Effects of oxytocin on cloprostenol-induced luteolysis, follicular growth, ovulation and corpus luteum function in heifers

ELSEVIER

EFFECTS OF OXYTOCIN ON CLOPROSTENOL-INDUCED LUTEOLYSIS, FOLLICULAR GROWTH, OVULATION AND CORPUS LUTFUM FUNCTION IN HEIFERS S. K. Tallam,’

J. S. Waltonla

and W. H. Johnson2

Departments of ‘Animal and Poultry Science and 2Population Medicine University of Guelph, Guelph, Ontario, NlG 2W 1, CANADA Received for publication: Accepted:

March September

2, 1 ggg 3,

1999

ABSTRACT Twenty-five normally cyclic Holstein heifers were used to examine the effects of oxytocin on cloprostenol-induced luteolysis, subsequent ovulation, and early luteal and follicular development. The heifers were randomly assigned to 1 of 4 treatments: Group SC-SC (n=6), Group SC-OT (n=6), Group OT-SC (n=6) and Group OT-OT (n=7). The SC-SC and SC-OT groups received continuous saline infusion, while Groups OT-SC and OT-OT received continuous oxytocin infusion (119 mg/d) on Days 14 to 26 after estrus. All animals received 500 pg, im cloprostenol 2 d after initiation of infusion (Day 16) to induce luteolysis. Groups SC-OT and OTOT received oxytocin twice daily (12 h apart) (0.33 USP units/kg body weight, SC) on Days 3 to 6 of the estrous cycle following cloprostenol-induced luteolysis, while Groups SC-SC and OT-SC received an equivalent volume of saline. Daily plasma progesterone (P4 ) concentrations prior to cloprostenol-induced luteolysis and rates of decline in P4 following the induced luteolysis did not differ between oxytocin-infused (OT-OT and OT-SC) and saline-infused (SC-SC and SC-OT) groups (P 9.1). Duration of the estrous cycle was shortened in saline-infused heifers receiving oxytocin daily during the first week of the estrous cycle. In contrast, oxytocin injections did not result in premature inhibition of luteal function and return to estrus in heifers that received oxytocin infusion (OT-OT). Day of ovulation, size of ovulating follicle and time of peak LH after cloprostenol administration for oxytocin and saline-treated control heifers did not differ (P >O.l). During the first 3 d of the estrous cycle following luteal regression, fewer (P 4.01) follicles of all classes were observed in the oxytocin-infused animals. Day of emergence of the first follicular wave in heifers treated with oxytocin was delayed (P < 0.05). The results show that continuous infusion of oxytocin during the mid-luteal stage of the estrous cycle has no effect on cloprostenol-induced luteal regression, timing of preovulatoty LH peak or ovulation. Further, the finding support that an episodic rather than continuous administration of oxytocin during the first week of the estrous cycle results in premature loss of luteal function. The data suggest minor inhibitory effects of oxytocin on follicular growth during the first 3 d of the estrous cycle following cloprostenol-induced luteolysis. 0 2000 by Elsevier Science Inc.

Acknowledgments This study was supported by the Natural Science and Engineering Research Council of Canada and by the Ontario Ministry of Agriculture, Food and Rural Affairs. We thank Sanofi Animal Health, Canada for oxytocin. aCorrespondence and reprint requests: Fax (519)767-0573; Email:[email protected]. Therfopnology 63:963-979,2000 0 2000 by Elsevier Science Inc.

0093-691WOO/$-eee front matter PII SOO93-691X(00)00243-0

964

Theriogenology INTRODUCTION

In addition to its role in luteolysis (8, 25, 30, 37, 39) oxytocin involvement in intraovarian events has been suggested. Oxytocin treatment has been shown to influence the timing of follicular development and gonadotrophin secretion (7, 32, 33, 34) in the rodent. Subcutaneous injections of oxytocin in rabbits shortly before mating blocks ovulation (4). The physiological role of oxytocin during the period around ovulation is further suggested by reports indicating that active immunization against oxytocin or infusion with oxytocin blocks establishment of pregnancy in ewes (41, 42). Estrus occurred earlier in oxytocin-treated ewes following cloprostenol treatment (42). The treatment of ewes with a slow-releasing oxytocin preparation at the onset of estrus has been shown to increase ovulation rates (20, 21). More recent reports of the presence of oxytocin receptors in bovine granulosa cells of developing and preovulatory follicles (28, 29) further suggest an intraovarian role for oxytocin. In a previous study (38) we demonstrated, in agreement with other studies (11, 17, 24), that continuous infusion of oxytocin during the mid-luteal phase of the estrous cycle delays luteal regression. We showed that a continuous infusion of oxytocin during the mid-luteal stage of the estrous cycle had only minor effects on follicular development. As a follow-up to this research, the present study investigated the effects of oxytocin infusion on ovarian function without the confounding influence of luteal-phase plasma progesterone and during a period of more rapid follicular growth. We chose to determine how continuous infusion of oxytocin would influence concomitant cloprostenol-induced luteolysis and subsequent follicular development and ovulation. Previous studies (2, 11) have shown that oxytocin injections during the early stage of the cycle (Days 2 or 3 to 6) cause premature return to estrus. It has been proposed that the observed effects of oxytocin injections on the CL are caused by the action of PGF2, released from the uterine endometrium in response to oxytocin administration (3, 12, 3 1). Some studies (27) tend to support the hypothesis that oxytocin given during this early stage of the cycle has inhibitory effects on luteotrophic processes. Additional objectives of the present study were to determine effects of exogenous oxytocin infusion on follicular and CL development during the first week of the estrous cycle. Assuming that exogenous oxytocin interacts with oxytocin receptors in the uterine endometrium and possibly in the CL, we proposed that a continuous infusion of oxytocin during this time would downregulate these receptors and block premature luteal regression induced by oxytocin injections in the subsequent cycle. We hypothesized that the episodic administration of oxytocin rather than continuous infusion was required for oxytocin-induced inhibition of luteal function during the first week of the estous cycle. MATERIALS Experimental

Animals

AND METHODS

and Treatments

A total of 25 nulliparous Holstein heifers (12 to 14 mo old) were used in 2 replicate trials. Following estrous synchronization using two intramuscular injections of 500 l.tg cloprostenol (Estrumate, Coopers Agropharm Inc., Willowdale, Ontario, Canada) given 11 d apart, the animals

Theriogenology

965

were randomly assigned to 1 of 4 treatment groups (Figure 1): SC-SC (n=6), SC-OT (n=6), OT-SC (n=6) and OT-OT (n=7). SC-SC and SC-OT received continuous saline infusion while OT-SC and OT-OT received continuous oxytocin (Sanofi Animal Health, Canada; oxytocin activity: 361 USP units/mg; vasopressin activity: 0.75 USP units/100 USP units of oxytocin) infusion for 12 d beginning with Day 14 of the cycle. The timing of oxytocin infusion was chosen based on results of a previous study (17) that indicated an oxytocin-induced delay in luteal regression during this period. Oxytocin or saline was administered using osmotic minipumps (Alzet-model 2ML.2, Alza Corporation, Palo Alto CA, USA) implanted subcutaneously on the lower shoulder region under local anaesthesia. Oxytocin minipumps contained 15 mg/mL of oxytocin in saline. Oxytocin was infused at a rate of 1.9 mg/d. All animals were given a single injection of 500 pg of cloprostenol (Estmmate) 2 d after initiation of infusion (Day 16) in order to induce luteolysis. The SC-OT and OT-OT groups received twice daily subcutaneous injections of oxytocin (0.33 USP units/kg body weight every 12 h (6) ) on Day 3,4, 5 and 6 of the subsequent estrous cycle, while Groups SC-SC and OT-SC received an equivalent volume of saline.

Saline iniections

PG

SC-SC I

0

Days

from

14

estrus

16

01234567 Oxytocin injections

PG

SC-OT IO

-__ Days

from

estrus

__--

I

14

16

I

I

i

I 8’

I

0

Days

from

estrus

14

1 oxytocin I

16

infusioJjT[‘i 1 I 1

I I Days

from

estrus

14

I

i

/ 1

I

1 1

1 I

Oxytocin injections

1 oxytocin i

16

I

01234567

PG

OT-OT I-0

II

Saline

PG

OT-SC

I

01234567

inf@sionl I I

1

1 I

1

01234567

Figure 1. Schematic diagram of experimental design for SC-SC, SC-OT, OT-SC and OT-OT treatments (PG = cloprostenol injection).

966

Theriogenology

Blood Sampling Blood samples were collected daily throughout the trial by jugular venipuncture for determination of plasma progesterone concentrations. All animals were equipped with an indwelling jugular cannula on Day 12 prior to initiation of infusion in order to facilitate frequent blood sampling. Window blood sampling was done at 30-min intervals for 6 h on Day 13 (1 d prior to infusion), Day 15 ( during infusion) and Day 17 ( early follicular phase 24 h after cloprostenol injection) to characterize the pattern of LH secretion. Beginning 24 h until 104 h after cloprostenol injection blood was collected at 3-h intervals for determination of plasma LH concentration and time of the preovulatoty LH surge. All minipumps were removed on Day 12 of infusion (Day 7 of the cycle following cloprostenol-induced luteolysis). Hormone Assays All blood samples were collected in heparinized vacutainer tubes (Beckton-Dickinson, Missisauga, Ontario, Canada), cooled on ice and centrifuged, and plasma stored at - 20 ‘C until RIA. Progesterone concentrations in plasma were determined using previously validated procedures (5). Plasma LH concentrations were determined using previously validated procedures (40). The sensitivities of the progesterone and LH assays were 0.125 ng/mL and 0.1 ng/mL, respectively. Inter- and inn-a-assay coefficients of variation were, respectively, 15.3 and 5.8% for progesterone, 19 and 5% for LH. Ultrasonography

of the Ovaries

Follicular development was monitored daily using transrectal ultrasonography (Aloka SSD210DX real-time B-mode linear array ultrasound scanner equipped with a 5.0 MHz transducer; Aloka Co. Ltd.; Tokyo, Japan) to determine size and number of follicles and CL. To determine the development of preovulatory follicles and the time of ovulation following cloprostenol injection, ultrasound examinations were conducted twice daily (12-h intervals). Images of the ovary were recorded with a graphics printer (Mitsubishi Video Copy Processor). These contained the size and relative position of follicles > 3 mm in diameter and the CL. Development of the dominant follicles and day of wave emergence were determined by retrospective examination of the ovarian images (13, 22, 36). The day of ovulation was defined as the day when the dominant follicle was last observed and confirmed by the observation of a CL at the same location as the ovulated follicle. The follicles observed were classified according to their diameters as Class 1 (3 to 5 mm), Class 2 (6 to 9 mm), or Class 3 (>9 mm). Data Analysis Daily plasma progesterone concentrations and number of follicles in different class categories were analyzed by repeated measurement analysis of variance using the GLM procedure of SAS (35). Development of the dominant follicle was analyzed by repeated measurement analysis of variance using GLM, RJZG and MANOVA procedures of SAS (35) as described by Allen et al. (1). Differences in treatment means were examined using pre-planned contrasts. Variables containing single observations in time were examined for treatment effects by two-way analysis of variance. Window plasma LH concentrations were subjected to the PULSAR algorithm

Theriogenology

967

(26) for determination of LH pulse’ frequency and amplitude, and mean and smoothed basal concentrations. These responses were evaluated for treatment effects by analysis of variance using the GLM procedure of SAS (35). Data for the period prior to the initiation of injections were analyzed for effects of oxytocin infusion by pooling data for SC-SC and SC-OT as saline control group (SC), and OT-SC and OT-OT were pooled as oxytocin-infused group (OT). Effects of oxytocin injections on first wave follicular development were examined by comparisons of follicular numbers on Days 3 to 1 I of the estrous cycle. In this analysis numbers in each follicular category (Class 1, 2 or 3) prior to initiation of injection regimens (Day 0 to 2) were used as covariates. Follicular dynamics were evaluated in 3-d periods: Period 1 (Days 3 to 5). E’eriod 2 (Days 6 to 8) and Period 3 (Days 9 to 11). Preplanned contrasts were used to compare treatment means. RESULTS Progesterone Concentrations

and CL, Development

The profiles of plasma progesterone concentrations during the time of induced luteolysis are shown in Figure 2. There were no treatment differences (PbO.05) in the profiles for plasma progesterone concentration before infusion (Days 0 to 14). During the period following initiation of infusion and prior to induced luteolysis (Days 14 to 16) progesterone concentrations did not change with days (P >o.l), and no treatment differences were detected. After induction of luteolysis, plasma progesterone declined (P ~0.05) rapidly to basal levels for both saline and oxytocin infusion treatments. Effects of treatment and treatment-by-day interaction were not significant (P >0.05). Plasma progesterone declined to less than 1 ng/mL within 24 h after induction of luteolysis. All heifers had fully developed corpora lutea (CL) at the initiation of infusion. Size of CL had begun to decrease by Day 16 when luteolysis was induced even though plasma progesterone had not declined. Oxytocin infusion had no effect on the development and regression of the CL (P > 0.1). The plasma progesterone concentrations during the cycle following cloprostenol-induced luteolysis are shown in Figure 3. Profiles of plasma progesterone concentrations from Days 0 to10 were not different (P>O.O5) among SC-SC, OT-OT and OT-SC treatments. There was a significant effect of day (P <0.05) and treatment-by-day interaction (PO.O5) during the first 10 d of the estrous cycle. The day when peripheral progesterone concentration was less than 1 ng/mL was not different among the treatments that did not experience premature luteal regression (Day 19.8k1.4, 20.3kl.4 and 17.3k1.4 for SC-SC, OT-OT and OT-SC, respectively). Oxytocin infusion had no significant effect on CL development. The Size of CL on Day 10 was not different (P%O5) among SC-SC, OT-SC and OT-OT treatments ( MeamkSEM: 23.3k1.8, 21.2k2.0 and 22.6k2.0 mm for SC-SC, OT-SC and OT-OT, respectively) In contrast, the size of the CL of SC-OT (16.3 f2.2 mm) on Day 10 was lower than that of the other treatments (P <0.05).

Theriogenology

INFUSION

:1;

I 0

2

4

6

8

10

12

14

16

18

20

22

24

Days from estrus

Figure 2. Plasma progesterone concentrations (Mean+SEM) of heifers receiving oxytocin (OT, 0) infusion and cloprostenol (PG) injection on Day 16.

saline (SC, +) or

Plasma LH Concentrations Characteristics of LH release prior to initiation of infusion (Day 13), during infusion (Day 15) and following luteolysis (Day 17) are summarized in Table 1. Mean baseline, pulse frequency and pulse amplitude LH values were not different between animals that received oxytocin and saline infusion (P >O.l). There was a significant effect of day (P O. 10). Baseline LH concentrations increased with time for both treatments, but change in LH pulse amplitude with day was not observed (P >O. 1). Differences in LH pulse frequency during infusion and following cloprostenol administration were not significant (P >O. 1). After luteolysis plasma LH concentration increased; with peak concentrations occurring at 63.Ok3.1 and 67.7k2.1 h (Table 2)

969

Theriogenology after cloprostenol treatment for OT and SC treatments, respectively. timing of LH peak and day of ovulation were not significant (P >0.05).

Treatment

differences

18

22

in

T _

9t 8 7 6 5 4 3 2 1 0 0

2

4

6

8

10 Days

12 from

14

16

20

24

26

estrus

Figure 3. Plasma progesterone concentrations (MeamkSEM) of heifers receiving oxytocin infusion with injections ( 4) of either oxytocin (OT-OT, 0) or saline (OT-SC, +) or saline infusion with injections of oxytocin SC-OT, W) or saline (SC-SC, IXi).

Follicular

Development

On the day of initiation of infusion, all heifers were on their second wave of follicular growth. Large (> 9 mm) second wave and medium (6 to 9 mm) first wave follicles were observed. Effects of oxytocin infusion on the size of the dominant follicle at the time of cloprostenol administration were not significant (P 9.05). The size of the first and second wave dominant follicles (Table 2) were not different (P > 0.05) between heifers that received oxytocin infusion (OT) and those that received saline (SC). Eleven of the 13 heifers infused with oxytocin and all SC animals ovulated between 2 and 5 d after induction of luteolysis (Table 2). The remaining 2 heifers infused with oxytocin ovulated 7 and 9 d after cloprostenol administration. Size of dominant first and second wave follicles at the time of cloprostenol administration had an effect (P ~0.05) on day

Theriogenology

970 of ovulation. The 2 animals that ovulated late had second wave largest follicles 10 mm diameter on the day of induction of luteolysis.

that were less than

Table 1. Characteristics of LH prior to (Day 13) and during (Day 15) saline (SC) or oxytocin (OT) infusion and 24 hours following cloprostenol administration (Day 17)a Treatment Saline (SC)

Parameters

LH mean (ng/mL)

Baseline LH (ng/mL)

LH pulse frequency/6

LH pulse amplitude

hours

(q&L)

Oxytocin (OT)

Day 13

0.62+0.07b

Day 15

0.80+0.07b

Day 17

1.12+0.07’

Day 13

0.36+0.06d

Day 15

0.5 1+0.05d

Day 17

0.74+0.05f

Day 13

2.7+0.2f

3 .4+0.2g

Day 15

2.7+o.2fi

2.3+0.2h

Day 17

2.220.2”

2.0+0.2h

Day 13

0.65+0.12’

0.61+0.12’

Day 15

0.68&O. 1 la

0.71+0.12’

Day 17

0.60+0.1 lg

0.83&I. 13g

b-hFor each item values with different superscripts within row or column differ (P ~0.05). ?ahres are means&EM.

Mean number of ovarian follicles of various class categories are summarized in Table 3. The number of Class 2 (6 to 9 mm) and Class 3 (> 9 mm) follicles was not different between treatments during the infusion period prior to ovulation. The OT group had a higher number of Class 1 (3 to 5 mm) follicles than SC on Days 14 to 16. The number of Class 1, 2 and 3 follicles after induction of luteolysis (Days 17 to 19) was not different between SC and OT treatments (P >O.l). The number of follicles of all categories was lower (P ~0.01) in oxytocin-infused heifers on Days 1 to 3 of the ensuing estrous cycle.

Theriogenology

971

Dynamics of first wave follicles The ovarian characteristics during the cycle resulting from cloprostenol-induced luteolysis are shown in Table 4 and Figure 4. The emergence of first wave follicular growth was delayed (PC 0.05) in oxytocin-infused treatments. Day of wave emergence for SC-SC and SC-OT was not different (P 9.05). The number of Class 1 (3 to 5 mm) follicles was not different among treatments (P >0.05); however, treatment-by-period interaction was significant (P <0.05). Whereas Class 2 (6 to 8 mm) follicles decreased from Period 1 (Days 3 to 5) to Period 2 (Days 6 to 8) for OT-OT and OT-SC treatments, the number of Class 2 follicles increased for OT-OT, and was greater (P ~0.01) during Period 3 (Days 9 to 11).

Table 2. Mean QSEM) of preovulatory and ovulatory parameters of saline (SC) and oxytocin (OT) infused heifers following cloprostenol-induced luteolysis Treatmen? Parameter Size of dominant

Saline infusion (SC)b

infusion (OT)’

follicle (mm)d - Wave I - Wave 2

Time of LH peak (h)e Day of ovulationf Size of ovulating

Oxytocin

follicle (mm)

Number of heifers ovulatingg

9.5H.4 10.9fl.4 67.7k2.1

9.1+0.4 11.6fl.5 63.Ok3.1

3.6fl.5

3.3H.5

14.8kO.6

15.OH.6

12

11

aTreatments not different (P >0.05). bSC treatment group is a pooling of SC-SC and SC-OT treatments. ‘OT treatment group is a pooling of OT-SC and OT-OT treatments. dOn day of cloprostenol(PG) administration. eHours from PG. fDay of PG=O. gWithin 2 to 5 d from PG.

Treatment and treatment-by-period effects on number of Class 3 (> 9 mm) follicles were significant (PcO.05). Heifers receiving oxytocin infusion (OT-OT and OT-SC) had a lower number of Class 3 follicles than those receiving saline only (SC-SC; P <0.05). No differences in the number of Class 3 follicles were detected between OT-OT and OT-SC (P >O. 1) or SC-SC and SC-OT treatments (P >0.05). During Period 1 (Days 3 to 5) no Class 3 follicles were detected in heifers receiving oxytocin infusion (OT-SC and OT-OT).

972

Theriogenology

Table 3. Mean (#EM) number of Class 1 (3 to 5 mm), Class 2 (6 to 9 mm) and Class 3 (> 9 mm) follicles of oxytocin (OT) or saline (SC) infused heifers on Days 14 to 16, Days 17 to 19 (first cycle) and Days 1 to 3 (cycle following cloprostenol-induced luteolysis) Day 14 to 16

Day 17 to 19

Treatment

Day 1 to 3 Treatment

Treatment

Follicles

SCa

OTh

SC

OT

SC

OT

Class 1

9.8k1.5

15.2+1.4*

13.5k1.6

10.7k1.5

14.5&l .7

7.9+1.6**

Class 2

9.5k1.5

7.3k1.5

7.1kl.l

5.0+1.1

12.521.5

5.1+1.4**

Class 3

4.7fl.5

4.2fl.4

3.8kO.4

3.4kO.4

1.8fl.4

0.3*0.4**

%C treatment group is a pooling of SC-SC and SC-OT ‘OT treatment group is a pooling of OT-SC and OT-OT *Treatment within follicle category and period different **Treatment within follicle category and period different

treatments. treatments. from SC (P ~0.05). from SC (P ~0.01).

Table 4. Ovarian characteristics of heifers receiving oxytocin infusion with injections of either oxytocin (OT-OT) or saline (OT-SC), or saline infusion with injections of oxytocin (SCOT) or saline (SC-SC) during the cycle following cloprostenol-induced luteolysis Treatment Characteristic

SC-SC

SC-OT

Size of first wave dominant follicle (mm)a

14.5kO.6

l&2+0.6

Size of CL (mm)a

23.3+1.8

Day of wave emergenceC Day plasma Pq
follicle

Treatment OT-SC

OT-OT

Contrast? 1

2

3

15.2k.6

16.2M.6

**

NS

NS

16.3k2.2

21.2k2.0

22.6k2.0

*

NS

NS

l.OkO.7

0.420.7

3.QO.7

2.020.7

NS

NS

*

19.8k1.4

9.Ok1.6

17.3k1.4

20.3k1.4

%**

NS

NS

NS

NS

NS

NS

14.5kO.6

17.QO.7

14.8k.6

16.2+0.6

**

22.Ok1.5

11.8+1.9

21.8+1.7

22.2k1.5

**

Significant difference: * P ~0.05, ** P ~0.01. NS no difference (P >0.05). aOn Day 10 (estrus=Day 0). ‘From Day 0 to 10. ‘From estrus. dContrasts 1: SC-SC vs SCOT; Contrast 2: OT-SC vs OT-OT; OT-OT.

Contrast 3: SC-SC vs OT-SC and

Theriogenology

973

Figure 4. Mean (+SEM) number of Class 1 (3 to 5 mm), Class 2 (6 to 9 mm) and Class 3 (> 9 mm) follicles of heifers receiving oxytocin infusion with injections of either oxytocin (OT-OT, n ) or saline (OT-SC, 0 ) or saline infusion with injections of oxytocin (SC-OT, q ) or saline (SC-SC, H ) during the cycle following cloprostenol-induced luteolysis (*Class 3 follicles not present for OT-OT and OT-SC; ab Treatment values with different superscripts within period and follicle class differ, P <0.05).

Theriogenology

974

Treatment effects on the growth of first wave dominant follicles were significant (P O.l). The size of the dominant follicle on Day 10 was, however, greater for SC-OT than for the rest of the treatments (P <0.005). The first wave dominant follicle became the ovulatory follicle in the SC-OT treatment. In contrast, the first wave dominant follicles of treatments that did not result in premature loss of luteal function regressed with the emergence of second wave dominant follicles. Comparison of SC-SC to OT-SC and OT-OT indicated that oxytocin infusion had no effect (P >0.05) on duration of estrous cycle, day of ovulation and the size of ovulating follicle.

18i 16-i

)NS -..

0

1

2

3

4

5

Days from

J

6

7

8

9

10

estrus

Figure 5. Profiles of first wave dominant follicle diameter of heifers receiving oxytocin infusion with injections of either oxytocin (OT-OT, 0) or saline (OT-SC, +), or saline infusion with injections of oxytocin (SC-OT, n ) or saline (SC-SC, B).

DISCUSSION The period had previous in infusion on

present study showed that a continuous infusion of oxytocin during the mid-luteal no effect on plasma progesterone concentrations. These results are in agreement with vivo studies that failed to show stimulatory or inhibitory effects of continuous oxytocin progesterone during the mid-luteal period (11, 17, 23). Exogenous oxytocin also had

Theriogenology no effect on cloprostenol-induced luteal regression, the LH surge and subsequent continuous infusion of oxytocin during the mid-luteal phase has been shown to inhibit both sheep (9) and cows (11, 17, 24, 38). Results of the present study are consistent studies in the ewe (9, 42) that show that the oxytocin-induced inhibition of luteolysis by exogenous administration of cloprostenol.

975 ovulation. A luteolysis in with similar is overcome

Oxytocin injections during early luteal development caused premature luteal regression in saline-infused animals. These animals also showed reduced growth of the CL and lower plasma progesterone compared to the oxytocin-infused and saline control treatments. These results agree with the earlier studies of Amstrong and Hansel (2) and Hansel and Wagner (15). Oxytocin injections superimposed on oxytocin infusion failed to shorten CL life span. Since continuous infusion of oxytocin has been shown to downregulate endometriaf oxytocin receptors in the ewe (9), it has been proposed that a similar phenomenon occurs in the cow. The results demonstrated in this study suggest involvement of oxytocin receptors. The generation of these receptors in the saline infusion treatment, presumably allow oxytocin injections to elicit the effects of shortening CL life span. In contrast, oxytocin-infused heifers show no response to oxytocin injections probably due to the downregulation of the receptors or lack of follicular development. The results of the present study support the concept that the episodic administration of oxytocin during early luteal development is necessary to shorten CL life span and that continuous infusion inhibits this effect. Conclusive evidence of downregulation of oxytocin receptors, however, needs to be confirmed by measurement of oxytocin receptors in the uterine endometrium during infusion. Oxytocin infusion itself had no effect on subsequent CL life span as shown by the similarity of estrous cycle duration for saline controls (SC-SC) and those treatments that received oxytocin infusion. Since the expression of the oxytocin gene in the CL is first detected during early luteal development (19) and oxytocin injections given during this period have been shown to inhibit CL development and progesterone synthesis, it has been proposed that oxytocin may have an intraovarian role in CL development (14). Miyamoto and Schams (27) have also suggested an intraovarian role of luteal oxytocin in progesterone secretion. On this basis, continuous infusion of a high dose of oxytocin during this period would be expected to compromise the role with the result that plasma progesterone concentrations would have been reduced during periods of infusion. This result was not, however, demonstrated in this study. The results do not indicate any effects of oxytocin on LH at any of the periods examined except for the decrease in pulse frequency immediately following the initiation of oxytocin infusion. These results were consistent with previous findings in the cow (6, 16, 43) and ewe (42) which found no effect of oxytocin on LH and FSH. Irvin et al. (18) demonstrated that oxytocin infusion in ovariectomized cows had no effect on basal concentrations, frequency and amplitude of LH pulses and the area under the LH curve following GnRH injection. Lutz et al. (24) also did not detect effect of oxytocin infusion on LH concentrations but reported elevated FSH in serum of the treated animals. In this case oxytocin infusion appeared to have influenced FSH secretion without affecting LH. Greater numbers of Class 1 (3 to 5 mm) follicles were observed in heifers receiving oxytocin infusion rather than saline during the initial period of infusion (Days 14 to 16). As we demonstrated in an earlier study (38). a similar infusion of oxytocin extended the luteal life span

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and resulted in an additional wave of follicular growth. In this second study, oxytocin-infused animals were beginning a new wave of follicular development during the initial period of infusion. It may be argued that the higher number of Class 1 follicles may be indicative of the emergence of a new wave of follicles rather than an oxytocin-induced enhancement of follicular development. It is significant to note, however, that animals receiving oxytocin infusion also had lower numbers of all classes of follicles on Days 1 to 3 of the ensuing cycle. This observation suggests a suppressive effect of oxytocin on follicular development during this period. Considering the results of our previous study (38) where oxytocin had minor effects on follicular growth, the current study suggests a more pronounced inhibition in the absence of mid-Meal progesterone secretion. It has been shown in heifers that the secondary FSH surge, which occurs 1 d after the preovulatory LH/FSH surge, may have a role in re-initiation of follicular development after ovulation. There is evidence that the suppression of this surge using steroid-free bovine follicular fluid delays the appearance of first wave follicular development and early luteal increase in plasma estradiol (10). Day of emergence of the first follicular wave was significantly delayed in the oxytocin-infused group, and a similar mechanism involving suppression of FSH surge could account for the lower number of follicles in this group during the early stage of the cycle. Conclusive evidence for such a mechanism would require a demonstration that the inhibition of follicular development was directly associated with suppression of the secondary FSH surge. In an earlier study (24), serum FSH concentrations of oxytocin-infused heifers were elevated. However, the study did not relate this oxytocin effect to follicular development. The premature loss of luteal function as a result of daily oxytocin injections of salineinfused (SC-OT) animals was followed by rapid growth of the first wave dominant follicle that subsequently ovulated. In contrast, the rest of the treatments maintained luteal phase concentrations of progesterone. In these treatments, the first wave dominant follicles regressed as the second wave dominant follicle emerged from the second wave of follicular growth. There was no clear explanation for the difference in number of Class 2 (6 to 9 mm) follicles of OT-OT and OT-SC during Period 3 (Days 9 to 11). Oxytocin infusion had inhibitory effects on growth of follicles into Class 3 as demonstrated by the failure to detect this class of follicles during Period 1 (Days 3 to 5). The first wave dominant follicle diameters on Day 10 were not different for saline and oxytocin-infused animals. It appears that even though there was a delay in wave emergence for the latter heifers, by Day 10 differences in size of the dominant follicle were not significant because the first wave dominant follicles in the saline group that had earlier emergence had begun to regress. Superimposing oxytocin injection on oxytocin-infusion had no effect on the growth of the first wave dominant follicle. These results have shown that heifers receiving continuous infusion of oxytocin during the mid-luteal period of the estrous cycle respond to cloprostenol-induced luteolysis in a manner similar to that of control animals. Twice-daily injections of oxytocin during the first week of the estrous cycle following cloprostenol-induced luteolysis resulted in premature return to estrus and ovulation in animals that received saline infusion. In contrast, oxytocin infusion alone or with twice-daily injections did not result in premature luteolysis. The results also support the hypothesis that episodic rather than continuous administration of oxytocin is necessary for oxytocin-induced inhibition of luteal function. This finding suggests that the infusion blocks the effects of oxytocin,

Theriogenology possibly by downregulation of endometrial oxytocin receptors. The results indicated inhibitory effects of oxytocin infusion on follicular growth during the first 3 d of the estrous cycle following cloprostenol-induced luteolysis. Taken together, these findings and those from our previous study (38) indicate that continuous infusion of oxytocin interferes slightly with folliculogenesis but without disturbing the normal processes required for luteolysis, follicular dominance, ovulation and subsequent luteal development. REFERENCES 1. Allen OB, Burton JH, Holt JD. Analysis of repeated measurements from animal experiments using polynomial regression. J Anim Sci 1983;57:765-770. 2. Armstrong DT, Hansel W. Alteration of the bovine estrus cycle with oxytocin. J Dairy Sci 1959;42:533-542. 3. Anderson KE, Bowerman AM, Melampy RM. Oxytocin on ovarian function in cycling and hysterectomized heifers. J Anim Sci 1965;24:964-968. 4. Brinkley HJ, Nalbandov AV. Effect of oxytocin on ovulation in rabbits and rats. Endocrinology 1963;73:515-515. 5. DeBoer G, Etches RJ, Walton JS. A solid-phase radioimmunoassay for progesterone in ovine plasma. Can J Anim Sci 1980;60:783-786. 6. Donaldson LE, Hansel W, van Vleck LD. Luteotrophic properties of luteinizing hormone and nature of oxytocin induced luteal inhibition in cattle. J Dairy Sci 1965;48:331-337. 7. Evans JJ, Hurd SJ, Mason DR. Oxytocin modulates the luteinizing hormone response of the rat anterior pituitary to gonadotrophin-releasing hormone in vitro. J Endocrinol 1995; 145: 113119. 8. Flint APF, Sheldrick EL.Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep. J Reprod Fertil 1983;67:215-225. 9. Flint APF, Sheldrick.EL. Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes. J Reprod Fertil 1985;75:623631. 10. Fortune JE, Sirois J, Turzillo AM, Lavoir M. Follicle selection in domestic ruminants. J Reprod Fertil 1991;43(Suppl.):187-198. 11. Gilbert CL, Lamming GE, Parkinson TJ, Flint APF, Wathes DC. Oxytocin infusion from d 10 after estrus extends the luteal phase in non-pregnant cattle. J Reprod Fertil 1989;86:203-210. 12. Ginther OJ, Woody CO, Mahajan S, Janakiraman K, Casida LE. Effect of oxytocin administration on the oestrus cycle of unilaterally hysterectomized heifers. J Reprod Fertil 1967;14:225-229. 13. Ginther OJ, Kastelic JP, Knopf L. Composition and characteristics of follicular waves during the bovine estrous cycle. Anim Reprod Sci 1989;20: 187-200. 14. Hansel W, Dowd JP. New concepts of the control of corpus luteum function. J Reprod Fertil 1986;78:755-768. 15. Hansel W, Wagner WC. Luteal inhibition in the bovine as a result of oxytocin injections, uterine dilatation, and intrauterine infusions of seminal and preputial fluids. J Dairy Sci 1960;43:796-805. 16. Harms PG, Nieswender GD, Malven PV. Progesterone and luteinizing hormone secretion during luteal inhibition by exogenous oxytocin. Biol Reprod 1969;1:228-233.

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