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Preovulatory LH profiles of superovulated cows and progesterone concentrations at embryo recovery
THERIOGENOLOGY
PREOWLATORY LH PROFILES OF SUPEROWLATED COWS AND PROGESTERONE CONCENTRATIONS AT EMBRYO RECOVERY A. Wubishet', D.J. Kesler, C.N. Graves, S.L. Spahr and R.J. Favero Department of Animal Sciences University of Illinois Urbana, IL 61801 Received for publication: November 3, 1988 Accepted: November 9, 1990 ABSTRACT Forty-two Holstein cows were randomly assigned to three superovulatory treatment groups of 14 cows each. Cows in Group I received follicle stimulating hormone (FSH; 50 mg i.m.); those in Group II received FSH (50 mg i.m.) along with GnRH (250 ug in 2 % carboxymethylcellulose s.c.) on the day of estrus; and cows in Group III were infused FSH (49 mg) via osmotic pump implants. FSH was administered over a 5-d period for cows in Groups I and II (twice daily in declining doses). Cows in Group III received FSH over a 7-d period (constantly at a rate of 7 mg/day). All cows received 25 mg PGF,a (prostaglandin F,a) 48 hours after initiation of the FSH treatment. Blood samples were collected from seven cows from each group at 2 hour intervals on the fifth day of superovulation for serum luteinizing hormone (LH) concentration analysis by radioimmunoassay, and blood samples were collected from all cows on the day of embryo recovery for plasma progesterone determination. The LH profile was not altered (P~0.05) by either GnRH administration or by the constant infusion of FSH as compared to FSH treatment alone. Plasma progesterone concentrations were highly correlated with the number of corpora lutea (CL) palpated (r=0.92; PcO.01) and with the number of ova and/or embryos recovered (r=0.88; PcO.01). The accuracy of predicting the number of recoverable ova and/or embryos by the concentration of plasma progesterone was 86%. Key words:
superovulation, LH surge, progesterone concentrations, recoverable embryos
1Present address: University of California-Extension, 777 E. Rilato Ave., San Bernardino, CA 92415
FEBRUARY 1991 VOL. 35 NO. 2
461
THERIOGENOLOGY
INTRODUCTION It has been reported (l-3) that gonadotropin releasing hormone (GnRH) treatment improves fertilization rates in cattle. It has also been shown that continuous infusion of FSH can bring about superovulation (4,5), but lower dosages of FSH result in a lower number of palpable corpora lutea (CL) and recoverable embryos (6). This study was undertaken to investigate if 1) multiple intramuscular injections of FSH alone, 2) multiple FSH injections along with the administration of GnRH on the day of expected estrus, or 3) the continuous infusion of FSH would affect the LH response. An additional objective of the study was to determine the relationship between concentration of progesterone in the plasma of superovulated dairy cows and the number of both palpable CL and recoverable embryos. MATERIALS AND METHODS Forty-two Holstein cows were randomly assigned to three Cows in Group I superovulatory treatments of 14 cows each. were administered FSH (FSH-P, 50 mg i.m., Burns-Biotech, Omaha, NE) twice daily in declining doses over a 5-d period. Cows in Group II were superovulated in the same manner as those in Group I, except they also received GnRH (250 ug s-c., Park, KS) Cystorelin, CEVA Laboratories Inc., Overland dissolved in 2% carboxymethylcellulose at 1400 h (54 h after prostaglandin F,a; PGF,a) on the day of expected estrus. cows in Group III received FSH in osmotic pumps (Alza Corporation, Palo Alto, CA) implanted under the skin anterio-dorsal to the The osmotic pumps delivered FSH at the rate of 7 scapula. mg/day and were left in situ for 7-d. All cows were treated with PGF,a (25 mg, Lutalyse, Upjohn Co., Kalamazoo, MI) 48 h after initiation of the FSH treatment. On Day 5, 20 ml of blood were collected at 2-h intervals for 24 h from the jugular vein via indwelling 14-g, 13.3-cm Angiocath catheter The catheters were flushed with 5 (Desert Co., sandy, UT). to 10 ml of concentrated sucrose solution to minimize injections blockage. When sampling and superovulatory coincided, samples were collected prior to injection. On the day of embryo recovery (8 d after insemination), 15 ml of blood were collected from all cows in each group into syringes containing 2 ml of 5% sodium citrate in saline. All blood samples were transferred into centrifuge tubes and cooled on ice immediately after collection and stored overnight at 4°C. All samples were processed to yield serum Serum and plasma samples were or plasma the following day. aliquoted into appropriately labeled 12 x 75-mm tubes and frozen at -20°C until analyzed. The LH concentration antibody radioimmunoassay
452
in serum was quantified by double according to procedures reported by
FEBRUARY 1991 VOL. 35 NO. 2
THERIOGENOLOGY Niswender et al. (7), except I251was used as the radioactive Anti-LH (B-225, first antibody), purified bovine LH label. (LER-1072) and NIH-LH-B10 (standard) were obtained from Drs. G.D. Niswender, L.E. Reichert, and the National Institute of Arthritis, Metabolism and Digestive Diseases, respectively. The second antibody, obtained from Dr. D.J. Kesler, was antirabbit gamma globulin generated in sheep at the University of Illinois at Urbana. The assay was validated in our laboratory as reported by Troxel et al. (8). Fifty to 100 ul of serum were collected in duplicate by an automatic pipette and The intra-assay coefficient of analyzed in one assay. variation was 6.5%. Plasma progesterone concentrations were determined by procedures describes and radioimmunoassay according to validated by Troxel et al. (8) and Wiseman et al. (9). Two assays were conducted on 50 to 100 ul of duplicate plasma samples. Intra- and inter-assay coefficients of variation were 5.2 and 14.9%, respectively. The LH data were analyzed by analyzed by analysis of variance as split-plot design as described by Gill and Hafs (10). Plasma progesterone data were analyzed by analysis of variance for a completely randomized design (11). RESULTS AND DISCUSSION Mean intervals from PGF,a injection to the occurrence of the LH surge were similar (P>O.O5) in the three treatment groups (Table 1). Only two (29%) cows in Group I, five (71%) in Group II and four (57%) in Group III had detectable LH surges. Table 1.
Interval (hours) from Lutalvse iniection to LH surge
Treatment
Mean
SEM
FSH (control)
58.0
5.9
FSH + GnRH
58.0
2.6
FSH in osmotic pump
64.6
2.4
Figure 1 depicts mean serum LH concentrations on Day 5 at 2-h intervals starting at 6 h before and ending at 6 h after the LH surge in cows in which the LH surge was detected. The LH surge in this study was defined as LH values two standard deviations above the mean LH concentration at 0800 h on Day In those cows in which an LH surge was detected, the 1. magnitudes of the peak values (34.1 f 17.2, 35.9 -+ 13.5 and 37.7 -+ 11.5 for Treatments I, II and III, respectively) were similar (P>O.O5). These results indicate that neither the
FEBRUARY 1991 VOL. 35 NO. 2
463
THERIOGENOLOGY injection of GnRH in Group II, nor the lower dosage of FSH, nor the manner in which it was administered in Group III during the first five days of superovulation affected the magnitude of the LH surge. These findings are similar to those of Kazmer et al. (12), and suggest that there is a threshold concentration of LH that is necessary to bring about ovulation and that this threshold is not the manner of administration of FSH. The GnRH also did not alter the profile of the preovulatory LH surge (Figure 1).
LH (ng/ml) 40
-6
-4
-2
0
2
+
FSH and QnRH
X
FSH in pump
4
6
Time Relative to LH Peak (h) Figure 1. Mean serum LH concentrations after the LH surge.
six h before to six h
The mean number of CL palpated and of embryos recovered from those in which the LH surge was detected and from those in which it was not detected are shown in Table 2. There was no difference (P>O.O5) in the number of ova or developing embryos recovered between the group of cows in which Lh surges were detected and those in which surges were not detected.
464
FEBRUARY 1991 VOL. 35 NO. 2
However, cows in which LH surges were detected tended to have more (P
Superovulatory ce
responses
in cows with detected and
petected
Undetected
Treatment
CL
Mea
FSH (control)
17.5
6.0
8.6
3.8
FSH + GnRH
14.8
2.7
13.0
4.2
8.8
2.4
6.0
2.8
9.
2.1
FSH in osmotic pump Class
Ova
.3 FSH
14.5
6.0
6.8
3.8
FSH + GnRH
13.2
2.7
10.0
4.3
7.0
2.5
5.0
2.9
FSH in osmotic pump "P
M
Class mean . . compared to cows with an undetected
7.3 LH surge
2.1
Plasma progesterone concentration was highly correlated with the number of CL palpated (r=0.92; PxO.01) and with the number of ova and/or embryos recovered (r=0.88; P
FEBRUARY 1991 VOL. 35 NO. 2
466
THERIOGENOLOGY Table 3. No. of cows
Accuracy of predicting the number of ova by nroaesterone concentrations
No. of Ova
6
0
Proaesterone concentration (na/ml) Selected No. of cows in Mean SEW Ranae selected ranue 1.78
0.33
0.5 to 1.9
5 (83%)
4
1 to 2
2.15
0.15
2.0 to 2.6
4 (100)
4
3 to 4
3.30
0.48
2.7 to 3.9
2 (50)
5 to 10
5.06
0.16
4.0 to 5.6
15 (88)
6.69
0.24
>5.7
10 (91)
17
>lO
11 Total 42
Total 36 186%)
The data from our study demonstrate that the preovulatory cows dairy were similar LH profiles of superovulated of the method of superovulation. Cows that regardless experienced an LH surge 48 to 72 h after PGF,a administration had a higher number of CL that subsequently developed than cows that did not have a LH surge 48 to 72 h after PGF,a. This could explain why the addition of GnRH treatment to superovulation procedures is efficacious. Furthermore, it is possible to predict the number of developing embryos via Further research is warranted progesterone concentrations. to develop a reliable method for predicting the number of ova present in commercial herds before superovulated cows are flushed. REFERENCES 1.
Humbolt, P. and Thibier, M. Effect of gonadotropin releasing hormone (GnRB) treatment during the midluteal phase in repeat breeder cows. Theriogenology &&:375-378 (1981).
2.
Nash, J. G., Jr., Ball, L. and Olson, J. D. Effects of reproductive performance on administration of GnRH to early postpartum dairy cows. J. Anim. Sci. =:1017-1021 (1980).
3.
Wubishet, A., Graves, C. N., Spahr, S. L., Kesler, D. J. and Favero, R. J. Effects of GnRH treatment on superovulatory responses of dairy COWS. Theriogenology 25~423-427 (1986).
456
FEBRUARY 1BBl VOL. 35 NO. 2
THERIOGENOLOGY
4.
Godke, R. A., Bercovitz, A. B. and Lambeth, V. A. Continuous infusion methods for superovulating beef and dairy cattle. The Louisiana Cattleman =:4 and 6 (1980).
5.
Wubishet, A., Graves, C. N., Spahr, S. L. and Kesler, D. J. Continuous subcutaneous infusion of follicle stimulating hormone as a method of superovulating dairy cows. Theriogenology =:809-811 (1986).
6.
Barnes, M. A., Castellano, A. M., Kazmer, G. W., Wade, R. J. and Halman, R. D. Effect of exogenous hormone release in dairy cows. Theriogenology =:311-323 (1982).
7.
Niswender, G. D., Reichert, L. E., Jr., Midgley, A. R., Jr. and Nalbandov, A. V. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology =:1166-1173 (1969).
8.
Troxel, T. R., Kesler, D. J., Nobel, R. C. and Carlin S. E. Ovulation and reproductive hormones following steroid pretreatment, calf removal and GnRH in postpartum suckled beef cows. J. Anim. Sci. =:652-659 (1980).
9.
Wiseman, B. S., Vincent, D. L., Thornford, P. J., Scheffrahn, N. S., Sargent, G. F. and Kesler, D. J. Changes in porcine, ovine, bovine and equine blood progesterone concentrations between collection and centrifugation. Anim. Reprod. Sci. 5:157-165 (1983).
10.
Gill, J. L. and Hafs, H. D. Analysis of repeated measurements on animals. J. Anim. Sci. =:331-336.
11.
Steel, R. G. D. and Torrie, J. H. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, NY. 1960.
12.
Kazmer, G. W., Barnes, M. A., Halman, R. D. and Dickey, J. F. Exogenous hormone response and fertility in dairy heifers after treatment with lutalyse and GnRH. Theriogenology =:575-585 (1981).
13.
Watson, E. D. and Munro, C. C. A reassessment of the technique of rectal palpation of corpora lutea in cows. Brit. Vet. J. =:555-560 (1980).
14.
Vaca, L. A., Galina, C., Fernandez-Baca, S., Escobar, J. and Ramirez, B. Progesterone levels and relationship with diagnosis of corpus luteum by rectal palpation during the estrous cycle in Zebu cows. Theriogenology x:67-76 (1983).
15.
Boyd, H. and Munro, C. D. Progesterone assays and rectal palpation in preservice management of a dairy herd. Vet. Rec. u:341-343 (1979).
FEBRUARY 1991 VOL. 35 NO. 2
457
PREOWLATORY LH PROFILES OF SUPEROWLATED COWS AND PROGESTERONE CONCENTRATIONS AT EMBRYO RECOVERY A. Wubishet', D.J. Kesler, C.N. Graves, S.L. Spahr and R.J. Favero Department of Animal Sciences University of Illinois Urbana, IL 61801 Received for publication: November 3, 1988 Accepted: November 9, 1990 ABSTRACT Forty-two Holstein cows were randomly assigned to three superovulatory treatment groups of 14 cows each. Cows in Group I received follicle stimulating hormone (FSH; 50 mg i.m.); those in Group II received FSH (50 mg i.m.) along with GnRH (250 ug in 2 % carboxymethylcellulose s.c.) on the day of estrus; and cows in Group III were infused FSH (49 mg) via osmotic pump implants. FSH was administered over a 5-d period for cows in Groups I and II (twice daily in declining doses). Cows in Group III received FSH over a 7-d period (constantly at a rate of 7 mg/day). All cows received 25 mg PGF,a (prostaglandin F,a) 48 hours after initiation of the FSH treatment. Blood samples were collected from seven cows from each group at 2 hour intervals on the fifth day of superovulation for serum luteinizing hormone (LH) concentration analysis by radioimmunoassay, and blood samples were collected from all cows on the day of embryo recovery for plasma progesterone determination. The LH profile was not altered (P~0.05) by either GnRH administration or by the constant infusion of FSH as compared to FSH treatment alone. Plasma progesterone concentrations were highly correlated with the number of corpora lutea (CL) palpated (r=0.92; PcO.01) and with the number of ova and/or embryos recovered (r=0.88; PcO.01). The accuracy of predicting the number of recoverable ova and/or embryos by the concentration of plasma progesterone was 86%. Key words:
superovulation, LH surge, progesterone concentrations, recoverable embryos
1Present address: University of California-Extension, 777 E. Rilato Ave., San Bernardino, CA 92415
FEBRUARY 1991 VOL. 35 NO. 2
461
THERIOGENOLOGY
INTRODUCTION It has been reported (l-3) that gonadotropin releasing hormone (GnRH) treatment improves fertilization rates in cattle. It has also been shown that continuous infusion of FSH can bring about superovulation (4,5), but lower dosages of FSH result in a lower number of palpable corpora lutea (CL) and recoverable embryos (6). This study was undertaken to investigate if 1) multiple intramuscular injections of FSH alone, 2) multiple FSH injections along with the administration of GnRH on the day of expected estrus, or 3) the continuous infusion of FSH would affect the LH response. An additional objective of the study was to determine the relationship between concentration of progesterone in the plasma of superovulated dairy cows and the number of both palpable CL and recoverable embryos. MATERIALS AND METHODS Forty-two Holstein cows were randomly assigned to three Cows in Group I superovulatory treatments of 14 cows each. were administered FSH (FSH-P, 50 mg i.m., Burns-Biotech, Omaha, NE) twice daily in declining doses over a 5-d period. Cows in Group II were superovulated in the same manner as those in Group I, except they also received GnRH (250 ug s-c., Park, KS) Cystorelin, CEVA Laboratories Inc., Overland dissolved in 2% carboxymethylcellulose at 1400 h (54 h after prostaglandin F,a; PGF,a) on the day of expected estrus. cows in Group III received FSH in osmotic pumps (Alza Corporation, Palo Alto, CA) implanted under the skin anterio-dorsal to the The osmotic pumps delivered FSH at the rate of 7 scapula. mg/day and were left in situ for 7-d. All cows were treated with PGF,a (25 mg, Lutalyse, Upjohn Co., Kalamazoo, MI) 48 h after initiation of the FSH treatment. On Day 5, 20 ml of blood were collected at 2-h intervals for 24 h from the jugular vein via indwelling 14-g, 13.3-cm Angiocath catheter The catheters were flushed with 5 (Desert Co., sandy, UT). to 10 ml of concentrated sucrose solution to minimize injections blockage. When sampling and superovulatory coincided, samples were collected prior to injection. On the day of embryo recovery (8 d after insemination), 15 ml of blood were collected from all cows in each group into syringes containing 2 ml of 5% sodium citrate in saline. All blood samples were transferred into centrifuge tubes and cooled on ice immediately after collection and stored overnight at 4°C. All samples were processed to yield serum Serum and plasma samples were or plasma the following day. aliquoted into appropriately labeled 12 x 75-mm tubes and frozen at -20°C until analyzed. The LH concentration antibody radioimmunoassay
452
in serum was quantified by double according to procedures reported by
FEBRUARY 1991 VOL. 35 NO. 2
THERIOGENOLOGY Niswender et al. (7), except I251was used as the radioactive Anti-LH (B-225, first antibody), purified bovine LH label. (LER-1072) and NIH-LH-B10 (standard) were obtained from Drs. G.D. Niswender, L.E. Reichert, and the National Institute of Arthritis, Metabolism and Digestive Diseases, respectively. The second antibody, obtained from Dr. D.J. Kesler, was antirabbit gamma globulin generated in sheep at the University of Illinois at Urbana. The assay was validated in our laboratory as reported by Troxel et al. (8). Fifty to 100 ul of serum were collected in duplicate by an automatic pipette and The intra-assay coefficient of analyzed in one assay. variation was 6.5%. Plasma progesterone concentrations were determined by procedures describes and radioimmunoassay according to validated by Troxel et al. (8) and Wiseman et al. (9). Two assays were conducted on 50 to 100 ul of duplicate plasma samples. Intra- and inter-assay coefficients of variation were 5.2 and 14.9%, respectively. The LH data were analyzed by analyzed by analysis of variance as split-plot design as described by Gill and Hafs (10). Plasma progesterone data were analyzed by analysis of variance for a completely randomized design (11). RESULTS AND DISCUSSION Mean intervals from PGF,a injection to the occurrence of the LH surge were similar (P>O.O5) in the three treatment groups (Table 1). Only two (29%) cows in Group I, five (71%) in Group II and four (57%) in Group III had detectable LH surges. Table 1.
Interval (hours) from Lutalvse iniection to LH surge
Treatment
Mean
SEM
FSH (control)
58.0
5.9
FSH + GnRH
58.0
2.6
FSH in osmotic pump
64.6
2.4
Figure 1 depicts mean serum LH concentrations on Day 5 at 2-h intervals starting at 6 h before and ending at 6 h after the LH surge in cows in which the LH surge was detected. The LH surge in this study was defined as LH values two standard deviations above the mean LH concentration at 0800 h on Day In those cows in which an LH surge was detected, the 1. magnitudes of the peak values (34.1 f 17.2, 35.9 -+ 13.5 and 37.7 -+ 11.5 for Treatments I, II and III, respectively) were similar (P>O.O5). These results indicate that neither the
FEBRUARY 1991 VOL. 35 NO. 2
463
THERIOGENOLOGY injection of GnRH in Group II, nor the lower dosage of FSH, nor the manner in which it was administered in Group III during the first five days of superovulation affected the magnitude of the LH surge. These findings are similar to those of Kazmer et al. (12), and suggest that there is a threshold concentration of LH that is necessary to bring about ovulation and that this threshold is not the manner of administration of FSH. The GnRH also did not alter the profile of the preovulatory LH surge (Figure 1).
LH (ng/ml) 40
-6
-4
-2
0
2
+
FSH and QnRH
X
FSH in pump
4
6
Time Relative to LH Peak (h) Figure 1. Mean serum LH concentrations after the LH surge.
six h before to six h
The mean number of CL palpated and of embryos recovered from those in which the LH surge was detected and from those in which it was not detected are shown in Table 2. There was no difference (P>O.O5) in the number of ova or developing embryos recovered between the group of cows in which Lh surges were detected and those in which surges were not detected.
464
FEBRUARY 1991 VOL. 35 NO. 2
However, cows in which LH surges were detected tended to have more (P
Superovulatory ce
responses
in cows with detected and
petected
Undetected
Treatment
CL
Mea
FSH (control)
17.5
6.0
8.6
3.8
FSH + GnRH
14.8
2.7
13.0
4.2
8.8
2.4
6.0
2.8
9.
2.1
FSH in osmotic pump Class
Ova
.3 FSH
14.5
6.0
6.8
3.8
FSH + GnRH
13.2
2.7
10.0
4.3
7.0
2.5
5.0
2.9
FSH in osmotic pump "P
M
Class mean . . compared to cows with an undetected
7.3 LH surge
2.1
Plasma progesterone concentration was highly correlated with the number of CL palpated (r=0.92; PxO.01) and with the number of ova and/or embryos recovered (r=0.88; P
FEBRUARY 1991 VOL. 35 NO. 2
466
THERIOGENOLOGY Table 3. No. of cows
Accuracy of predicting the number of ova by nroaesterone concentrations
No. of Ova
6
0
Proaesterone concentration (na/ml) Selected No. of cows in Mean SEW Ranae selected ranue 1.78
0.33
0.5 to 1.9
5 (83%)
4
1 to 2
2.15
0.15
2.0 to 2.6
4 (100)
4
3 to 4
3.30
0.48
2.7 to 3.9
2 (50)
5 to 10
5.06
0.16
4.0 to 5.6
15 (88)
6.69
0.24
>5.7
10 (91)
17
>lO
11 Total 42
Total 36 186%)
The data from our study demonstrate that the preovulatory cows dairy were similar LH profiles of superovulated of the method of superovulation. Cows that regardless experienced an LH surge 48 to 72 h after PGF,a administration had a higher number of CL that subsequently developed than cows that did not have a LH surge 48 to 72 h after PGF,a. This could explain why the addition of GnRH treatment to superovulation procedures is efficacious. Furthermore, it is possible to predict the number of developing embryos via Further research is warranted progesterone concentrations. to develop a reliable method for predicting the number of ova present in commercial herds before superovulated cows are flushed. REFERENCES 1.
Humbolt, P. and Thibier, M. Effect of gonadotropin releasing hormone (GnRB) treatment during the midluteal phase in repeat breeder cows. Theriogenology &&:375-378 (1981).
2.
Nash, J. G., Jr., Ball, L. and Olson, J. D. Effects of reproductive performance on administration of GnRH to early postpartum dairy cows. J. Anim. Sci. =:1017-1021 (1980).
3.
Wubishet, A., Graves, C. N., Spahr, S. L., Kesler, D. J. and Favero, R. J. Effects of GnRH treatment on superovulatory responses of dairy COWS. Theriogenology 25~423-427 (1986).
456
FEBRUARY 1BBl VOL. 35 NO. 2
THERIOGENOLOGY
4.
Godke, R. A., Bercovitz, A. B. and Lambeth, V. A. Continuous infusion methods for superovulating beef and dairy cattle. The Louisiana Cattleman =:4 and 6 (1980).
5.
Wubishet, A., Graves, C. N., Spahr, S. L. and Kesler, D. J. Continuous subcutaneous infusion of follicle stimulating hormone as a method of superovulating dairy cows. Theriogenology =:809-811 (1986).
6.
Barnes, M. A., Castellano, A. M., Kazmer, G. W., Wade, R. J. and Halman, R. D. Effect of exogenous hormone release in dairy cows. Theriogenology =:311-323 (1982).
7.
Niswender, G. D., Reichert, L. E., Jr., Midgley, A. R., Jr. and Nalbandov, A. V. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology =:1166-1173 (1969).
8.
Troxel, T. R., Kesler, D. J., Nobel, R. C. and Carlin S. E. Ovulation and reproductive hormones following steroid pretreatment, calf removal and GnRH in postpartum suckled beef cows. J. Anim. Sci. =:652-659 (1980).
9.
Wiseman, B. S., Vincent, D. L., Thornford, P. J., Scheffrahn, N. S., Sargent, G. F. and Kesler, D. J. Changes in porcine, ovine, bovine and equine blood progesterone concentrations between collection and centrifugation. Anim. Reprod. Sci. 5:157-165 (1983).
10.
Gill, J. L. and Hafs, H. D. Analysis of repeated measurements on animals. J. Anim. Sci. =:331-336.
11.
Steel, R. G. D. and Torrie, J. H. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, NY. 1960.
12.
Kazmer, G. W., Barnes, M. A., Halman, R. D. and Dickey, J. F. Exogenous hormone response and fertility in dairy heifers after treatment with lutalyse and GnRH. Theriogenology =:575-585 (1981).
13.
Watson, E. D. and Munro, C. C. A reassessment of the technique of rectal palpation of corpora lutea in cows. Brit. Vet. J. =:555-560 (1980).
14.
Vaca, L. A., Galina, C., Fernandez-Baca, S., Escobar, J. and Ramirez, B. Progesterone levels and relationship with diagnosis of corpus luteum by rectal palpation during the estrous cycle in Zebu cows. Theriogenology x:67-76 (1983).
15.
Boyd, H. and Munro, C. D. Progesterone assays and rectal palpation in preservice management of a dairy herd. Vet. Rec. u:341-343 (1979).
FEBRUARY 1991 VOL. 35 NO. 2
457