b Values with different letters in rows and columns are different
(P
516
Theriogenology
-
Group
80~llicular
L
---=+ Group H
-C
Group
LI
score (mm)
19
40
days after immunoglobulin
Figure 4. Effect of underfeeding and passive immunization against testosterone on follicular score in dairy cows. Values are means f SD. In our study underfeeding had no effect on the occurence of large follicles (2 10 mm) on the ovaries of dairy cows two days before immunoglobulin treatment (Table 5). Six days after the administration of normal immunoglobulins, the number of females in Groups H and L with large follicle did not differ significantly, whereas passive immunization against testosterone increased (P
Effect of underfeeding and passive immunization against testosterone on presence of large follicles (2 10 mm) on the ovary of postpartum dairy COWS a, 2 days before and 6 days after immunoglobulin administration
Time from treatment
Group H
Group L
Group LI
-2 days
4/1ob
z/lob
l/lob
+6 days
5/1obc
z/lob
8/1OC
a number of females with large follicles/number of females treated. b*c values with different letters are different (P
Theriogenology
517
Thereafter, the number of follicles did not differ among the 3 Groups. Up to 6 days after immunoglobulin treatment cystic structures were not detected on the ovaries of any cow. Later, between Day 12 and 18 after treatment ovarian cysts were detected in similar frequency among all the Groups, totalling about 1 in 10 cows per group. The observed cysts did not affect ovarian activity as measured by progesterone. Estrous behaviour was detected earlier in Group H than in Groups L and LI (26 f 11, 39 + 18 and 36 f 19 days post partum in Groups H, L LI respectively) although the difference was significant only between Group H and Group L. Concentrations of progesterone indicative of formation of a functional corpus luteum (> 1 ng/ml) were detected sooner than estrous behaviour and at about the same period in the three groups (20 f 15, 24 + 13 and 22 + 10 days post partum for Groups H, L and LI respectively). The duration of the first luteal phase was shorter (P
518
Theriogenology -+--
40-
+-
Group C
nrrwl [3Hltestosterone
Group
bound /
I
I
30. 20. 10-
0’ -10
Figure 5.
I a a z m m 10 15 20 25 30 -5 0 5 days post-immunological treatment
0
35
Tritiated
testosterone bound in vitro by plasma from Group C and androgen immunized heifers of Group I during a 52 days period. Values are means f SD.
Before immunization, the mean and basal concentrations of LH were not different between the Group C and the Group I (0.4 f 0.1 nglml), but the median number of LH pulses was higher for the Group C than for Group I (P
Time from treatment
-12 hours Mean concentration Baseline Pulse number Amplitude
+9
hours Mean concentration Baseline Pulse number Amplitude
Group
0.4 0.4 2 0.4
C
i: 0.1 f 0.1 f 0.3
0.4 + 0.1 0.4 f 0.2 1 1.2 It1.7
a Different from Group C (PXO.05).
Group I
0.4 0.4
oa
* 0.1 t 0.1
1.3 + 1.1
0.4 f 0.1 0.4 f 0.1 2 0.6 + 0.8
Theriogenology
519
Amplitude of pulses were variable in the two groups (between 75 and 85% of coefficient of variation). Secretory patterns of LH were not significantly modified by injection of nurmal or anti-testosterone bovine immunoglobulins, despite an increase in the number of LH pulses in the Group I which was close to being significant (P = 0.07). Table 7. Median number of follicles t 9 mm diameter in Groups C and I first LH after treatment which was after 7 days immunoglobulin administration
0
24
48
72
96
Group C
0
0
0
0
0
Group I
0
0
o-5
ia
ia
Hours after first LH
a different from Group C
(P
The number of large follicles (2 9 mm) present on the ovary was determined (Table 7). Throughout experimental period the median number of large follicles was 0 in Group C, whereas it was 1 in Group I (P
.--e...-
c
Group
,2Domlnantfollicle
0
Hoursafter
Group
-
I
diameter (mm)
24 the first
48
72
100
LH injection
Figure 6. Growth of the dominant follicle (mm, mean f SD) after stimulation with LH injections every 2 hours for 48 hours in Group C and androgen immunized heifers in Group I.
520
Theriogenology
The mean diameter reached was 7.5 f 1 mm 5 days after beginning of treatment. In the Group I each heifer developed a dominant follicle but this occured earlier than in the Group C since the follicle appeared 24 h after the first injection of LH. The mean diameter reached was 9 f: 2 mm 4 days after beginning of treatment. None of the heifers in the two groups did ovulate following this treatment. However in two heifers of the Group I, the dominant follicle disappeared 5 days after treatment without formation of a corpus luteum (plasma progesterone <1 ng/ml). DISCUSSION The restricted level of nutrition induced a marked difference in body weight, milk yield and energy balance during the first 56 days of the postpartum period in the Groups L and LI when compared to the Group H. Undernutrition had no effect on secretory pattern of LH one week after initiation of regimen, nevertheless passive immunization against testosterone increased significantly the number of pulses and mean plasma levels of LH in underfed cows. This was consistent with results of studies in active immunization against either estradiol176, progesterone or androgen in cows (31-34). In this experiment frequency of secretion of LH was similar to that seen in milked dairy cows before spontaneous recovery of ovarian function (16, 35). In milked dairy cows "normal" pituitary gonadotrophin secretion, which is a prerequisite to resume cyclic function post partum. can be achieved as early as days lo-15 post partum (35). In our experiment one week post partum was too late for undernutrition to have an effect on LH secretion and on duration of the ovarian inactivity, despite the possibility of increased number of LH pulses in underfed cows caused by immunization. At this stage secretion of LH did not seem to be a factor limiting restoration of cyclicity post partum in dairy cows. This explains why reactivation of ovarian function was similar across the two nutritional groups. In the present experiment post partum interval to first ovulation was in agreement with a number of studies in dairy cows (7, 36, 37). It came as a surprise to note that the first cycle was shorter in Group H than in Group L, although reduced luteal function occurs frequently during early post partum period in dairy cows and can be attributed to a functionally deficient corpus luteum (37-40). However, we observed a period of supresssed estrous behavior although underfed cows initiated cycles of nearly normal duration. Gringer and Wilhelms (41) reported delayed estrous behavior in dairy cows underfed during the first five weeks of lactation; Holness and Hopley (4) and Gauthier et al (5) observed the same phenomenon in underfed beef cows. These findings emphasize the subjective character of heat detection which could result in a diagnosis of ovarian inactivity. Passive immunization against testosterone did not hasten the onset of normal ovarian activity in the present experiment. Walton (36) showed that dairy cows actively immunized against androstenedione before calving, failed to indicate a pronounced effect of immunization upon resumption of ovarian function during the postpartum period. But it seems that immunization against testosterone decreased the interval
Theriogenology
521
from calving to first estrus, because this interval was not significantly different between Group H and Group LI in our experiment. Moreover, immunization against testosterone increased progesterone secretion during the first luteal phase, which was consistent with results of other studies (34, 42, 43). The increase of LH secretion in underfed testosterone immunized cows increased the corpus luteum secretion suggesting better ovarian function before or after ovulation. An increase in steroidogenic activity of luteal tissue in immunized cows may confer some advantages for progesterone mediated response in early pregnancy. During the early postpartum period the (9 to 13 days), follicular population in our study consisted predominantly of 3-4 mm follicles which was in agreement with data from Dufour and Roy (44). Large follicles (2 10 mm) were also observed before immunoglobulin treatment, from ovaries of 1 to 4 cows of each group. Similarly Savio et al (37) showed that the mean post partum interval to detection of the first dominant follicle in dairy cows was 11.6 i 8.9 days and Spicer et al (45) reported that large follicles (t 8 mm) were present by day 7 post partum in acyclic, suckled beef cows. Thus follicular growth is active early post partum. Passive immunization against testosterone increased follicular score and the number of underfed dairy cows with large follicles. In mature heifers, follicular development was also increased after passive (48) and active (43) immunization against testosterone and active immunization against estradiol-lj'b (46)) as well as in prepubertal heifers actively immunized against androstenedione (34) * But no abnormal ovarian response, such as cystic follicles or anovulatory period, was recorded; this is different of that reported after active immunization Furthermore restoration of ovarian activity (32. 33, 36, 43. 46-48). and pregnancy rate were comparable in the three groups, although there was a trend for calf number to be higher in immunized than in not immunized underfed cows, which was in agreement with results from Wise and Schanbacher (42). All beef heifers became anestrous as a result of restricted dietary energy consumption, a finding which agrees with previous observations (8-10). Mean levels and frequency of LH were low and in agreement with secretory pattern of LH found in underfed anestrous in heifers with nutritionally induced anestrus postpartum cows (5), and in underfed cyclic beef heifers (9, 50). Imakawa et al. (10) showed that in underfed heifers suppression of LH occurred long before cessation of the estrous cycle. The low nunber of LH pulses in underfed cattle appears to be due to a lack of GnRH secretion by the hypothalamus, rather than the inability of the pituitary to respond to GnRH (49,50). Imakawa et al (9) demonstrated that heifers that were anestrous due to undernutrition had an increased negative feedback of estradiol-17b which resulted in a decreased secretion of LH. Pituitary gonadotrophin secretion is a prerequisite to resume ovarian function. In control group the lack of ovulatory follicle development after pulsatile injections of porcine LH. demonstrated that these females were in profound ovarian inactivity (18). In the present
Theriogenology
522
study. LH pulses number showed a slight but not significant increase In the immunized after passive immunization against testosterone. group, follicular growth was stimulated during LH treatment although no ovulation occurred. However, in two heifers of the Group I, the dominant follicle disappeared without forming a corpus luteum. Immunization against testosterone was not sufficient enough to move heifers from profound to weak ovarian inactivity allowing LH stimulation to induce ovulation. The dose of anti-testosterone was either too small to counteract steroid negative feedback and to promote gonadotrophin secretion and follicular development, or too high thus inhibiting the positive feedback of E2 on the hypothalamopituitary axis. Indeed in a previous experiment (22) the injection of anti-testosterone bovine serum increased LH pulsatility in underfed ovariectomized heifers treated with E2, by cross-reacting with and neutralizing circulating E2, despite antibodies that had a very low affinity for E2. Thus if E2 is bound by antibodies it cannot trigger LH discharge and ovulation. Undernutrition in cattle induces changes in sensitivity of the hypothalamus-pituitary axis to the negative feedback action of gonadal steroids. In a previous study (21) it has been shown that the 5adihydrotestosterone binding levels to sex steroid binding protein were lower in underfed ovariectomized heifers than in normal fed ones, and lower in anestrous heifers than in cvclic ones. Passive immunization against testosterone would compensate this lack of sex steroid binding protein in underfed cattle and results in a reduced concentration of biologically active steroid and diminished negative feedback leading to an increase in LH levels and in follicular development. REFERENCES 1.
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19.
Martin, B., Fouchet, C. and Thibier, M. Steroid-protein interactions in bovine plasma. J. Reprod. Fertil. !&: 143-149 (1976).
20. Suzuki, Y., Itagaki, E., Mori, H. and Hosoya, T. Isolation of testosterone-binding globulin from bovine serum by affinity chromatography and its molecular characterization. J. Biochem. 8.l: 1721-1731 (1977). il. Lermite, V. and Terqui, M. Plasma sex-steroid-binding protein (SBP) in mature heifers: effects of reproductive status, nutritional levels, porcine growth hormone and oestradiol-176 treatments. Biol. Reprod., !&: 864-870 (1991). 22. Lermite, V., Thimonier, J. and Terqui, M. Effect of passive immunization against testosterone on steroid negative feedback luteinizing hormone secretion in regulation of (LH) ovariectomized dairy heifers. Reprod. Nutr. Devel. a: 541-550 (1991). 23.
Pelletier, J., Garnier, D.H., De Reviers, M.T., Terqui, M. and Ortavant, R. Seasonal variation in LH and testosterone release in rams of two breeds. J. Reprod. Fertil. 6!&: 341-346 (1982).
Changes in 24. Montgomery, G.W., Martin, G.B. and Pelletier, J. pulsatile LH secretion after ovariectomy in Ile de France ewe in two seasons. J. Reprod. Fertil. W: 173-183 (1985). 25.
Merriam, G.R and Watchter, K.W. Algorithms for the study of 310-318 (1982). episodic hormone secretion. Am. J. Physiol. &:
26.
Palmer, E. Urinary and Jousset, B. plasma progesterone levels in non-pregnant Fertil. 9: 121-122 (1975).
27.
Giraud, J.M. et Petit, M. Determination et Agabriel, J., utilisation de la note d'etat d'engraissement en elevage allaitant. Bull. Tech. C.R.Z.V. Theix, INRA. 66: 43-50 (1986).
oestrogen and ares. J. ,Reprod.
Theriogenology 28.
INRA. Alimentation des bovins, ovins et Jarrige). INRA Publications - Versailles, 1988.
525
caprins (ed. R. pp 96-120.
29. Kobilinsky, A. and Decoux, G. MCDLI un programme d'analyse de variance et de covariance. in MODULAD, une bibliotheque Fortran 77 pour l'analyse des don&es, INRIA , Paris, 1986. for the behavioral Non parametric statistics 30. Siegel, S. sciences. New York, MC Graw-Hill Book Company, 1956. pp 111-127. 31.
Hoskinson, R.M. Campbell, B.K., Scaramuzzi, R.J., Avenell, J., and Evans, G. LH levels in the androgen-immune cow. Proc. Ann. Meet. Endocr. Sot. Aust. 28: abstr 114 (1985).
32.
Chang, C., Roberts, A.J. and Reeves, J.J. Increased luteinizing hormone secretion and ovarian function in heifers actively immunized against estrogen and progesterone. J. Anim. Sci. a: 771-776 (1987).
Morris, B.A. and Reproductive and Webb, R. 33. Price, C.A., endocrine effects of active immunization against a testosterone conjugate in the heifer. J. Reprod. Fertil. 81: 149-160 (1987). 34.
D'Occhio. M.J., Gifford, D.R., Hoskinson, R.M., Weatherly,T. and Setchell, B.P. Gonadotrophin secretion and ovarian responses in prepubertal heifers actively immunized against androstenedione and oestradiol-176. J. Reprod. Fertil. B:l59-168 (1988).
and Walter, D.L. mechanisms Endocrine 35. Schallenberger, E. contributing to postpartum anoestrus in dairy and beef cattle. In: Ellendorff F. and F. Elsaesser F., (eds). Endocrine Causes of Seasonal and LactationalAnestrus in Farm Animals. Martinus Nijhoff, The Hague,1985, pp 206-220. J.S. Effect of immunization active 36. Walton, against androstenedione on ovarian function in dairy cows and prepubertal gilts. Anim. Prod. Sci. 8: 349-364 (1985). 37. Savio, J.D., Boland, M.P., Hynes, N. and Roche, J.F. Resumption of follicular activity in the early post-partum period of dairy cows. J. Reprod. Fertil. 88: 569-579 (1990). 38. Schams, D, Schallenberger, E., Menzer, C, Stangl, J., Zottmeier, K .. Hoffman, B. and Karg, H. Profiles of LH, FSH and progesterone in post-partum dairy cows and their relationship to the commencement of cyclic functions. Theriogenology 1(1: 453-468 (1978). 39. Eger, S., Shemesh, M., Schindler, H., Amir, S. and Foote, R.H. Characterisation of short luteal cycles in the early post-partum period and their relation to reproductive performance of dairy cows. Anim. Reprod Sci. Ih: 215-224 (1988).
Theriogenology
526
40. Short,
R.E.. Bellows, R.A., Staigmiller, R.B., Berardinelli. J.G. and Custer, E.E. Physiological mechanisms controlling anestrus and fertility in postpartum beef cattle. J. Anim. Sci. 68: 799-816 (1989).
41. Gringer,C. and Wilhelms, G. Effect of duration and pattern of lactation on underfeeding in milk production and early reproduction of dairy cows. Aust. J. Exp. Agric. Anim. Husb. 19:
395-401
(1979).
42. Wise, T.H. and Schanbacher, B.D. Reproductive effects of immunizing heifers against androstenedione and oestradiol-176. J. Reprod. Fertil. 69: 605-612 (1983).
43. D'Occhio, M-J.,
Gifford, D.R., Cox, R.I., Weatherly. T. and Setchell, B.P. Ovarian responses of beef heifers to a bovine serum albumin-testosterone based immunogen. Proc. Aust. Sot. Reprod. Biol. fi: abstract 38 (1986).
44. Dufour, J.J and Roy, G.L. Distribution of ovarian follicular populations in the dairy cow within 35 days after parturition. J. Reprod. Fertil. 23: 229-235 (1985). 45. Spicer, L.J., Leung. K., Convey, E.M., Gunther, J.. Short, R.E and Tucker, B.A. Anovulation in postpartum suckled beef cows. 1. Associations among size and numbers of ovarian follicles, uterine involution and hormones in serum and follicular fluid. J. Anim. Sci. 66: 727-734 (1986).
46. Martin,
T.E.,Henricks, D.M., Hill, J.R. and Rawlings, N.C. Active immunization of the cow against oestradiol-17S. J. Reprod. Fertil. s: 173-178 (1978).
47. Wise, T. and Ferrell, C. Effects of immunization of heifers against estradiol on growth, reproductive traits, and carcass characteristics (41886). Proc. Sot. Exp. Biol. Med. fi: 243-248
(1984). 48. Sreenan, J.M., Morris, D., Tait, A. and Diskin, M.G. Steroid immunization in cows: potential for increasing ovulation and twinning rates. Proc. 10th Int. Congr. Anim. Reprod. & A.I., Urbana-Champaign, 1984, u,-VIII-Z-VIII-27. 49.
Beal, W.E, Short, R.E., Staigmiller, R.B., Bellow, R.A., Kaltenbach, C.C. and Dunn, T.G. Influence of dietary energy intake on bovine pituitary and luteal function. J. Anim. Sci. 4.4: 181-188 (1978).
50. Gauthier, D. and Mauleon, P. Influence of dietary intake and weight variation on LH release after a gonadotrophin-releasing hormone (GnRH) injection during the post-partum period of the nursing cow. Reprod. Nutr. Develop. a: 829-835 (1983).
39:507-526,1993
EFFECT OF PASSIVE IMMUNIZATION AGAINST TESTOSTERONE ON REPRODUCTIVE HORMONE SECRETION AND OVARIAN FUNCTION IN DAIRY COWS AND PUBERTAL BEEF HEIFERS V, Lermitel, L. Delaby2, J. Thimonierx, R. Dufour4 and M. Terquil 1 INRA Station de Physiologie de La Reproduction Centre de Recherches de Tours - 37380 Nouzilly, France 2 INRA Station de Recherches sur la vache laitiere 35590 Saint Gilles, France 3 Ecole Nationale Superieure Agronomique Chaire de Zootechnie Place Viala 36060 Montpellier CEDEX, France 4 Laboratoires IFFA, Rhone-Merieux, BP 7009 69342 Lyon CEDEX, France Received for publication: August 1, 1991 Accepted: November 14, 1992 ABSTRACT Multiparous dairy cows were divided in 3 groups from Day 5 up to Day 56 post partum: high energy level (Group H, n=lO), low energy level (Group L, n=lO) and low energy level plus anti-testosterone bovine immunoglobulins (Group LI, n= 10). Undernutrition decreased body weight, body condition score, milk yield and energy balance in Groups L and LI compared to Group H (P
Acknowledgements V. Lermite was a recipient of a RhBne-Merieux Doctoral Fellowship. The authors thank D. ANDRE and his staff for help in LH and Progesterone radioimmunoassay, M. PETIT for providing Salers heifers and J.L. TOUZE and his staff for ultrasound examinations and management of the experimental animals.
Copyright 0 1993 Butterworth-Heinemann
Theriogenology
508 INTRODUCTION
Failure to reproduce is a major factor limiting production efficiency of cattle (1). The deleterious effect of undernutrition and of negative energy balance on postpartum rebreeding performance of beef cattle (2-6) and of high producing dairy cattle (7) has been well recognized. Ovarian inactivity has been induced by limiting energy intake in post-pubertal heifers (8-10). Endocrine studies (11) suggest two main conditions resulting in ovarian inactivity in ewe and cow: normal FSH secretion and low LH secretion result in weak ovarian inactivity, while low FSH and low LH secretion result in profound ovarian inactivity. In dairy and beef cows during postpartum, weak ovarian inactivity has been described by various groups (12-15). Deep ovarian inactivity is observed in underfed suckling beef cows (5, 16, 17). In the ewe during seasonal inactivity the gonadotrophin requirement to induce ovulation have been determined by Oussaid (18): in profound ovarian inactivity, ovulation induction requires both pulsatile FSH and LH injections; when ewes are in weak inactivity only pulsatile injections of LH are needed. In the cow the sequence seems to be the same (Terqui, personal communication). Steroid negative feedback variations are possible modulators of these events, since undernutrition in cattle induces changes in sensitivity of the hypothalamo-pituitary axis to the negative feedback action of estradiol-17P (9, 10). A sex steroid-binding-protein (SBP) exists in plasma of bull and cow and binds mainly the 5a-dihydrotestosterone and testosterone and weakly estradiol-17b (19-21). Underfeeding decreased the steroid binding capacity by SBP in heifers (21); thus antibody to steroid could be a way to restore steroid binding levels and reduce the amount of steroid available for feedback effects. Our experiment was designed to test: (I) the influence of passive immunization against testosterone on LH secretory pattern and ovarian activity in postpartum underfed dairy cows;. (II) the influence of passive immunization against testosterone on LH secretory pattern, and on ovarian activity when exogenous LH stimulation is combined with immunization, in heifers that were maintained in anestrus by undernutrition and presumed to have profound ovarian inactivity. MATERIALS AND METHODS Passive immunization was performed by using anti-testosterone bovine immunoglobulins. The cows were injected subcutaneously with 100 + 9 ml and heifers with 56 f 4 ml of bovine antiserum. Further details concerning the preparation and characterization of the antiserum have been described elsewhere (22). Plasma LH concentrations were measured in duplicate by a double antibody-radioimmunoassay developed in sheep by Pelletier et al (23) and modified by Montgomery et al (24). All samples were included in the same assay to avoid problems of inter-assay variation. The limit of detection was 0.1 ng/ml and the intra-assay coefficients of variation were 9.4 and 11.5 % for 0.4 and 4 ng/ml, respectively.
Theriogenology Serial blood samples were analyzed to determine mean and basal concentrations (ng/ml) of LH, frequency (pulses / 6h) and amplitude (ng/ml) of pulses of LH, through the use of Pulsar algorithms developed by Merriam and Watchter (25). Plasma progesterone was measured in duplicate by a antibody-radioimmunoassay developed by Palmer and Jousset (26) extraction from plasma with hexan. All samples were included same assay. The limit of detection was 6.25 pg per tube and the assay coefficients of variation were 6 and 11.5 % for 1.5 and 5 respectively.
double after in the intrang/ml.
Blood samples were collected from the jugular vein into heparinized tubes and immediately centrifuged. Aliquots of plasma were stored at -20°C. Heifers and cows were weighed before feeding on two consecutive days at two weeks intervals throughout the experiment; the average weight on two consecutive days was used to determine weight changes. To describe level and variation of body reserve (fatness) the 5-points BCS system (body condition score system) was used (27). It is a combination of visual appraisal plus palpation (l=very thin, 5= very fat). Ovaries were observed in all females using ultrasound echotomography. The instrument used was an ultrasound scanner (Toshiba SAL 32B) equipped with a linear-array, 5 MHz transducer designed for intrarectal placement. Follicles > 3mm in diameter were detectable and a large follicle was defined as a follicle ;?10mm in diameter for cows and t qmm for heifers, while a cystic follicle was defined as one that exceeded 20 mm in diameter. Estrous behavior was observed twice daily. Experiment 1 This experiment involved 30 multiparous postpartum dairy cows (Friesian x Holstein), all calving in autumn. All the cows were weighed at Day 5 post partum and averaged 614 f 49 kg body weight (mean t SD). The mean milk yield of these cows was 6265 kg in 309 days during the preceding lactation. During the first week post partum, cows were fed a high plane of nutrition. From Week 2 until Week 8 post partum, cows were divided in 3 groups of 10 cows on the basis of their previous milk production, live weight, age and calving date. Ten cows remained on a high energy diet (Group H), while the 20 remaining cows were on a low energy diet (Group L). cows in Groups H and L were fed a fixed amount (13 kg/cow/day) of forage dry-matter consisting of maize silage and lucerne hay. The amount of concentrate (soya bean meal and cereal) was apportioned individually by milk production and experimental level of
510
Theriogenology
nutrition (H or L). Nutritional values in net energy of the total ration per cow and per day are described in table 1. Nitrogen supply was adequate at all production levels, only an energy deficit was applied. After Week 8 post partum, all cows were fed a high plane of nutrition. All the cows received minerals and vitamins as a supplement according to recommendations of INRA (Institut National de Recherche Agronomique. 28). throughout the study. Table 1. Energy values of the high and low nutrition plane
Milk production
High nutrition
Low nutrition
Group H (n=lO)
Group L (n=20)
25-29
kg
30-37 kg
25-29
kg
30-37&z
Weeks post partum +l +2 +3 +4 +5 +6 +7
+a
24a
25
24
29 29
;z
24
28
Z?
24 26
27 27 27
;76 36
24 24
28
25
26 26 27 27 27 27 26
a Net energy (Meal) per cow per day. Between Days 11 and 15 post partum, cows of the Group H and 10 cows of the Group L received sub-cutaneously a single injection of normal bovine immunoglobulins. The remaining cows of the Group L received anti-testosterone bovine immunoglobulins (Group LI, n=lO). From Day 0 post partum, daily blood samples were collected to monitor the anti-testosterone immunoglobulin titre, plasma progesterone to determine resumption of ovarian activity designated in our study to be when plasma progesterone rose above lng/ml). Serial blood samples were collected at 20-minute intervals for 12 hours during the day before and the day after administration of immunoglobulins, to determine secretory patterns of LH. Ultrasound examination of ovarian follicles was performed once weekly until Day 56 post partum, starting 48 hours before administration of immunogloglobulin. Cows were milked twice daily and milk yield was recorded once weekly. Cows were inseminated artificialy at the first estrus period following Week 8 post partum. Three inseminations were permitted to ensure pregnancy.
51-l
Theriogenology Experiment 2
The second experiment involved 15 cyclic heifers from a rustic breed (Salers) that were 16 months old and averaged 378 i: 31 kg body weight (mean f SD) when underfeeding was initiated. Each heifer was subjected to a high energy diet (10 Meal net energy per animal per day) before the beginning of the experiment. Then heifers received a low energy diet (3 Meal net energy per animal per day) to induce anestrus. This low energy diet was maintained for the remainder of the experiment. The composition of diets is described in Table 2. Nitrogen supply was adequate and all heifers received minerals and vitamins as a supplement according to INRA recommendations (28). Anestrus was defined as an absence of ovulation. Onset of anestrus was determined by finding progesterone concentrations < lng/ml for 2 weeks. Table 2. Composition and Experiment 2
energy
values
Group H
Net energyb
diets
fed
to heifers in
Amount (DM)a
Ingredients
Straw Maize silage Pasture hay Soya bean meal
of
1.8 3.5 1.7 0.4 10
Group L
3.5 0.9 0.2 3
a DM : kg dry-matter per heifer per day. b Net energy (Meal) per heifer per day.
Blood samples were collected at 3-day intervals throughout the study to monitor plasma progesterone. Heifers in Group C (n = 7) were given normal bovine immunoglobulin 83 f 24 days (mean + SD) after ovarian activity had stopped as determined by plasma progesterone. Treated heifers (Group I, n = 8) received a single dose antitestosterone bovine immunoglobulin. Daily blood samples were collected to follow anti-testosterone immunoglobulins titre after injection. Twelve hours before and 96 hours after immunoglobulin treatment, blood samples were collected from each heifer over a 12 hours period every 20 mn for LH assay. Seven days after immunoglobulin administration, 30 of porcine luteinizing hormone (pLH) dissolved in 0.9% (w/v) w saline solution, were given intravenously to each heifer every 2 hours for 48 hours. PLH (lmg pLH CY 1300 = 0.2 mg pure pLH) was obtained from Dr. Y. Combarnous Nouzilly, France). Ultrasound (INRA,
512
Theriogenology
examination of ovarian follicles was performed 8 days before and 3 days after immunoglobulin, and for five days after first pLH injection. Data for body weight, antiserum titre, LH and progesterone concentrations, milk yield, energy balance and follicular score were subjected to an analysis of variance with split plots for repeated measurements (29). Number of LH pulses, body condition score, number of follicles and resumption of ovarian activity were assessed by a non parametric test (30). RESULTS Postpartum dairy cows Between Days 5 and 56 post partum changes of mean body weight were different (P
Body weight loss (kg) Body condition score
Day 5 Day 56
Total net energy deficit (MCal)b
Group H
Group L
29 t 24
53 f 30a
60 f 3oa
4.2 * 0.4 1.8 f o.6a
4.5 + 0.6 1.9 f o.6a
4.3 f 0.4 2.8 + 0.4 -77
-266a
Group LI
-283a
a Different from Group H (P
Milk yield (figure 1) was identical for all cows during the first 2 weeks post partum (31 + 3 kg per day, mean + SD). Cows in Group H showed an increase in milk yield up to Week 5 post partum, after that it remained stable during the remaining 3 weeks. In cows of Groups L and LI milk yield declined after Week 3 post partum (Figure 1).
513
Theriogenology +-
--.-0.-- Group L
Group H
-
Group LI
Milk yield (kg/day)
34 c 32. 30282624’ 0
1
2
3
4
Weeks
Figure 1.
5
8
I a
7
postpartum
Milk yield changes in the 3 Groups. Values are means.
Changes in mean energy balance (Figure 2) differed between the cows in Group H and those in Group L and LI (P
----D-- Group L
-+- Group H 2Net
energy
balance
-
Group LI
(M&/day)
0 -2 -4 -6 -8 0
2
4
Weeks
Figure 2. Net energy means.
balance
6
8
10
postpartum
changes in
the
3
Groups. Values
are
514
Theriogenology
Administration of bovine anti-testosterone immunoglobulin into Group LI resulted in an increase (P
-
Group LI
40nmoI [SHltestosterone
-
Groups H+L bound /
I
i 3020. IO-
0’ -5
0
I I v 5 10 15 20 25 30 days after immunoglobulin
m 35
’ 40
Figure 3. Tritiated testosterone binding in vitro by plasma from Groups H and L and from Group LI shown during a 32 days period anti-testosterone normal after or bovine immunoglobulins injection. Values are means f SD. Before immunoglobulin treatment mean and basal concentrations, median number of pulses and amplitude of pulses of LH were not different between the Group H and the Groups L and LI. Injection of normal immunoglobulins did not modify the secretory pattern of LH in Groups H and L. After injection of anti-testosterone immunoglobulins in Group LI, mean concentration of LH increased and was 57% higher (PCO.05) than in Group L. Baseline was not modified by immunization, but median number of pulses increased in Group LI (P
515
Theriogenology
passive immunization against underfeeding and Table 4. Effect of testosterone on the secretory pattern LH in dairy cows, 24 hours before and 24 hours after immunoglobulin injection. Values are means f SD (ng/ml). Pulse number values are reported as medians.
Time from treatment
Group H
Group L
Group LI
-24 hours Mean concentration Baseline Pulse number Amplitude
0.7 f 0.4 0.6 f 0.2 7 0.7 + 0.4
0.8 0.7
f. 0.5 t 0.4 8
0.6 i:0.3
0.9 0.7
f 0.3 f 0.3 8
0.7 * 0.2
+24 hours Mean concentration Baseline Pulse number Amplitude
0.8 + o.4ab 0.7 t o.3a 0.7
f 0.4 8a 0.7 + o.4ab
0.6
t 0.3 7a 0.5 t o.2a
1.1 f 0.3 0.7 f 0.2 lob 1.0 f 0.4
a,b Values with different letters in rows and columns are different
(P
516
Theriogenology
-
Group
80~llicular
L
---=+ Group H
-C
Group
LI
score (mm)
19
40
days after immunoglobulin
Figure 4. Effect of underfeeding and passive immunization against testosterone on follicular score in dairy cows. Values are means f SD. In our study underfeeding had no effect on the occurence of large follicles (2 10 mm) on the ovaries of dairy cows two days before immunoglobulin treatment (Table 5). Six days after the administration of normal immunoglobulins, the number of females in Groups H and L with large follicle did not differ significantly, whereas passive immunization against testosterone increased (P
Effect of underfeeding and passive immunization against testosterone on presence of large follicles (2 10 mm) on the ovary of postpartum dairy COWS a, 2 days before and 6 days after immunoglobulin administration
Time from treatment
Group H
Group L
Group LI
-2 days
4/1ob
z/lob
l/lob
+6 days
5/1obc
z/lob
8/1OC
a number of females with large follicles/number of females treated. b*c values with different letters are different (P
Theriogenology
517
Thereafter, the number of follicles did not differ among the 3 Groups. Up to 6 days after immunoglobulin treatment cystic structures were not detected on the ovaries of any cow. Later, between Day 12 and 18 after treatment ovarian cysts were detected in similar frequency among all the Groups, totalling about 1 in 10 cows per group. The observed cysts did not affect ovarian activity as measured by progesterone. Estrous behaviour was detected earlier in Group H than in Groups L and LI (26 f 11, 39 + 18 and 36 f 19 days post partum in Groups H, L LI respectively) although the difference was significant only between Group H and Group L. Concentrations of progesterone indicative of formation of a functional corpus luteum (> 1 ng/ml) were detected sooner than estrous behaviour and at about the same period in the three groups (20 f 15, 24 + 13 and 22 + 10 days post partum for Groups H, L and LI respectively). The duration of the first luteal phase was shorter (P
518
Theriogenology -+--
40-
+-
Group C
nrrwl [3Hltestosterone
Group
bound /
I
I
30. 20. 10-
0’ -10
Figure 5.
I a a z m m 10 15 20 25 30 -5 0 5 days post-immunological treatment
0
35
Tritiated
testosterone bound in vitro by plasma from Group C and androgen immunized heifers of Group I during a 52 days period. Values are means f SD.
Before immunization, the mean and basal concentrations of LH were not different between the Group C and the Group I (0.4 f 0.1 nglml), but the median number of LH pulses was higher for the Group C than for Group I (P
Time from treatment
-12 hours Mean concentration Baseline Pulse number Amplitude
+9
hours Mean concentration Baseline Pulse number Amplitude
Group
0.4 0.4 2 0.4
C
i: 0.1 f 0.1 f 0.3
0.4 + 0.1 0.4 f 0.2 1 1.2 It1.7
a Different from Group C (PXO.05).
Group I
0.4 0.4
oa
* 0.1 t 0.1
1.3 + 1.1
0.4 f 0.1 0.4 f 0.1 2 0.6 + 0.8
Theriogenology
519
Amplitude of pulses were variable in the two groups (between 75 and 85% of coefficient of variation). Secretory patterns of LH were not significantly modified by injection of nurmal or anti-testosterone bovine immunoglobulins, despite an increase in the number of LH pulses in the Group I which was close to being significant (P = 0.07). Table 7. Median number of follicles t 9 mm diameter in Groups C and I first LH after treatment which was after 7 days immunoglobulin administration
0
24
48
72
96
Group C
0
0
0
0
0
Group I
0
0
o-5
ia
ia
Hours after first LH
a different from Group C
(P
The number of large follicles (2 9 mm) present on the ovary was determined (Table 7). Throughout experimental period the median number of large follicles was 0 in Group C, whereas it was 1 in Group I (P
.--e...-
c
Group
,2Domlnantfollicle
0
Hoursafter
Group
-
I
diameter (mm)
24 the first
48
72
100
LH injection
Figure 6. Growth of the dominant follicle (mm, mean f SD) after stimulation with LH injections every 2 hours for 48 hours in Group C and androgen immunized heifers in Group I.
520
Theriogenology
The mean diameter reached was 7.5 f 1 mm 5 days after beginning of treatment. In the Group I each heifer developed a dominant follicle but this occured earlier than in the Group C since the follicle appeared 24 h after the first injection of LH. The mean diameter reached was 9 f: 2 mm 4 days after beginning of treatment. None of the heifers in the two groups did ovulate following this treatment. However in two heifers of the Group I, the dominant follicle disappeared 5 days after treatment without formation of a corpus luteum (plasma progesterone <1 ng/ml). DISCUSSION The restricted level of nutrition induced a marked difference in body weight, milk yield and energy balance during the first 56 days of the postpartum period in the Groups L and LI when compared to the Group H. Undernutrition had no effect on secretory pattern of LH one week after initiation of regimen, nevertheless passive immunization against testosterone increased significantly the number of pulses and mean plasma levels of LH in underfed cows. This was consistent with results of studies in active immunization against either estradiol176, progesterone or androgen in cows (31-34). In this experiment frequency of secretion of LH was similar to that seen in milked dairy cows before spontaneous recovery of ovarian function (16, 35). In milked dairy cows "normal" pituitary gonadotrophin secretion, which is a prerequisite to resume cyclic function post partum. can be achieved as early as days lo-15 post partum (35). In our experiment one week post partum was too late for undernutrition to have an effect on LH secretion and on duration of the ovarian inactivity, despite the possibility of increased number of LH pulses in underfed cows caused by immunization. At this stage secretion of LH did not seem to be a factor limiting restoration of cyclicity post partum in dairy cows. This explains why reactivation of ovarian function was similar across the two nutritional groups. In the present experiment post partum interval to first ovulation was in agreement with a number of studies in dairy cows (7, 36, 37). It came as a surprise to note that the first cycle was shorter in Group H than in Group L, although reduced luteal function occurs frequently during early post partum period in dairy cows and can be attributed to a functionally deficient corpus luteum (37-40). However, we observed a period of supresssed estrous behavior although underfed cows initiated cycles of nearly normal duration. Gringer and Wilhelms (41) reported delayed estrous behavior in dairy cows underfed during the first five weeks of lactation; Holness and Hopley (4) and Gauthier et al (5) observed the same phenomenon in underfed beef cows. These findings emphasize the subjective character of heat detection which could result in a diagnosis of ovarian inactivity. Passive immunization against testosterone did not hasten the onset of normal ovarian activity in the present experiment. Walton (36) showed that dairy cows actively immunized against androstenedione before calving, failed to indicate a pronounced effect of immunization upon resumption of ovarian function during the postpartum period. But it seems that immunization against testosterone decreased the interval
Theriogenology
521
from calving to first estrus, because this interval was not significantly different between Group H and Group LI in our experiment. Moreover, immunization against testosterone increased progesterone secretion during the first luteal phase, which was consistent with results of other studies (34, 42, 43). The increase of LH secretion in underfed testosterone immunized cows increased the corpus luteum secretion suggesting better ovarian function before or after ovulation. An increase in steroidogenic activity of luteal tissue in immunized cows may confer some advantages for progesterone mediated response in early pregnancy. During the early postpartum period the (9 to 13 days), follicular population in our study consisted predominantly of 3-4 mm follicles which was in agreement with data from Dufour and Roy (44). Large follicles (2 10 mm) were also observed before immunoglobulin treatment, from ovaries of 1 to 4 cows of each group. Similarly Savio et al (37) showed that the mean post partum interval to detection of the first dominant follicle in dairy cows was 11.6 i 8.9 days and Spicer et al (45) reported that large follicles (t 8 mm) were present by day 7 post partum in acyclic, suckled beef cows. Thus follicular growth is active early post partum. Passive immunization against testosterone increased follicular score and the number of underfed dairy cows with large follicles. In mature heifers, follicular development was also increased after passive (48) and active (43) immunization against testosterone and active immunization against estradiol-lj'b (46)) as well as in prepubertal heifers actively immunized against androstenedione (34) * But no abnormal ovarian response, such as cystic follicles or anovulatory period, was recorded; this is different of that reported after active immunization Furthermore restoration of ovarian activity (32. 33, 36, 43. 46-48). and pregnancy rate were comparable in the three groups, although there was a trend for calf number to be higher in immunized than in not immunized underfed cows, which was in agreement with results from Wise and Schanbacher (42). All beef heifers became anestrous as a result of restricted dietary energy consumption, a finding which agrees with previous observations (8-10). Mean levels and frequency of LH were low and in agreement with secretory pattern of LH found in underfed anestrous in heifers with nutritionally induced anestrus postpartum cows (5), and in underfed cyclic beef heifers (9, 50). Imakawa et al. (10) showed that in underfed heifers suppression of LH occurred long before cessation of the estrous cycle. The low nunber of LH pulses in underfed cattle appears to be due to a lack of GnRH secretion by the hypothalamus, rather than the inability of the pituitary to respond to GnRH (49,50). Imakawa et al (9) demonstrated that heifers that were anestrous due to undernutrition had an increased negative feedback of estradiol-17b which resulted in a decreased secretion of LH. Pituitary gonadotrophin secretion is a prerequisite to resume ovarian function. In control group the lack of ovulatory follicle development after pulsatile injections of porcine LH. demonstrated that these females were in profound ovarian inactivity (18). In the present
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study. LH pulses number showed a slight but not significant increase In the immunized after passive immunization against testosterone. group, follicular growth was stimulated during LH treatment although no ovulation occurred. However, in two heifers of the Group I, the dominant follicle disappeared without forming a corpus luteum. Immunization against testosterone was not sufficient enough to move heifers from profound to weak ovarian inactivity allowing LH stimulation to induce ovulation. The dose of anti-testosterone was either too small to counteract steroid negative feedback and to promote gonadotrophin secretion and follicular development, or too high thus inhibiting the positive feedback of E2 on the hypothalamopituitary axis. Indeed in a previous experiment (22) the injection of anti-testosterone bovine serum increased LH pulsatility in underfed ovariectomized heifers treated with E2, by cross-reacting with and neutralizing circulating E2, despite antibodies that had a very low affinity for E2. Thus if E2 is bound by antibodies it cannot trigger LH discharge and ovulation. Undernutrition in cattle induces changes in sensitivity of the hypothalamus-pituitary axis to the negative feedback action of gonadal steroids. In a previous study (21) it has been shown that the 5adihydrotestosterone binding levels to sex steroid binding protein were lower in underfed ovariectomized heifers than in normal fed ones, and lower in anestrous heifers than in cvclic ones. Passive immunization against testosterone would compensate this lack of sex steroid binding protein in underfed cattle and results in a reduced concentration of biologically active steroid and diminished negative feedback leading to an increase in LH levels and in follicular development. REFERENCES 1.
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