- Email: [email protected]
The control of superovulation in the bovine with a PMSG-antiserum
THERIOGENOLOGY
THE CONTROL OF SUPEROVULATION
IN
THE BOVINE WITH A PMSG-ANTISERUM
D. Dhondt, R. Bouters, J. Spincemaille, M. Coryn and M. Vandeplassche. Clinic for obstetrics and reproduction Veterinary Faculty State University Casinoplein 24, B-9000 Ghent - Belgium Received
for publication:
May 2, 1978
ABSTRACT A PMSG antiserum prepared in turkeys has been injected on the first day of standing heat in 20 superovulated cows (2000-3000 1-U. PMSG on DlO, 2x500 mcg Estrumate on D12). The treatment did not reduce the individual variability in superovulatory response but resulted in a higher ovulation (15,7 CL vs 9,4), a decreased number of unruptured follicles (2,8 vs 6,5), a better fertilisation (80% vs 60%) and a shorter external heat with less inseminations to be performed. ACKNOWLEDGMENT The financial support from assistance of A. Geldhof, C. supply of the PMSG IGe 270.1 the antiserum by the ORGANON
JUNE 1978 VOL. 9 NO. 6
IWONL-CSVH and the technical De Bondt & R. Verbeke and the Hst.) for the preparation of Co. are gratefully acknowledged.
529
THERIOGENOLOGY
INTRODUCTION Superovulation in cattle is usually induced by injection of PMSG (pregnant mare serum gonadotrophin) in the mid cycle, followed by prostaglandins 48 hrs later. With this procedure an average of 10 CL (corpora lutea) per donor are induced (1). Superovulation however is characterised by an enormous individual variability and frequently by ovarian overstimulation with the persistence of many unruptured follicles (1, 4). These variablesof SUperovulation might be due to the long half-life time of PMSG compared with endogenous pituitary gonadotrophins (5). Recently it has been demonstrated that PMSG can still be detected 10 days after i.m. injection in cows (6). The purpose of this experiment was to obtain a more predictable superovulatory response by the use of a specific PMSG-antiserum which might cut abruptly the activity of PMSGand avoid the risk for protracted ovarian stimulation.
MATERIAL AND MEX'HODS 1. Preparation of the antiserum. --------~~~-~~~-~~__~~-~~_^--~~~ Adult turkeys were injected S.C. at 3 weeks interval with 6000 I.U. PMSG in complete Freund's adjuvant. After a series of injections with PMSG 1 turkey produced antibodies against PMSG. Fifty ml blood were weekly One ml of pooled anticollected and the serum pooled. serum neutralised 2000 I.U. PMSG in vitro, as tested by the rat ovarian weiqht increase (7). 2. Experimental animals -B-B_-_-_-_-_-_-_-_-_-_-r_-_-_-_-__-l A group of 20 dairy cattle (8 heifers, 12 cows) received 2000 I.U. (heifers) or 3000 I.U. (cows)PMSG 1.m. on DlO of the oestrous cycle, followed by 2 injections of 500 mcg Estrumate (ICI) 48 hrs and 54 hrs Later. At the first signs of heat the animals were inseminated with deep frozen semen and injected immediately after insemination with I ml PMSG antlserum diluted - 9 ml. Inseminations were repeated every 12 hrs as Long as oestrous behaviour was present. A group of 102 dairy cows received only 2000-3000 1-U. PMSG on DlO and Estrumate on D12 of the cycle. The results in these 102 animals have been published elsewhere (3). Embryos and unfertilized eggs were recovered with a transcervical method (3) on D6-D7. Eleven animals were slaughtered within 24 hrs following embryo recovery and the number of CL and follicles with a diameter over 1 cm were counted. In 9 animals these numbers had to be estimated at rectal palpation.
530
JUNE 1978 VOL. 9 NO. 6
'I'MERIOGENOLOGY
Two more cows were injected on DlO with 3000 1-U. PMSG i.m. immediately followed by 2 ml PMSG antiserum I.V. The same batch of PMSG (FOLLIGON CH 1858, Organon) was used throughout the experiment both in experimental and in control animals. RESULTS The number of CL and of ruptured large follicles, the number of eggs recovered and the fertilisation rate in 20 cows injected with 1 ml PMSG antiserum on the first day of standing heat are summarised in Table I. From this table it is obvious that 313 CL (average 15.7), and 56 stimulated but unruptured follicles (average 2.8) were present on both ovaries. From these 313 CL. 116 (35%) or an average of 5.8 eggs per donor have been recovered of which 93 (80%) were fertilised. In the control group (102 animals) an average of 9.4 CL and 6.5 unruptured follicles, a recovery of 30% or 2.9 eggs per donor and a fertilisation rate of 60% have been recorded (table I). As a result of PMSG-antiserum injection on the first day of standing heat a significant shortening of the external heat period was observed (average 24 hrs vs 48 hrs for the 102 controls). In this way only 2 inseminations per donor were necessary while in the control group the great majority of animals had to be inseminated 4 times. The 2 cows which received PMSG and antiserum against PMSG at the same time showed normal oestrous behaviour on the 3rd day following p rostaglandin injection. On rectal palpation 6 and 10 days later 1 single CL could be palpated in each cow and the presence of active luteal tissue was confirmed by progesterone measurements in the peripheral blood. DISCUSSION The observation that superovulation in cattle can be blocked by a simultaneous injection of PMSG and of an antiserum against PMSG confirms the findings of SPINCEMAILLE in vitro and in vivo in rats (7) and from BINDON & PIPER (2) in vivo in sheep and cattle. Furthermore the antiserum obviously does not interfere with the endogenous release of bovine L,H since spontaneous single ovulation occurred in both experimental animals. The same PMSG-antiserum however shows a clear cross
JUNE 1978 VOL. 9 NO. 6
531
THERIOGENOLOGY
reaction with equine LH (7). The administration of PMSG antiserum on the day of standing heat, i.e. 4 days after PMSG injection resulted in : a) a higher ovulation rate as compared with control animals which received only PMSG (15.7 vs 9.4 per stimulated animal). b) a decrease in the number of stimulated but unruptured follicles (2.8 vs 6.5 per donor). c) an increased fertilisation rate of the recovered eggs (80% vs 60%) d) a shorter oestrous period (24 hrs vs 28 hrs). This resulted in a reduction of the number of inseminations required and saving of valuable semen. These beneficial effectsof the antiserum are most probably due to its neutralising effect on the PMSG still circulating in the donor animal on the day of standing heat. This would interrupt any further stimulation of growing follicles on the ovaries, and create a more favourable hormonal environment for ovulation and for fertilisation by decreasing the relative oestrogen level. The use of the PMSG antiserum however had no effect on the great variability in ovulatory response between individual animals. Further experiments on the optimal dose and time of injection of PMSG antiserum might give better possibilities for more the cow.
532
predictable
superovulatory
reaction
In
JUNE 1978 VOL. 9 NO. 6
T3ERIOGEMKOGY
REFERENCES 1. BETTERIDGE, K.J. (1977). Superovulation. In : Embryo transfer in farm animals. A review of Ed. Betteridge, K.J. techniques and applications. Canada Department of Agriculture, Monograph, N"l& l-9 . 2. BINDON, B.M. 61 PIPER, L.R. (1977) Induction of ovulation in sheep and cattle by injections of PMSG and ovine anti-PMSG immune serum. Theriogenology, 4, 171. 3. BOUTERS, R., DHONDT, D., VANDEPLASSCHE, M & CORYN, M. (1976). Het niet-chirurgisch opvangen van bevruchte eieren bij het rund. Vlaams Diergeneeskundig Tijdschrift, l.2, 426-432. 4. DHONDT, D. & BOUTERS, R. (1977). Eitransplantatie bij het rund : literatuuroverzicht. Communications of the Fat. of Vet. Med., state Univ. Ghent, 20, N'l-2 PP 78. 5. MCINTOSH, J.E.A., MOOR, R.M. & ALLEN, W.R. (1975). Pregnant mare serum gonadotrophin : rate of clearance from the circulation of sheep. J. Reprod. Fert., 44, 95-100. 6. SCHAMS, D., MEMZER, Ch., CHALLENBERGER, E., HOFFMANN, B. HAHN, J. & HAHN, R. (1977). Some studies on pregnant mare gonadotrophin (PMSG) and on endocrine responses after application for superovulation in cattle. E.E.C.-Seminar "Control of reproduction in the cow", Galway, 27-29 September. 7. SPINCEMAILLE, J. (1978) Ontwikkeling van een Hemagglutinatie-inhibitie test als drachtdiagnose bij de merrie. In druk : Vlaams Diergeneeskundig Tijdschrift.
JUNE 1978
VOL. 9 NO. 6
533
THERKKENOLOCY
TABLE I No unovulatec No recovered eggs follicles
No
-r
No fertilised eoqs
1
12b
0
8
7
2
qb
0
4
3
3
lib
0
5
4
4
12b
0
4
4
5
lib
0
7
6
6
12b
0
0
0
7
2ob
5
7
8
3b 85 x = 10.6 22
0 5 ;; = 0.6 3
10
lob
0
9
9
11
11
1
3
0
12
9
5
6
5
13
24
4
5
1
9
7
0 35 j; = 4.4. 18
0 31 ;; = 3.! 17
14
15
4
2
0
15
18
6
1
L
16
25
4
24
24
17
1
10
0
0
18
58
6
5
5
19
30
4
5
0
20 otal OTAL ONTROL
5 228 x = 19.0
4 51 x = 4.2
3 81 ii = 6.7
62 ;; = 5.2.
313 x = 15.7 258 x = 9,4
56 z = 2.8 $63 x = 6,5
116 ii = 5.8 296 x = 2,9
;; = 4.6 178 x = 1.8
0
93
: heifers : 2000 I.E. PMSG : 3000 I.E. PMSG : cows b : no. of CL estimated by rectal palpation: in all other cases animals were slaughtered after egg recovery. a:
animal no. l-8 9-20
534
JUNE 1978 VOL. 9 NO. 6
THE CONTROL OF SUPEROVULATION
IN
THE BOVINE WITH A PMSG-ANTISERUM
D. Dhondt, R. Bouters, J. Spincemaille, M. Coryn and M. Vandeplassche. Clinic for obstetrics and reproduction Veterinary Faculty State University Casinoplein 24, B-9000 Ghent - Belgium Received
for publication:
May 2, 1978
ABSTRACT A PMSG antiserum prepared in turkeys has been injected on the first day of standing heat in 20 superovulated cows (2000-3000 1-U. PMSG on DlO, 2x500 mcg Estrumate on D12). The treatment did not reduce the individual variability in superovulatory response but resulted in a higher ovulation (15,7 CL vs 9,4), a decreased number of unruptured follicles (2,8 vs 6,5), a better fertilisation (80% vs 60%) and a shorter external heat with less inseminations to be performed. ACKNOWLEDGMENT The financial support from assistance of A. Geldhof, C. supply of the PMSG IGe 270.1 the antiserum by the ORGANON
JUNE 1978 VOL. 9 NO. 6
IWONL-CSVH and the technical De Bondt & R. Verbeke and the Hst.) for the preparation of Co. are gratefully acknowledged.
529
THERIOGENOLOGY
INTRODUCTION Superovulation in cattle is usually induced by injection of PMSG (pregnant mare serum gonadotrophin) in the mid cycle, followed by prostaglandins 48 hrs later. With this procedure an average of 10 CL (corpora lutea) per donor are induced (1). Superovulation however is characterised by an enormous individual variability and frequently by ovarian overstimulation with the persistence of many unruptured follicles (1, 4). These variablesof SUperovulation might be due to the long half-life time of PMSG compared with endogenous pituitary gonadotrophins (5). Recently it has been demonstrated that PMSG can still be detected 10 days after i.m. injection in cows (6). The purpose of this experiment was to obtain a more predictable superovulatory response by the use of a specific PMSG-antiserum which might cut abruptly the activity of PMSGand avoid the risk for protracted ovarian stimulation.
MATERIAL AND MEX'HODS 1. Preparation of the antiserum. --------~~~-~~~-~~__~~-~~_^--~~~ Adult turkeys were injected S.C. at 3 weeks interval with 6000 I.U. PMSG in complete Freund's adjuvant. After a series of injections with PMSG 1 turkey produced antibodies against PMSG. Fifty ml blood were weekly One ml of pooled anticollected and the serum pooled. serum neutralised 2000 I.U. PMSG in vitro, as tested by the rat ovarian weiqht increase (7). 2. Experimental animals -B-B_-_-_-_-_-_-_-_-_-_-r_-_-_-_-__-l A group of 20 dairy cattle (8 heifers, 12 cows) received 2000 I.U. (heifers) or 3000 I.U. (cows)PMSG 1.m. on DlO of the oestrous cycle, followed by 2 injections of 500 mcg Estrumate (ICI) 48 hrs and 54 hrs Later. At the first signs of heat the animals were inseminated with deep frozen semen and injected immediately after insemination with I ml PMSG antlserum diluted - 9 ml. Inseminations were repeated every 12 hrs as Long as oestrous behaviour was present. A group of 102 dairy cows received only 2000-3000 1-U. PMSG on DlO and Estrumate on D12 of the cycle. The results in these 102 animals have been published elsewhere (3). Embryos and unfertilized eggs were recovered with a transcervical method (3) on D6-D7. Eleven animals were slaughtered within 24 hrs following embryo recovery and the number of CL and follicles with a diameter over 1 cm were counted. In 9 animals these numbers had to be estimated at rectal palpation.
530
JUNE 1978 VOL. 9 NO. 6
'I'MERIOGENOLOGY
Two more cows were injected on DlO with 3000 1-U. PMSG i.m. immediately followed by 2 ml PMSG antiserum I.V. The same batch of PMSG (FOLLIGON CH 1858, Organon) was used throughout the experiment both in experimental and in control animals. RESULTS The number of CL and of ruptured large follicles, the number of eggs recovered and the fertilisation rate in 20 cows injected with 1 ml PMSG antiserum on the first day of standing heat are summarised in Table I. From this table it is obvious that 313 CL (average 15.7), and 56 stimulated but unruptured follicles (average 2.8) were present on both ovaries. From these 313 CL. 116 (35%) or an average of 5.8 eggs per donor have been recovered of which 93 (80%) were fertilised. In the control group (102 animals) an average of 9.4 CL and 6.5 unruptured follicles, a recovery of 30% or 2.9 eggs per donor and a fertilisation rate of 60% have been recorded (table I). As a result of PMSG-antiserum injection on the first day of standing heat a significant shortening of the external heat period was observed (average 24 hrs vs 48 hrs for the 102 controls). In this way only 2 inseminations per donor were necessary while in the control group the great majority of animals had to be inseminated 4 times. The 2 cows which received PMSG and antiserum against PMSG at the same time showed normal oestrous behaviour on the 3rd day following p rostaglandin injection. On rectal palpation 6 and 10 days later 1 single CL could be palpated in each cow and the presence of active luteal tissue was confirmed by progesterone measurements in the peripheral blood. DISCUSSION The observation that superovulation in cattle can be blocked by a simultaneous injection of PMSG and of an antiserum against PMSG confirms the findings of SPINCEMAILLE in vitro and in vivo in rats (7) and from BINDON & PIPER (2) in vivo in sheep and cattle. Furthermore the antiserum obviously does not interfere with the endogenous release of bovine L,H since spontaneous single ovulation occurred in both experimental animals. The same PMSG-antiserum however shows a clear cross
JUNE 1978 VOL. 9 NO. 6
531
THERIOGENOLOGY
reaction with equine LH (7). The administration of PMSG antiserum on the day of standing heat, i.e. 4 days after PMSG injection resulted in : a) a higher ovulation rate as compared with control animals which received only PMSG (15.7 vs 9.4 per stimulated animal). b) a decrease in the number of stimulated but unruptured follicles (2.8 vs 6.5 per donor). c) an increased fertilisation rate of the recovered eggs (80% vs 60%) d) a shorter oestrous period (24 hrs vs 28 hrs). This resulted in a reduction of the number of inseminations required and saving of valuable semen. These beneficial effectsof the antiserum are most probably due to its neutralising effect on the PMSG still circulating in the donor animal on the day of standing heat. This would interrupt any further stimulation of growing follicles on the ovaries, and create a more favourable hormonal environment for ovulation and for fertilisation by decreasing the relative oestrogen level. The use of the PMSG antiserum however had no effect on the great variability in ovulatory response between individual animals. Further experiments on the optimal dose and time of injection of PMSG antiserum might give better possibilities for more the cow.
532
predictable
superovulatory
reaction
In
JUNE 1978 VOL. 9 NO. 6
T3ERIOGEMKOGY
REFERENCES 1. BETTERIDGE, K.J. (1977). Superovulation. In : Embryo transfer in farm animals. A review of Ed. Betteridge, K.J. techniques and applications. Canada Department of Agriculture, Monograph, N"l& l-9 . 2. BINDON, B.M. 61 PIPER, L.R. (1977) Induction of ovulation in sheep and cattle by injections of PMSG and ovine anti-PMSG immune serum. Theriogenology, 4, 171. 3. BOUTERS, R., DHONDT, D., VANDEPLASSCHE, M & CORYN, M. (1976). Het niet-chirurgisch opvangen van bevruchte eieren bij het rund. Vlaams Diergeneeskundig Tijdschrift, l.2, 426-432. 4. DHONDT, D. & BOUTERS, R. (1977). Eitransplantatie bij het rund : literatuuroverzicht. Communications of the Fat. of Vet. Med., state Univ. Ghent, 20, N'l-2 PP 78. 5. MCINTOSH, J.E.A., MOOR, R.M. & ALLEN, W.R. (1975). Pregnant mare serum gonadotrophin : rate of clearance from the circulation of sheep. J. Reprod. Fert., 44, 95-100. 6. SCHAMS, D., MEMZER, Ch., CHALLENBERGER, E., HOFFMANN, B. HAHN, J. & HAHN, R. (1977). Some studies on pregnant mare gonadotrophin (PMSG) and on endocrine responses after application for superovulation in cattle. E.E.C.-Seminar "Control of reproduction in the cow", Galway, 27-29 September. 7. SPINCEMAILLE, J. (1978) Ontwikkeling van een Hemagglutinatie-inhibitie test als drachtdiagnose bij de merrie. In druk : Vlaams Diergeneeskundig Tijdschrift.
JUNE 1978
VOL. 9 NO. 6
533
THERKKENOLOCY
TABLE I No unovulatec No recovered eggs follicles
No
-r
No fertilised eoqs
1
12b
0
8
7
2
qb
0
4
3
3
lib
0
5
4
4
12b
0
4
4
5
lib
0
7
6
6
12b
0
0
0
7
2ob
5
7
8
3b 85 x = 10.6 22
0 5 ;; = 0.6 3
10
lob
0
9
9
11
11
1
3
0
12
9
5
6
5
13
24
4
5
1
9
7
0 35 j; = 4.4. 18
0 31 ;; = 3.! 17
14
15
4
2
0
15
18
6
1
L
16
25
4
24
24
17
1
10
0
0
18
58
6
5
5
19
30
4
5
0
20 otal OTAL ONTROL
5 228 x = 19.0
4 51 x = 4.2
3 81 ii = 6.7
62 ;; = 5.2.
313 x = 15.7 258 x = 9,4
56 z = 2.8 $63 x = 6,5
116 ii = 5.8 296 x = 2,9
;; = 4.6 178 x = 1.8
0
93
: heifers : 2000 I.E. PMSG : 3000 I.E. PMSG : cows b : no. of CL estimated by rectal palpation: in all other cases animals were slaughtered after egg recovery. a:
animal no. l-8 9-20
534
JUNE 1978 VOL. 9 NO. 6