A study of ovarian activity in the ewe using chronic catheterization of the utero-ovarian vein

THERIOGENOLOGY

A STUDY OF OVARIAN ACTIVITY IN THE EWE USING CHRONIC CATHETERIZATION OF THE UTERO-OVARIAN VEIN.

F. GAYERIE, Y. COGNIE, A. LOCATELLI and J. SAUMANDE

Station de Physiologie de la Reproduction. INRA, 37380 NOUZILLY, FRANCE.

Received for publication: Accepted:

November 30, 1982 March 1, 1983

ABSTRACT Eighty-six adult, cyclic, anestrous and postpartum Tie-de-France ewes were placed under general anaesthesia. A catheter was inserted in the utero-ovarian vein (u.o.v.) of one or both ovaries in each ewe. The animals were kept unrestrained in pens and allowed to experience normal maternal behaviour, including suckling. Oestrus was detected with rams. out of 167 catheters inserted, 70.7% allowed repetitive blood collection, 24.0% were obstructed for short periods and 5.4% remained completly blocked. Oestradiol- 17/Jwas assayed without extraction from the U.O.V. plasma samples. From these results, we conclude that this technique is suitable for investigating the mechanisms involved in the recovery of cyclical ovarian activity after parturition, particularly because the follicular response to endogenous gonadotropins can be assessed. This is not possible when plasma samples are obtained from a jugular vein.

INTRODUCTION The serial and continuous collection of peripheral blood is routinely achieved in most domestic animals using a catheter introduced into the jugular vein (j.v.>. However, because the concentration of the ovarian steroid oestradiol-17p (E2/3) is low in the peripheral plasma (1, 21, only the preovulatory increase can be followed accurately and minor changes such as responses to LH pulses are missed. The actual ovarian secretion of this steroid should be studied by sampling the blood draining from the ovary. Long term access to the ovarian vasculature of unstressed animals is provided by the autotransplantation of the whole genital tract to a more accessible position such as the neck (3) or by the anastomosis of the utero-ovarian vein (u.o.v.) with the midline mammary vein (4). However, the necessity to prepare jugulocarotid skin loops several weeks before transplanting the uterus and ovary (3), the 4 days required to complete the anastomosis of the utero-ovarian vein to the mammary vein after the first part of the procedure (4) and the time necessary to perform the operations have prevented experiments on large numbers of animals, particularly when blood sampling must be initiated a few days after a physiological event such as parturition. Various techniques have already been used for the repetitive collection of samples from the -in situ ovarian effluent from

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THERIOGENOLOGY animals (5, 6, 7). A simple technique for chronic unstressed in situ is described in this paper. Results catheterization of U.O.V. -obtained in cyclic and post-partum anovular ewes are presented.

MATERIALS AND METHODS Ile-de-France ewes were used in various Eighty-six adult experiments. They were all kept inside on the Institute farm under conditions of natural light, temperature and humidity. They were fed rations appropriate for their reproductive states. The states of the various groups are shown in Table I. Throughout the experiments, the ewes were kept free in the pens and the lactating animals suckled their young normally.

Table I: Reproductive states of the ewes used for ovarian activity studies and results of surgical intervention.

Reproductive states

Number of ewes

Number of catheters*

% of catheters _~~-----~~~---~~~~~--~~~~~~~~~~~~~ without with permanently problems interruptions blocked

Post partum

69

138

68.8

26.8

4.4

Cyclic

12

21

81.0

9.5

9.5

5

8

75.0

12.5

12.5

86

167

70.7

24.0

5.4

Seasonally acyclic

Total

* Catheters were inserted during 3 to 4 days in seasonally and postpartum anoestrous ewes and during 3 to 8 days in cyclic ewes.

After the ewes were given a general anaesthetic (Pentothal and Nembutal), the catheters were inserted to allow endotracheal intubation and to maintain inhalation (fluothane, 02) using a closed circuit apparatus. With the animal lying on its back, the abdomen was shaved and sterilized. The uterine horns were drawn up to the edges of a 15cm mid-line incision. A large branch of the uterine vein was isolated for a length of 2cm from the utero-tubal end of the broad ligament. A ligature was tied around the cranial branch of the uterine vein and a clamp was placed distally. A small incision was made in the dilated part of the vein. The clamp was removed from the vein and silastic tubing (lmm i.d.), previously filled with heparinized physiological saline (600 I.U. heparinfml) and clamped at its free tip, was progressively inserted

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THERIOGENOLOGY through the incision and advanced inside the uterine vein towards its junction with the ovarian vein until it reached the caudal vena cava. The catheter was then retracted to place its tip in the utero-ovarian vein half-way between the junction with the vena cava and the junction with the ovarian vein, as indicated in Figure 1.

Figure 1: Insertion of the catheter into the utero-ovarian vein

A ligature was placed around the vein and the catheter next to the incision. A loop was made with the catheter and several additional ligatures were tied to secure it to the broad ligament. To assess the potency of the catheter, the clamp was removed from the tip and the blood allowed to flow. If the test was satisfactory, the catheter was cleared of blood by pushing through heparinized saline from a syringe and its distal tip was stoppered and brought outside the body cavity through a small incision in the flank. It was secured to the skin; no protection was necessary. In an early experiment, the animals received one injection of 12,000 I.U. heparin in physiological saline immediately after the operation and two injections per day throughout the experiment. In a following experiment, the ewes received only one injection immediately after the operation which was found to be satisfactory. Within 45 minutes, the animals were standing and blood collection began 24 h later. For each sample run, the dead space of saline in the catheter was discarded and 3 ml of blood were then collected by simply allowing the blood to flow into the collection tubes. After collection, the blood was centrifuged and the plasma was stored at -15OC until assay. At the end

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of the experiment, the catheters were removed; the position of their tips and the ovarian status of the ewes were recorded before the animals were returned to the flock. The high concentration of E2/? (8) in U.O.V. plasma and the absence of proteins with high affiriizy fcr E2/3(9) made it possible to assay the steroid without extraction. Such an assay has been described recently in detail (10) and compared with an extraction reference assay (11). The steroTd was assayed in 100 ul of plasma and all the reagents were added simultaneously. Each sample was assayed in duplicate and all the sampling and dispensing, including those for the standards, were done using an automatic sample processor. Using this method, the non-specific binding never exceeded 4%. The smallest concentration significantly different from zero was 16 pg/ml. The intra-assay variation was 6.5%. The inter-assay variation was less than 10%.

RESULTS Technical results As shown in Table I, 138 catheters were inserted in post-partum ewes between day 5 and day 78 after parturition. Catheters were also inserted in non-pregnant ewes: 10 in the middle of and 11 at the end of the breeding season. These catheters were maintained in the veins from 3 to 8 days. Of the inserted catheters, 70.7% allowed repetitive collection of blood without problems. In 24.0 % of the cases, some samples were missed and 5.4% of the inserted catheters never allowed blood collection. At removal, these catheters were found to be ruptured or blocked by a blood clot. The main reasons for the short:interruptions were small blood clots and kinked catheters. When the interval between two samples did not exceed 4 hours, it was possible to collect blood from both catheters at the same time in less than 1 min. The injection of heparinized physiological saline in the general circulation was found to be unnecessary. The length of catheter lying in the utero-ovarian vein was measured for 106 catheters. This length ranged from 7 to 13 cm in 89% of the cases. Only 7.5% were between 3 and 7cm and 3.5% were between 13 and 15cm. These figures were not always related to the length of the uterus, the involution of which was at different stages of completion: 30cm 2 5 (m + sd) for the pregnant horns one week after parturition, 16cm 5 3 for the- non-pregnant horns and the pregnant horns four weeks after parturition. Even when the catheters were inserted as early as 5 days after parturition, no signs of lassitude, no infections and no deaths were observed. The presence of catheters did not impair the ovulatory response of the ovaries to PMSG (Table II). Twenty-four hours after the operation, no effect of anaesthesia on hypothalamo-hypophyseal function was evident. In non-cyclic animals, FSH concentrations were in the usual range and the frequency and amplitude of LH pulses appeared normal (COGNIE, GAYERIE, in preparation). Ovulations ranged from one to five and only three ewes out of 24 did not respond in a Spring trial after an i.m, injection of 600 I.U. of PMSG, This response is similar to responses in non-catheterised animals treated in the same conditions.

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THERIOGENOLOGY

Table 11: Ovulations induced with 600 I.U. of PMSG in catheterized postpartum ewes in Spring. Serial samples were collected for 36 hours after PMSG and daily samples for the 17 days following. The increasing frequency of short luteal phases observed at D40 may have been due to the onset of seasonal anoestrus.

Ewe No

Interval PMSG LH surge (hours)

1 2 3 4 5 6 7 8

> 35 21 > 35 16 29 23 > 35 16

9 10 11 12 13 14 15 16

> 35

17 18 19 20 21 22 23 24

LH surge duration (hours)

10 8 8 9 9

Number of ovulations

Induced luteal phase (days)

l(a) 1 l(a) 1 1 2 l(a) 2

> 16 O(b) > 15 12 > 15 14 12 12

l(a)

> 14

18 22 19 11 12 17

8 12 6 8 9 6

1 1 1 2 1 3

11 11 11 10 7 3

28

7

1

6

18 23 18 > 35 16

12 10 8 9

2 5 1 l(a) 2

7 13 7 9 10

a: delayed ovulation b: progesterone remained very low

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THERIOGENOLOGY

9

4

+

a-. 76sf’-

-29

-25

-21

-17

-13

-9

0

8 hours

.R.

2 WUUTIONS

OL.

0

10

. .. .

32 24 4ft.r start

40 of

58 48 LH surge

Figure 2: FSH, LH, E 2/Jand progesterone in the right (0) and E /31n the left (01 U.O.V. plasma collected from a conscious 2ewe (N'4) around the time of a spontaneous preovulatory LH surge. Samples were collected every 20 min and every 2 hours, respectively, in the first and latter parts of the profile.

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THERIOGENOLOGY

Investigation of ovarian activity To illustrate the possibilities opened up by the technique, a few individual results are presented. Ewe No. 4 was a cyclic ewe studied during a natural oestrus and ovulation period (Figure 2). When the catheters were inserted, one and two fourteen-day old corpora lutea were found on the right and the left ovary, respectively, and two new corpora lutea were found on the right ovary when the catheters were removed. E2/3(10), LH (12). FSH (13) and progesterone (14) were all assayed by radioimmuncassay in the same U.O.V. plasma samples. During the 30 hours preceeding the LH surge, E levels in the right U.O.V. showed large fluctuations between l~Opg/ml and 2012 pglml. In the left u.o.v., the level of E /7 remained at the limit of detection of the assay for most of the sampfing period. After the start of the LH surge, E2P concentration dropped dramatically and stayed low for 34 hours. It only rose again in response to LH pulses. At the same time, E &concentration rose in the left U.O.V. and progesterone level increase2’. Nineteen days after parturition, concentration of E2/rose in ewe No . 17 in the right U.O.V. 20 min after each LH pulse was observed in the j.v. plasma (Figure 3). When the catheters were inserted, the uterine horns were fully regressed and two medium follicles (4mm in diameter) were found on the right ovary; the highest E2rconcentration was lOOOpg/ml. In ewe No. 3 (Figure 4), E2P concentration was measured in the j.v. and in the U.O.V. plasma using the reference extraction method over a period of 12 hours, 5 days after parturition. The concentration of E never exceeded the limit of detection of this assay in the j.v., bu 6 showed small fluctuations from 2.2pg/ml to 23.3pg/ml in the U.O.V. plasma. .50;\50-

240. $30. ‘20. s z lo:

, 9

Figure 3: Concentrations of E2/5 (0) measured in the right U.O.V. plasma in reponse to LH pulses (4) observed in the jugular vein plasma of ewe No. 17 (day 19 postpartum). E2,gassayed without extraction.

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,

. 11

,

, 13

.

. 15

,

. 17

,

,

,

19

. 21

HOW8

Figure 4: Concentration of E2p in the j.v. plasma (.I)and in the right (R.O.) and left (L.O.) U.O.V. plasma of ewe No. 3 (day 5 post-partum) E2/3assayed without extraction.

745

THERIOGENOLOGY DISCUSSION In these experiments, the animals were kept free in pens, which presented at least two advantages. Firstly, as soon as possible after recovery from anaesthesia and never more than 2 hours later, ewe:lamb relations including free suckling were allowed again. Secondly, oestrus could be easily detected with rams. Providing the diameter of the catheter was appropriate, the success of the surgical intervention did not depend upon the reproductive states of the ewes. This suggests that this technique can be used to investigate cyclicity, seasonal and post partum acyclicity, puberty and gestation. During our period of observation, no complications were observed resulting from the presence of the catheters in the abdomen. This surgical approach (from one branch of the uterine vein) could also be used to collect blood specifically from the uterine vein, from the U.O.V.. or from the vena cava without dramatically affecting the vascularisation of both uterus and ovary. However, changes in blood flow (15) may need to be investigated. In addition, the damage to the tissues, especially to the wall of the uterus, was minimal with this approach compared with other techniques (6). This is expected to reduce artificial modifications of the endocrine environment (16). In catheterized animals, preovulatory surge of LH, ovulation rate and progesterone secretion were similar to those reported previously in the same breed of sheep and in the same physiological situations (17, 18), which shows that reproductive function was not disturbed. The presence of a measurable quantity of steroid in the U.O.V. was always associated with the presence of a large(diameter larger than 5 mm) follicle on the adjacent ovary. However, some follicles, even those which were large, were found not to The size and the number of follicles were not always secrete E 2P' related to the concentration of E measured in the U.O.V. Possibly, ti blood from the ovarian and uterine veins might not be completely mixed by the time it enters the catheter(l). Physiological factors may also contribute. It was impossible to know whether the follicles observed on the ovary were healthy or atretic. In the latter case, no estrogens are secreted (19, 20). However, the assay results did not appear to depend on the distance to which the catheter had been inserted when examined at the end of the experiment. In conclusion, this technique of catheterization makes possible the investigation of ovarian steroid release related to gonadotrophin secretion using serial blood sample collection from unstressed animals in any reproductive state. Compared with other methods, this technique is relatively quick (30min). simple (laparotomy) and under good conditions does not affect the reproductive potential of the ewe. In contrast, techniques involving autotransplantation are suitable for very long term studies on subjects such as seasonality.

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THERIOGENOLOGY REFERENCES 1. Pant, H.C., Hopkinson, C.R.N. and Fitzpatrick R.J. Concentration of oestradiol, progesterone, luteinizing hormone and follicle-stimulating hormone in the jugular venous plasma of ewes during the oestrous cycle, J. Endocr. -73~247-255 (1977). 2. Cahill, L.P., Saumande, J., Ravault, J.P. Blanc, M.R. Thimonier, J., Mariana, J.C. and Mauleon, P. Hormonal and follicular relationships in ewes of high and low ovulation rate, J. Reprod. Fert. -62:141-150 (1981). 3. Harrison, F.A., Heap, R.B. and Linzell, J.L. Ovarian function in the sheep after autotransplantation of the ovary and uterus to the neck, J. Endocr. -40:X111 (Abstr.) (1968). 4. Thorburn, G.D. and Mattner, P.E. Anastomosis of the utero-ovarian and anterior mammary veins for collection of utero-ovarian venous blood: progesterone secretion rates in cyclic ewes, J. Endocr. -50:307-320 (1971). 5. Lindner, H.R., Sass, M.B. and Morris, B. Steroids in the ovarian lymph and blood of conscious ewes, J. Endocr. -30:361-376 (1964). 6. Eiler, H. Effect of luteinizing hormone on ovarian release of steroids: ovarian vein sampling technique in conscious gilt, Ann. J. Vet. Res. -39:1695-1698 (1978). 7. Thorburn, G.D., Cox, R.I., Currie, W.B., Restall, B.J. and Schneider, W. Prostaglandin F and progesterone concentrations in the utero-ovarian venous plasma of the ewe during the oestrous cycle and early pregnancy, J. Reprod. Fert. Suppl. -18:151-158 (1973). 8. Cox, R.I., Mattner. P.E. and Thorburn, G.A. Changes in ovarian secretion of oestradiol-17/3around oestrus in the sheep, J. Endocr. -49:345-346 (1971). 9. Cook, B., Hunter, R.H.F. and Kelly, A.S.L. Steroid-binding proteins in follicular fluid and peripheral plasma from pigs, cows and sheep, J. Reprod. Fert. -51:65-71 (1977). 10. Saumande, J. Radioimmunoassay of oestradiol-17p in unextracted ewe plasma, Steroids -38:425-437 (1981). 11. Thibier, M. and Saumande, J. Oestradiol-17/3 progesterone and 1% hydroxyprogesterone concentrations in jugilar venous plasma in cows prior to and during oestrus, J. Steroid. Biochem. -6:1433-1437 (1975). 12. Pelletier, J., Kann, G., Dolais, J. and Rosselin, G. Dosage radioimmunologique de l'hormone luteinisante plasmatique chez le mouton. Mise au point de la technique de dosage, C.R. Acad. Sci. Paris, Ser. D, 266:2291-2294 (1979).

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THERIOGENOLOGY 13. Blanc, M.R. and Poirier, J.C. A new homologous radioimmunoassay for ovine follicle stimulating hormone:development and characterization, Ann. Biol. anim. Bioch. Biophys. -19:1011-1026 (1979). 14. Palmer, E. and Jousset, B. Urinary oestrogen and plasma progesterone levels in non pregnant mares, J. Reprod. Fert. Suppl. -23:213-221 (1975). 15. Mattner, P.E., Stracy, B.D. and Brown, B.W. Changes in total ovarian blood flow during anaesthesia and cannulation of utero-ovarian veins, J. Reprod. Fert. -46:517 (abstract) (1976). 16. Greenwood, B. and Kerry, P.J. Prostaglandin production by a mild inflammatory lesion in sheep, Br. J. Pharmac. -53:305-307 (1975). 17. Thimonier, J. Hormonal control of oestrous cycle in the ewe (a review), Livest. Prod. Sci. 6:39-50 (1979). 18. Cognie, Y. in dry and during the -16:529-536

and Pelletier, J. Preovulatory LH release and ovulation in lactating ewes after progestagen and PMSG treatment seasonal anoestrus, Ann. Biol. anim. Biochim. Biophys. (1976).

19. Hay, M.F. and Moor, R.M. Distribution ofb5-3P-hydroxysteroid dehydrogenase activity in the Graafian follicles of the sheep, J. Reprod. Fert. -43:313-322 (1975). 20. Moor, R.M., Hay, M.F., Dott, H.M. and Cran, D.G. Macroscopic identification and steroidogenic function of atretic follicles in sheep. J. Endocr. -77:309-318 (1978).

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