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Progesterone profile in buffaloes during various stages of oestrous cycle using radioimmunoassy technique
THERIOCENOLOGY
PROQESTERONE PROFIU IN BUFFMOW DURING VARIOUS STAQ?XS OF OBSTROUS CYCLE USINQ RADIOIMMUNOASSYTEXXNI@JE
O.P. Takkar, Mshar Singh and P.N. Varmana Departmsnt of Anhal Science, Punjab Agricultural University Ludhiana, 141004, Punjab, India. Received
for Publication: Accepted:
December 4, 1980 March 25, 1982
ABSTRACT Investigation were carried out to study the norms of progesterone concentration in the blood serum of buffaloes during various phases of oestrous cyale. Twenty four animals ( 12 heifers and 12 cows) were used. The blood serum samples were stored at -2OoC until processed for progesterone asaay. The progesterone concentrations were measured bv the radioimunoassav technicrue. The progesterone levels were 0;360 + 0.062 and 6.33i + 0.068 ng/ml on the day of oestrus in buffalo-herfers and buffalo-&m, respectively. The values were around 1 ng/ml till day 6, followed by a gradual increase to a peak average value of 4.880 t 0.399 and 5.119 + 0.415 ng/ml on day 15 of the cycle in heifers ati cow6, respZctively. Thereafter, the progesterone concentration fell abruotlv to a level sLtilar to that at oestrus. The msan progesterbne-valuea day before osstrus was 0.488 f 0.067 and 0.577 2 0.053 ng/m.lin buffalo-heifers and buffalo-cass, respectively. The man progesterone concentration on different &ya of the cycle ( except day 16 1 did not differ significantly (PLO.01) between heifers and cows. IWRODUCJXON
The control of reproduotive cycle in sexually mature and adult female is an interplay of oestrogen and progesterom hormones liberated from the ovary under the influence of pituitary gonadotrophins. Sufficient information about the status of these hormones in cattle is already available ( 1, 2, 3, 4, 5, 6 1. But very few systematic studies appear to have been made in buffaloes which hava attained the position of important farm livestock in many parks of the world ( 7, 8 1. However, the data presented by
aDepartment of Veterinary Physiology. A&nowledaemeata: The authors exprass their thanks to Dr. S.S. gill Professor and Head, Department of imlmal Science and Dr. H.S. Giwaua, Senior Animal Qeneticlst for providing necessary facilities.
MAY 1982VOL. 17 NO. 5
565
THERIOGENOLOGY
these authors are based on a limited number of animals and frequency of sampling. Therefore, studies were undertaken to establish progesterone profile in sexually mature buffalo-heifers and buffalo-cows during different phases of oestrous cycle using radioimmunoassaytechnique. MATERIALS AND METHODS In other to study progesterone profile during various phases of oestrous cycles, 24 clinically healthy Murrah buffaloes were taken. These comprised 12 cycling-sexuallymature buffalo heifers aged 2 to 34 years and weighinq between 310 to 375 kq and 12 buffalo cows aged 6 to 8 years 7 between their 2nd to-4th lactation 1. All were maintained at the University Dairy Farm. Oestrus detection was done by parading vasectomised bulls four times a day at 0500, 0800, 1700 and 2000 hours. Rectal palpation and blood collections were made daily between 0700 to 0900 hours. Sequential changes were confirmed rectally by the presence of tone in the uterus, graffian follicle, ovulation and development of corpus luteum (cl). The blood serum samples were stored at -2OOC until processed for progesterone assay. Highly specific antisera@ames Hixon and William Hansel, Cornell University, Ithaca, bTewYork, U.S.A.)for progesterone with least cross reactivity was used and the sensitivity was 1OOpg per tub. The blood serum was analysed for progesterone by radlolmmunoassay technique ( 7, 9, 10). Data were analysed by analysis of variance (11). RESUIA'SAND DISCUSSION The overall mean progesterone concentration for the twelve buffalo heifers and twelve buffalo cows during the oestrous cycle are summarised in Table 1. The mean concentration of progesterone in buffalo heifers and buffalo cows was 0.360 + 0.062 and 0.334 + 0.066 nslhrlserum at oestrus. These fTndinas are in close agreement with e&rlier reports ( 7, 8, 12, 13 1, which reported blood progesterone level between 0.16 to 0.5 n&ml on day of oestrus. A very lw progesterone concentration at oestr& has been observed in water and swamp buffaloes respectively ( 14, 15 1. Low progesterone in these buffaloes may be due to type variance. The level remained around 1.0 ng/ml during the first few days of the cycle in both the groups. It increased steadily to 1.418 2 0.295, 1.312 2 0.166 and 2.935 + 0.355, 3.187 i 0.424 ng/ml on day 7 and 10 in the buffalo heifers and buffalo cows, respectively. The peak values of 4.888 + 0.399 and 5.119 2 0.415 ng/ml serum, was obtained on day 15 of the cycle followed by a gradual decline. Similar mid cycle-peak values between day 14 to 16 of the cycle have been reported by other workers ( 8, 12 1. However, a lower peak progesterone value was elso observed ( 7, 15 1. The lo& values were probably because of chromatography step used in radioimnunoassay procedure.
566
MAY
1982 VOL. 17 NO. 5
THERIOCENOLOCY Table & Mean Progesterone level (ng/inl)during Oestrous Cycle in the Blood serum of Buffalo heifers & Buffalo cows. Day of Cvcle
Buffalo heifers (N = 12)
Buffalo sows (N - 12
0.
0.36OiO.062
0.33420.066
1.
0.386~0.087
0.384+0.056
2.
0.483&0.046
0.489~0.085
3.
0.56420.095
0.597~0.101
4.
0.656~0.087
0.697~0.090
5.
0.859$0.149
0.805~0.123
6.
1.13920.277
0.99820.086
7.
1.418fO.295
1.312f0.166
8.
1.78520.391
1.81220.333
9.
2.53920.575
2.386L0.266
10.
2.93520.355
3.18720.424
11.
3.372;tO.550
3.69320.378
12.
4.044~0.380
4.23520.326
13.
4.27720.400
4.606+0.471
14.
4.808~0.448
4.910f0.412
15.
4.88820.399
5.11920.415
16.
3.73420.436**
4.54220.309**
17.
2.79740.540
3.382iO.348
18.
1.62920.436
1.668~0.198
19.
0.89220.132
0.82320.084
20.
0.488+0.067
0.57720.053
21.
0.32120.072
0.53620.066
** Significantly different
( PLO.01 1
N- Number of animals MAY 1982 VOL. 17 NO. 5
567
THERIOGENOLOGY
A precipitous fall in progesterone level then occurred in 3 to 7 days in buffalo heifers and 4 to 6 days in buffalo cows. The mean values a day before oestrus were 0.321 + 0.072 and 0.536 + 0.066 ng/ml in buffalo heifers and buffalo cows, respectively. These findings compare favourably with results obtained by other workers ( 7, 8, 12 1. The level of progesterone in blood serum rose and fell in coincidence with the growth and regression of the CL as confirmed by alternate day rectal examination of these animals during the oestrous cycle. The statistical analysis of data by *Students" t-test between mean progesterone values in buffalo heifers and buffalo cows on corresponding days were not significant except day 16. The foregoing results indicate that the progesterone level was significantly low at the beginning and towards the end of the cycle as against the luteal phase. In cows, the progesterone concentration of 0.5 to 1.0 ng/ml at oestrus and 4 to 7 ng/ml at mid cycle have been reported ( 4, 16, 17, 18 ). The trend in progesterone profile in buffaloes were therefore, similar, but not same as compared to cows. REFERENCBS 1.
Agarwal,
2.
Christensen, D.S., Hop~ood, M.L. and Wiltbank, J.N. Levels of hormones in the serum of cycling beef cows. J. Anfm. sci. 2 t 577-584 ( 1974 1.
3.
Henricks, D.M. Dickey, S.F. and Niswender, C.D. serum L H and plasma progesterone levels during the estrous cyale and early pregnancy in cows. Biol. Reprod. 2 I 346-351 l 1979 1.
4.
Rajamahendran, R., Lague, P.C. and Baker, R.D. Plasma progesterone levels in cycling and gonadotropfn-prostaglandintreated heifers. Can. J. Anim. SC).56 f 37-42 ( 1976 1.
5.
Saba, N., Cunningham, N.F. and Millar, P.0. Plasma Progesterone Androstenedione and Testosterone concentration in free martin heifers. J. Reprod. Fart. 42 : 37-45 ( 1975 1.
6.
Stabenfeldt, Q.B., Ewing, L.L., Patton, J.P. and Mc Donald, L.E. Peripheral plasma progesterone level during the bovine oestrous cycle. J. Reprod. Fert. 19 : 433-442 ( 1969 1.
568
Ratman, S.A., Laumas, K.R., Agarwal, V-K. and Ahmad, A. Studies on steroid hormones: Progesterone concentration in blood serum of Eebu cows during oestrous cycle. Indian J. Anim. Sci. -47 : 715-719 ( 1977 1.
S.P.,
MAY
1982 VOL. 17 NO. 5
THERIOCENOLOGY
7.
Ahmad, A.,
8.
Pandav. -_- R.S. Hozmonal status of female and induced breeding in Murrah buffaloes. Proc. Seminar sponsored by FAO/EIDA-on "Buffalo Reproduction and Artificial Insemination" held at NDRI, Karnal-India. ( 1979 1.
Agarwal, S.P. Aganral, V.K., Rahman, S.A., and Laumas, K.R. Steroid Hormones: Part II-Serum progesterone concentration in buffaloes. Indian J. Exp. Bio. a: 591-593 ( 1977 1.
Spleler, J.M., Webb, R.L. Ealdarini, R.J., and Coppola, J-R. A radioinununoassay for progesterone. Steroids M : 433-442 (1972). lo. Thorneycroft, I.H. and Stone, S.C. Radioimmunoassayof serum progesterone In woman receiving oral contraceptive steroids. Contraception,2 t 129-146 ( 1972 1. 11. Snedecor, 6.W., and Ccchran, W.G., Statistical methods. The Iowa State University Press Ames, Icwa, U.S.A. 6th edition 1967 p 59-62. 12. Kaur, H. and Arora, S.P. Studies on progesterone levels In blood plasma of normal cycling buffaloes as Influenced by different levels of nutrition. Progress Report, NDRI, Karnal, India. ( 1978 1. 13. Singh, Q.B., Dugwekar, Y.G.. Chauhan, F.S., Shanna, Ri;phr;nSingh, M. Luteolytlc effect of prostaglandins F buffaloes. Vet. Rec. 104 : 412-413 ( 197;4). Z4. Kamonpatana, M., Kunawongkrit, A., Bodhlpaksha, P., Schams, D ., Van dwiel, D.F.M. Problems of Reproduction in Female Swamp Buffalo. Proc. Seminar sponsored by FAO/SIDA on "8uffalo Reproduction and Artificial Inseminationheld at NDRI, Karnal-India ( 1979 1. 15. Perera, B.M.A.O. EYnohroniaation of oestrus and fertility in buffaloes of Sri Lanka. Proc. Seminar sponsored by FAO/SIDA on "Buffalo Reproduction and Artificial Insemination"held at NDRI, Karnal-India ( 1979 1.
9.
16. Brltt, J.H. and Ulberg, L.C. Melengesterol acetate administration to dairy heifers and progestaqen levels In the ?:;;%I blood plasma. J. Reprod. Fert. 29 : 119-122 . 17. DonaldSOn, L.B., Bassett, J.H. and Thornburn, G.D. Peripheral Plasma progesterone and concentration of cows during puberty. oestrous cycle, pregnancy and lactation and effect of under nutrition or exogenous oxytocln on progesterone concentration. J. Endocrlnol. e : 599 ( 1970 1. 18. Nakao. Tar One, H., Sato, K., and Miyake, M. Studies On Cystic ovarian disease in dairy cattle. 1. Adrenocortical function, serum concentration of sex steroid and serum protein Pattern In cows with normal oestrus cycles and cows with cystic ovaries. Res. Bull. Obihlro Univ., 2: 397-416 ( 1975 ).
MAY 1982VOL. 17 NO. 5
569
PROQESTERONE PROFIU IN BUFFMOW DURING VARIOUS STAQ?XS OF OBSTROUS CYCLE USINQ RADIOIMMUNOASSYTEXXNI@JE
O.P. Takkar, Mshar Singh and P.N. Varmana Departmsnt of Anhal Science, Punjab Agricultural University Ludhiana, 141004, Punjab, India. Received
for Publication: Accepted:
December 4, 1980 March 25, 1982
ABSTRACT Investigation were carried out to study the norms of progesterone concentration in the blood serum of buffaloes during various phases of oestrous cyale. Twenty four animals ( 12 heifers and 12 cows) were used. The blood serum samples were stored at -2OoC until processed for progesterone asaay. The progesterone concentrations were measured bv the radioimunoassav technicrue. The progesterone levels were 0;360 + 0.062 and 6.33i + 0.068 ng/ml on the day of oestrus in buffalo-herfers and buffalo-&m, respectively. The values were around 1 ng/ml till day 6, followed by a gradual increase to a peak average value of 4.880 t 0.399 and 5.119 + 0.415 ng/ml on day 15 of the cycle in heifers ati cow6, respZctively. Thereafter, the progesterone concentration fell abruotlv to a level sLtilar to that at oestrus. The msan progesterbne-valuea day before osstrus was 0.488 f 0.067 and 0.577 2 0.053 ng/m.lin buffalo-heifers and buffalo-cass, respectively. The man progesterone concentration on different &ya of the cycle ( except day 16 1 did not differ significantly (PLO.01) between heifers and cows. IWRODUCJXON
The control of reproduotive cycle in sexually mature and adult female is an interplay of oestrogen and progesterom hormones liberated from the ovary under the influence of pituitary gonadotrophins. Sufficient information about the status of these hormones in cattle is already available ( 1, 2, 3, 4, 5, 6 1. But very few systematic studies appear to have been made in buffaloes which hava attained the position of important farm livestock in many parks of the world ( 7, 8 1. However, the data presented by
aDepartment of Veterinary Physiology. A&nowledaemeata: The authors exprass their thanks to Dr. S.S. gill Professor and Head, Department of imlmal Science and Dr. H.S. Giwaua, Senior Animal Qeneticlst for providing necessary facilities.
MAY 1982VOL. 17 NO. 5
565
THERIOGENOLOGY
these authors are based on a limited number of animals and frequency of sampling. Therefore, studies were undertaken to establish progesterone profile in sexually mature buffalo-heifers and buffalo-cows during different phases of oestrous cycle using radioimmunoassaytechnique. MATERIALS AND METHODS In other to study progesterone profile during various phases of oestrous cycles, 24 clinically healthy Murrah buffaloes were taken. These comprised 12 cycling-sexuallymature buffalo heifers aged 2 to 34 years and weighinq between 310 to 375 kq and 12 buffalo cows aged 6 to 8 years 7 between their 2nd to-4th lactation 1. All were maintained at the University Dairy Farm. Oestrus detection was done by parading vasectomised bulls four times a day at 0500, 0800, 1700 and 2000 hours. Rectal palpation and blood collections were made daily between 0700 to 0900 hours. Sequential changes were confirmed rectally by the presence of tone in the uterus, graffian follicle, ovulation and development of corpus luteum (cl). The blood serum samples were stored at -2OOC until processed for progesterone assay. Highly specific antisera@ames Hixon and William Hansel, Cornell University, Ithaca, bTewYork, U.S.A.)for progesterone with least cross reactivity was used and the sensitivity was 1OOpg per tub. The blood serum was analysed for progesterone by radlolmmunoassay technique ( 7, 9, 10). Data were analysed by analysis of variance (11). RESUIA'SAND DISCUSSION The overall mean progesterone concentration for the twelve buffalo heifers and twelve buffalo cows during the oestrous cycle are summarised in Table 1. The mean concentration of progesterone in buffalo heifers and buffalo cows was 0.360 + 0.062 and 0.334 + 0.066 nslhrlserum at oestrus. These fTndinas are in close agreement with e&rlier reports ( 7, 8, 12, 13 1, which reported blood progesterone level between 0.16 to 0.5 n&ml on day of oestrus. A very lw progesterone concentration at oestr& has been observed in water and swamp buffaloes respectively ( 14, 15 1. Low progesterone in these buffaloes may be due to type variance. The level remained around 1.0 ng/ml during the first few days of the cycle in both the groups. It increased steadily to 1.418 2 0.295, 1.312 2 0.166 and 2.935 + 0.355, 3.187 i 0.424 ng/ml on day 7 and 10 in the buffalo heifers and buffalo cows, respectively. The peak values of 4.888 + 0.399 and 5.119 2 0.415 ng/ml serum, was obtained on day 15 of the cycle followed by a gradual decline. Similar mid cycle-peak values between day 14 to 16 of the cycle have been reported by other workers ( 8, 12 1. However, a lower peak progesterone value was elso observed ( 7, 15 1. The lo& values were probably because of chromatography step used in radioimnunoassay procedure.
566
MAY
1982 VOL. 17 NO. 5
THERIOCENOLOCY Table & Mean Progesterone level (ng/inl)during Oestrous Cycle in the Blood serum of Buffalo heifers & Buffalo cows. Day of Cvcle
Buffalo heifers (N = 12)
Buffalo sows (N - 12
0.
0.36OiO.062
0.33420.066
1.
0.386~0.087
0.384+0.056
2.
0.483&0.046
0.489~0.085
3.
0.56420.095
0.597~0.101
4.
0.656~0.087
0.697~0.090
5.
0.859$0.149
0.805~0.123
6.
1.13920.277
0.99820.086
7.
1.418fO.295
1.312f0.166
8.
1.78520.391
1.81220.333
9.
2.53920.575
2.386L0.266
10.
2.93520.355
3.18720.424
11.
3.372;tO.550
3.69320.378
12.
4.044~0.380
4.23520.326
13.
4.27720.400
4.606+0.471
14.
4.808~0.448
4.910f0.412
15.
4.88820.399
5.11920.415
16.
3.73420.436**
4.54220.309**
17.
2.79740.540
3.382iO.348
18.
1.62920.436
1.668~0.198
19.
0.89220.132
0.82320.084
20.
0.488+0.067
0.57720.053
21.
0.32120.072
0.53620.066
** Significantly different
( PLO.01 1
N- Number of animals MAY 1982 VOL. 17 NO. 5
567
THERIOGENOLOGY
A precipitous fall in progesterone level then occurred in 3 to 7 days in buffalo heifers and 4 to 6 days in buffalo cows. The mean values a day before oestrus were 0.321 + 0.072 and 0.536 + 0.066 ng/ml in buffalo heifers and buffalo cows, respectively. These findings compare favourably with results obtained by other workers ( 7, 8, 12 1. The level of progesterone in blood serum rose and fell in coincidence with the growth and regression of the CL as confirmed by alternate day rectal examination of these animals during the oestrous cycle. The statistical analysis of data by *Students" t-test between mean progesterone values in buffalo heifers and buffalo cows on corresponding days were not significant except day 16. The foregoing results indicate that the progesterone level was significantly low at the beginning and towards the end of the cycle as against the luteal phase. In cows, the progesterone concentration of 0.5 to 1.0 ng/ml at oestrus and 4 to 7 ng/ml at mid cycle have been reported ( 4, 16, 17, 18 ). The trend in progesterone profile in buffaloes were therefore, similar, but not same as compared to cows. REFERENCBS 1.
Agarwal,
2.
Christensen, D.S., Hop~ood, M.L. and Wiltbank, J.N. Levels of hormones in the serum of cycling beef cows. J. Anfm. sci. 2 t 577-584 ( 1974 1.
3.
Henricks, D.M. Dickey, S.F. and Niswender, C.D. serum L H and plasma progesterone levels during the estrous cyale and early pregnancy in cows. Biol. Reprod. 2 I 346-351 l 1979 1.
4.
Rajamahendran, R., Lague, P.C. and Baker, R.D. Plasma progesterone levels in cycling and gonadotropfn-prostaglandintreated heifers. Can. J. Anim. SC).56 f 37-42 ( 1976 1.
5.
Saba, N., Cunningham, N.F. and Millar, P.0. Plasma Progesterone Androstenedione and Testosterone concentration in free martin heifers. J. Reprod. Fart. 42 : 37-45 ( 1975 1.
6.
Stabenfeldt, Q.B., Ewing, L.L., Patton, J.P. and Mc Donald, L.E. Peripheral plasma progesterone level during the bovine oestrous cycle. J. Reprod. Fert. 19 : 433-442 ( 1969 1.
568
Ratman, S.A., Laumas, K.R., Agarwal, V-K. and Ahmad, A. Studies on steroid hormones: Progesterone concentration in blood serum of Eebu cows during oestrous cycle. Indian J. Anim. Sci. -47 : 715-719 ( 1977 1.
S.P.,
MAY
1982 VOL. 17 NO. 5
THERIOCENOLOGY
7.
Ahmad, A.,
8.
Pandav. -_- R.S. Hozmonal status of female and induced breeding in Murrah buffaloes. Proc. Seminar sponsored by FAO/EIDA-on "Buffalo Reproduction and Artificial Insemination" held at NDRI, Karnal-India. ( 1979 1.
Agarwal, S.P. Aganral, V.K., Rahman, S.A., and Laumas, K.R. Steroid Hormones: Part II-Serum progesterone concentration in buffaloes. Indian J. Exp. Bio. a: 591-593 ( 1977 1.
Spleler, J.M., Webb, R.L. Ealdarini, R.J., and Coppola, J-R. A radioinununoassay for progesterone. Steroids M : 433-442 (1972). lo. Thorneycroft, I.H. and Stone, S.C. Radioimmunoassayof serum progesterone In woman receiving oral contraceptive steroids. Contraception,2 t 129-146 ( 1972 1. 11. Snedecor, 6.W., and Ccchran, W.G., Statistical methods. The Iowa State University Press Ames, Icwa, U.S.A. 6th edition 1967 p 59-62. 12. Kaur, H. and Arora, S.P. Studies on progesterone levels In blood plasma of normal cycling buffaloes as Influenced by different levels of nutrition. Progress Report, NDRI, Karnal, India. ( 1978 1. 13. Singh, Q.B., Dugwekar, Y.G.. Chauhan, F.S., Shanna, Ri;phr;nSingh, M. Luteolytlc effect of prostaglandins F buffaloes. Vet. Rec. 104 : 412-413 ( 197;4). Z4. Kamonpatana, M., Kunawongkrit, A., Bodhlpaksha, P., Schams, D ., Van dwiel, D.F.M. Problems of Reproduction in Female Swamp Buffalo. Proc. Seminar sponsored by FAO/SIDA on "8uffalo Reproduction and Artificial Inseminationheld at NDRI, Karnal-India ( 1979 1. 15. Perera, B.M.A.O. EYnohroniaation of oestrus and fertility in buffaloes of Sri Lanka. Proc. Seminar sponsored by FAO/SIDA on "Buffalo Reproduction and Artificial Insemination"held at NDRI, Karnal-India ( 1979 1.
9.
16. Brltt, J.H. and Ulberg, L.C. Melengesterol acetate administration to dairy heifers and progestaqen levels In the ?:;;%I blood plasma. J. Reprod. Fert. 29 : 119-122 . 17. DonaldSOn, L.B., Bassett, J.H. and Thornburn, G.D. Peripheral Plasma progesterone and concentration of cows during puberty. oestrous cycle, pregnancy and lactation and effect of under nutrition or exogenous oxytocln on progesterone concentration. J. Endocrlnol. e : 599 ( 1970 1. 18. Nakao. Tar One, H., Sato, K., and Miyake, M. Studies On Cystic ovarian disease in dairy cattle. 1. Adrenocortical function, serum concentration of sex steroid and serum protein Pattern In cows with normal oestrus cycles and cows with cystic ovaries. Res. Bull. Obihlro Univ., 2: 397-416 ( 1975 ).
MAY 1982VOL. 17 NO. 5
569