- Email: [email protected]
Incidence of delayed ovulation in Holstein heifers and its effects on fertility and early luteal function
Theriogenology40:1073-1081,1993
INCIDENCE OF DELAYED OVULATION IN HOLSTEIN HEIFERS AND ITS EFFECTS ON FERTILITY AND EARLY LUTEAL FUNCTION J. Hernandez-Cerdn,
L. Zarco and V. Lima-Tamayo
Departamento de Reproduccicjn Facultad de Medicina Veterinaria y Zootecnia Universidad National Autonoma de Mexico Apartado Postal 22-256, Mexico, D. F. 14000, Mexico Received for publication: flovember 3, 1992 Accepted: JuZ;y 23, 1993 ABSTRACT In the first of 3 experiments 134 first-service and 108 repeat-breeder Holstein heifers were palpated at 12-hour intervals starting 24 hours after insemination to compare the incidence of delayed ovulation in the 2 groups. Delayed ovulation was defined as failure to ovulate within the first 24 hours after insemination. Ovulation occurred within 24 hours post insemination in 92.1% of the animals and was delayed in 7.9% of the cases, with no differences between first-service and repeat-breeder heifers, indicating that the subfertility of the repeat-breeder animals was not due to delayed ovulation. The duration of the delay was at most 12 hours since all the animals had ovulated by 36 hours post insemination. Conception rate of the 19 animals with delayed ovulation (42.1%) was not different (P>O.O5) from that of the 223 heifers that ovulated on time (44.8%). In a second experiment, no differences were detected between 15 heifers with delayed ovulation and 15 animals that ovulated on time with respect to their progesterone concentrations during the first 8 days post insemination, indicating that delayed ovulation is not associated with delayed luteinization or subnormal early luteal function. In the third experiment, the conception rate of 126 repeat-breeder heifers that were treated with hCG at the time of insemination was 26.7%; the conception rate of 101 repeat-breeder heifers that were inseminated twice, at 12 and 24 hours after the onset of es&us, was 34.6%; and the conception rate of 105 repeat-breeder heifers which were not treated with hCG and which were inseminated only once was 30.5% (P>O.OS) It is concluded that delayed ovulation is not an important cause of infertility and does not constitute an impo~nt com~nent of the repeat-breeding s~drome in Holstein heifers. Key words: delayed ovulation, repeat breeder, heifer, progesterone,
infertility
INTRODUCTION In Bos taurus females ovulation normally takes place between 26 and 36 hours after the onset of estrus, with few individu~s ovulating outside this range (1). It is generally considered that animals that have not ovulated by 36 hours after the onset of estrus, or by 24 hours after insemination, have delayed ovulation (l-4). Although some authors have reported very low incidences of delayed ovulation in dairy cattle (l), others have found incidences ranging from 18 % (35) to more than 30% (2,6). Some authors have reported low conception rates in cows with delayed ovulation (2,5), and many consider delayed ovulation as a significant factor in the repeatbreeding syndrome (2,4,5,7,8). It has been suggested that in animals with delayed ovulation, the application of artificial insemination 12 hours after the onset of es&us
Copyright 0 1993 Bu~e~orth-Heinemann
1074
Theriogenology
could result in a large interval between in~m~ation and ovulation, resulting in sperm aging and a lack of fertilizing ability (2,5). Other authors have suggested that the hormonal asynchrony associated with delayed ovulation results in delayed or subnormal luteal function (g-12), which can in turn result in abnormal embryo development (10) or in alterations of the uterine environment that lead to early embryonic death (9,11,-13). To improve fertility in repeat-breeder cows through the correction of delayed ovulation or subnormal luteal function, some authors have resorted to the administration of ovulation-inducing hormones such as gonadotropin releasing hormone (GnRH; 4,7,8,12,14,15) or human chorionic gonadotropin (hCG; 4,14,16). Others have reported the use of double insemination in repeat-breeder animals (12,17) or in animals with’diagnosed delayed ovulation (2,5). The degree of success with these approaches has been variable, and no consensus exists on their effectiveness. All the information presented above has been obtained from experiments with adult cows. There is almost no info~ation about the incidence. effects on fertility and treatment of delayed ovulation in dairy heifers. There is only’one experiment which indirectly suggests that delayed ovulation is present in repeat-breeder heifers, since the heifers had a preovulatory release of LH that was delayed with respect to the onset of estrus (18). Results from adult cows should not be extrapolated to heifers, since lactation imposes a burden on the energy balance of the cow that affects the neuroendocrine control of ovarian activity (19-22), and the heifer is not exposed to such burden. As an example of the potential impact of this difference between cows and heifers, it can be mentioned that other ovulation disorders such as ovarian cysts are characteristic of lactating cows (23) and are strongly associated with ketosis (24,25), a metabolic disorder of cows in strong negative energy balance due to lactation. The objectives of this study were to determine the incidence of delayed ovulation and its importance as part of the repeat-brewing syndrome in Holstein heifers, to compare the progesterone profdes of heifers with delayed or normal ovulation during the first 7 days post-insemination, and to evaluate the use of hCG or reinsemination to improve fertility in repeat-breeder heifers. MATERIALS AND METHODS This study was carried out on a large dairy cattle farm located in the Mexican state of Hidalgo, on the central plateau of the country. The animals were kept stabled and were fed according to NRC requirements (26), using mainly alfalfa hay, corn silage and concentrates. Experiment I We used 108 repeat-breeder and 134 first-service Holstein heifers. To be classified as a repeat breeder, the heifer had to have been unsuccessfully in~minated at Least 3 times, should have estrous cycles of normal length (19 to 22 days), and should not have genital abnormalities. During the study, estrus detection was carried out by continuous observation of homosexual activity 24 hours a day, and artificial insemination was carried out 12 hours after the onset of standing e&us. All animals were palpated per rectum at the time of artificial insemination to determine the location of the preovulatory follicle. They were palpated again 24 hours after in~mination (36 hours after estrns onset) to
Theriogenology
1075
determine if ovulation had taken place. Heifers that had not ovulated at that time were classified as delayed ovulators and continued to be palpated every 12 hours until the occurrence of ovulation was determined. A comparison was made between the incidence of delayed ovulation in first-service heifers and in repeat-breeder animals. Also, in order to determine the effects of delayed ovulation on fertility, the conception rate of the animals with delayed ovulation was compared with that of the animals that ovulated at a normal time. Both comparisons were done using Chi-square analysis. Experiment II To determine if the animals with delayed ovulation had an endocrine or follicular disturbance that would result in abnormal function of the corpus luteum during the first 8 days post insemination, blood was collected every day for 8 days from 15 animals in which ovulation had not taken place by 24 hours after insemination and 15 animals in which it had occurred on time. The blood was collected into heparinized tubes and was centrifuged within 15 minutes to separate the plasma, which was kept frozen until being assayed for progesterone using a solid-phase radioimmunoassay (27). The concentrations of progesterone were compared by split-plot analysis of variance for repeated measures, with the type of ovulation (normal or delayed) as the whole plot factor, the day post insemination (1 to 8 days) as sub-plot factor, and the subject nested within type of ovulation as replication. Experiment III Three groups of repeat-breeder heifers were randomly formed to determine if either hCG administration or reinsemination imnroved fertilitv in such animals. The hCG group consisted of 126 heifers which were inseminated’12 hours after the onset of estrus and which were intramuscularly injected with 5,000 IU hCG (Gonaforte, Parfarm, Mexico) at the time of insemination. The reinseminated group consisted of 101 heifers that were inseminated twice, 12 and 24 hours after the onset of estrus. The control group consisted of 105 heifers not treated with hCG and inseminated only once, 12 hours after the onset of estrus and. The conception rates of the 3 groups were compared by X’ test. RESULTS Experiment I All the heifers had a large preovulatory follicle at the time of artificial insemination. As expected, the conception rate at the service under study was lower IP
Theriogenology
1076
Table 1.
Incidence of delayed ovulationa in repeat-breeder heifers
and first-service Holstein
Type of heifer
Time of ovulation
Repeat-breede? n=lOS
First-servicec n=134
Within 24 hours post insemination 24 to 36 hours post insemination More than 36 hours post insemination
10
(9.3%)
9
(6.7%)
0
(0.0%)
0
(0.0%)
Row values did not differ (P > 0.05). a Estrus detection was carried out by continuous observation of homosexual activity 24 hours a day, and artificial insemination was carried out 12 hours after the onset of standing estrus. Heifers that had not ovulated by 24 hours after insemination were classitied as delayed ovulators. Normal ovulation ocurred during the first 24 hours post insemination. b Repeat-breeder heifers were animals which had been unsuccesfully inseminated at least 3 times, had estrous cycles of normal length (19 to 22 days), and did not have genital abnormalities. ’ First-service heifers were inseminated during their first estrus after reaching 15 months of age and 320 kg of body weight.
Table 2. Conception rate” of heifers with delayed and normal ovulation
Type of ovulationb
Total number of heifers
Pregnant
Nonpregnant
Conception rate
Delayed
19
8
11
42.1%
Normal
223
100
123
44.8%
Total
242
108
134
44.6%
‘Conception rates did not differ (P > 0.05) b Estrus detection was carried out by continuous observation of homosexual activity 24 hours a day, and artificial insemination was carried out 12 hours after the onset of standing estrus. Heifers that had not ovulated by 24 hours after insemination were classified as delayed ovulators. Normal ovulation ocurred during the first 24 hours post insemination.
1077
Theriogenology
Experiment II Table 3 shows that the progesterone concentrations of 15 animals with delayed ovulation and of 15 heifers with normal ovulation did not differ between the 2 groups of animals in any of the first 8 days post insemination (P> 0.05). Table 3.
Progesterone concentrations in plasma (nglml) during the first 8 days post insemination in Holstein heifers with delayed and normal ovulation Type of ovulation’
Day post insemination
Delayed (n = 15)
; 3 4 5 6 7 8
Normal (n = 15)
Mean
Mean
Pooled SEM
0.44 0.40 0.36 0.57 0.77 1.13 1.69 2.09
0.29
0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17
0.37 0.35 0.58 0.83 1.47 1.87 2.28
Row values did not differ (p > 0.05). ’ Estrus detection was carried out by continuous observation of homosexual activity 24 hours a day, and artificial insemination was carried out 12 hours after the onset of standing es&us. Heifers that had not ovulated by 24 hours after insemination were classified as delayed ovulators. Ovulations ocurring by 24 hours post insemination were classified as normal.
Experiment III Neither insemination
hCG treatment at the time of insemination nor use of double improved the conception rate of repeat-breeder heifers (Table 4). DISCUSSION
The overall incidence of delayed ovulation found in dairy heifers in this study (8%) was much lower than the incidences reported by other authors in adult dairy cows (2,3,5,6). Furthermore, we did not find a single case of ovulation taking place more than 36 hours after artificial insemination, whereas Bostedt (2) found that 9.8% of the cows had not yet ovulated 48 hours after insemination, and Van Rensburg and De Vos (5) found that in some cows ovulation was delayed for more than 72 hours. This difference between heifers and cows may be even greater than it appears, since in the present study, estrus detection was carried out 24 hours a day, and thus artificial insemination was performed exactly 12 hours after the onset of estrus;
1078
Theriogenology
whereas in the other studies, estrus detection was carried out by the farmers following traditional practices; thus, insemination could have been performed in some cows up to 24 hours after the actual onset of estrus. It is likely that the higher incidence of delayed ovulation and the longer intervals to ovulation in adult cows as compared with heifers are due to metabolic and neuroendocrine alterations associated with lactation (19-25). Table 4.
Conception rates of repeat-breedela double insemination
Holstein heifers treated with hCG or
Treatmentb
Total number of heifers
hCG
126
33
93
26.7%
Double AI
101
35
66
34.6%
Control
105
32
73
30.5%
Pregnant
Nonpregnant
Conception rate
Conception rates did not differ (P > 0.05). a Repeat-breeder heifers were animals which had been unsuccesfully inseminated at least 3 times, had estrous cycles of normal length (19 to 22 days), and did not have genital abnormalities. b hCG = Intramuscularly injected with 5,000 IU hCG at the time of insemination, 12 hours after the onset of es&us; double AI = inseminated twice, 12 and 24 hours after the onset of estrus; control = not treated with hCG and inseminated only once, 12 hours after the onset of estrus.
The results of this experiment suggest that in heifers delayed ovulation is not associated with infertility, since the incidence of the condition was similar in firstservice and repeat-breeder animals, and the conception rate of heifers with delayed ovulation was not different from that in heifers ovulating at a normal time. In contrast, Bostedt (2) reported that the conception rate was reduced from 63.1% in cows that ovulated on time to 9.5% in cows with delayed ovulation. Van Rensburg and De Vos (5) reported 0% conception in cows with delayed ovulation that were inseminated only once. It is possible that the different effects of delayed ovulation on the fertility of heifers and cows is due to the much shorter delay that occurs in cases of delayed ovulation in heifers. Although in our present study palpations were performed only every 12 hours, and thus no precise estimate of the magnitude of the delay exists, the interval between insemination and ovulation in the delayed ovulators ranged between 24 and 36 hours, and was never longer than 36 hours. Erb et al. (9), working with adult cows, found that the conception rate of cows ovulating 2 days after the onset of estrus (42%) was similar to that of cows ovulating on time (40%), whereas the conception rate of cows in which ovulation was delayed more than 2 days was 0%. Some authors have suggested that delayed ovulation may be associated with delayed luteinization or subnormal luteal function during the early luteal phase, thus leading to early embryonic death (9-13). The results of the present study do not support this concept, since the conception rates of delayed ovulators were similar to those of animals ovulating on time. In addition, progesterone profiles during the first 8 days post insemination were essentially identical in animals with delayed ovulation
Theriogenology
and in normal ovulators. Although some authors have found a relation between early luteal function and fertility in cows (9-12), we have previously reported that progesterone profiles during the first 8 days post insemination are not different between first-service and repeat-breeder heifers or between pregnant and non-pregnant heifers (28). Also, Linares et al. (29) found no significant relationship between embryonic morphology and progesterone levels during the first 7 days post insemination in dairy heifers. Subnormal luteal function could be more common in lactating cows than in heifers, since luteal function is affected by the energy balance of the animal (19). Some authors have suggested that the administration of hCG (4) or GnRH (4,7,8,12) may improve the conception rate of repeat-breeder cows through the prevention of delayed ovulation. Lee et al. (7), however, found that the administration of GnRH significantly improved the conception rates of first-service and repeat breeder cows that were producing more than 7000 kg of milk per lactation, whereas no improvement occurred in heifers. They suggested that the effect of GnRH in cows could result from the correction of delayed ovulation, and that the lack of effect in heifers could be due to the fact that they were not lactating. The results of our present study support this concept, since the administration of hCG to repeat-breeder heifers at the time of insemination did not improve conception rates. This outcome could be expected if one considers the low incidence and short duration of delayed ovulation in repeat-breeder heifers, as well as the lack of detrimental effect of delayed ovulation on luteal function. These same factors could explain the lack of effect of reinsemination in heifers, in contrast with cows, in which Bostedt (2) reported that the conception rate of delayed ovulators improved from 9.5 % in those inseminated only once to 52.9% in animals that were reinseminated. Van Rensburg and De Vos (5) reported that conception rate in cows with delayed ovulation increased from 0% to 62.7% when reinsemination was performed. It is concluded that delayed ovulation is not a major cause of infertility in dairy heifers, and is not a significant component of the repeat-breeder syndrome in such animals. Thus, treatments or management practices oriented toward preventing delayed ovulation in heifers are not likely to be successful. REFERENCES Zemjanis, R. Repeat-breeding or conception failure in cattle. In: Morrow, D. A. (ed.), Current Therapy in Theriogenology. W. B. Saunders Co., Philadelphia, 1980. pp. 205-213. Bostedt, H. Delayed ovulation as a cause of sterility in the AI of cattle. 8th. Intl. Congress on Anim. Reprod. pp. 552-554 (1976). Leidl, W., Bostedt, H., Lamprecht, W., Prinzen, R. and Went, V. The influence of GnRH and hCG on the time of ovulation in cows artificially inseminated. Tieraerztl Umsch, x:546-555 (1979). Nakao, T., Shirakawa, J., Tsurubayashi, M., Ohboshi, K., Abe, T., Sawamukai, Y., Saga, N., Tsunoda, N. and Kawata, K. A preliminary report on the treatment of ovulation failure in cows with gonadotropin-releasing hormone analog or human chorionic gonadotropin combined with insemination. Anim. Reprod. Sci. 2:489-495 (1984).
T~ef~ogenoiogy
1080 5.
Van Rensburg, S.W.J. and De Vos, W.H. Ovulatory Onderstepoort J. Vet. Res. 29:55-78 (1962).
failure
in bovines.
6.
Hancock, J.L. The clinical analysis of reproductive @:513-517 (1948).
7.
Lee, C.N., Maurice, E., Ax, R.L., Pennington, J.A., Hoffman, W.F. and Brown, M.D. Efficacy of gonadotropin-releasing hormone administered at the time of artificial insemination of heifers and postpartum and repeat-breeder dairy cows. Am. J. Vet. Res. @:2160-2163 (1983).
8.
Lucy, M.C. and Stevenson, J.S. Gonadotropin-releasing hormone at estrus: luteinizing hormone, estradiol and progesterone during the periestrual and postinsemination periods in dairy cattle. Biol. Reprod. 3.&300-311 (1986).
9.
Erb, R-E., Garverick, H.A., Randell, RD., Brown, B.L. and Callahan, C.J, Profiles of reproductive hormones associated with fertile and nonfertile inseminations of dairy cows. Theriogenology 5:27-41 (1976).
failure in cattle. Vet Rec.
10.
Maurer, R.R. and Echternkamp, S.E. Hormonal development. The~og~nology u: 1l-22 (1982).
11.
Shelton, K., Gayerie de Abreu, M.F., Hunter, M.G., Parkinson, T. J. and Lamming, G.E. Luteal inadequacy during the early luteal phase of subfertile cows. J. Reprod. Fertil. 90: l-10 (1990).
12.
Stevenson, J.S., Call, E.P. and Scoby, gonadotropin-releasing hormone treatment Dairy Sci. a: 1766-1772 (1990).
13.
Kimura, M., Nakao, T., Moriyoshi, M. and Kawata, K. Luteal phase deficiency as a possible cause of repeat breeding in dairy cows. Br. Vet. J. ,&$:560-566 (1987).
14.
Swanson, L.V. and Young, A.J. Failure of gonadotropin-releasing hormone or human chorionic gonadotropin to enhance the fertility of repeat breeder cows when administered at the time of insemination. Theriogenology $&955-963 (1990).
15.
Bon Durant, R-H., Revah, I., Franti, C., Harman, R.J., Hird, D., Klingborg, D., McCloskey, M., Weaver, L. and Wilgenberg, B. Effect of gonadotropinreleasing hormone on fertility in repeat-breeder California dairy cows. Theriogenology %:365-374 (1991).
16.
Hansel, W., Spalding, R.W., Larson, L.L., Laster, D.B., Wagner, J.F. and Braun, R.K. Influence of human chononic gonadotropin on pregnancy rates in lactatmg dairy and beef cows. J. Dairy Sci. 59:751-759 (1976).
17.
Drost, M. A practice approach to the infertile cow. The Bovine Practitioner 12: 117-121 (1980).
18.
Gustafsson, H., Larsson, K., Kindahl, H. and Madej, A. Sequential endocrine changes and behavior during oestrus and metoestrus in repeat breeder and virgin heifers. Anim. Reprod. Sci. u:261-273 (1986).
asynchrony
and embryonic
R.K. Double insemination and of repeat-breeding dairy cattle. J.
1081
Theriogenology 19.
Villa-Godoy, A., Hughes, T.L., Emery, R.S., Chapin, L.T. and Fogwell, R.L. Association between energy balance and luteal function in lactating dairy cows. J. Dairy Sci. 21:1063-1072 (1988).
20.
Staples, C.R. and Thatcher, J.H. Relationship between ovarian activity and energy status during the early postpartum period of high producing dairy cows. J. Dairy Sci. 73:938-947 (1990).
21.
Lucy, M.C., Staples, C.R., Michel, EM. and Thatcher, W. W. Energy balance and size and number of ovarian follicles detected by ultrasonography in early postpartum dairy cows. J. Dairy Sci. z:473-482 (1991).
22.
Canfield, R.W. and Butler, W.R. Energy balance, first ovulation and the effects of naloxone on LH secretion in early postpartum dairy cows. 5. Anim. Sci. 69: 740-746 (1991).
23.
Arthur, G.H., Noakes, D.E. and Pearson, H. Veterinary Obstetrics. Bailliere Tindall, London, 1982, pp. 295-327.
24.
Andersson, L. and Emanuelson, U.: An epidemiological study hyperketonemia in Swedish dairy cows. Prev. Vet. Med. 3~449-462 (1985).
25.
Andersson, L., Gustafsson, A.H. and Emanuelson, U.: Effect of hyperketonemia and feeding on fertility in dairy cows. Theriogenology 36:521536 (1991).
26.
National Research Council. Nutrient Requirements Academy Press, Washington, 1988.
27.
Pulido, A., Zarco, L., Galina, C.S., Murcia, C., Flores, G. and Posadas, E. Progesterone metabolism during storage of blood samples from Gyr cattle: effects of anticoagulant, time and temperature of incubation. Theriogenology 3:965-975 (1991).
28.
Hernandez, C.J., Zarco, Q.L. and Lima, T.V. Niveles de progesterona plasmatica durante 10s primeros 7 dias posinseminacidn en vaquillas Holstein repetidoras y de primer servicio. Vet. Mex. a:189-192 (1992).
29.
Linares, T., Larsson, K. and Edqvist, L.-E. Plasma progesterone levels from estrus through Day 7 after AI in heifers carrying embryos with normal or deviating morphology. Theriogenology 17: 125-132 (1982).
Reproduction
of Dairy Cattle.
and of
National
INCIDENCE OF DELAYED OVULATION IN HOLSTEIN HEIFERS AND ITS EFFECTS ON FERTILITY AND EARLY LUTEAL FUNCTION J. Hernandez-Cerdn,
L. Zarco and V. Lima-Tamayo
Departamento de Reproduccicjn Facultad de Medicina Veterinaria y Zootecnia Universidad National Autonoma de Mexico Apartado Postal 22-256, Mexico, D. F. 14000, Mexico Received for publication: flovember 3, 1992 Accepted: JuZ;y 23, 1993 ABSTRACT In the first of 3 experiments 134 first-service and 108 repeat-breeder Holstein heifers were palpated at 12-hour intervals starting 24 hours after insemination to compare the incidence of delayed ovulation in the 2 groups. Delayed ovulation was defined as failure to ovulate within the first 24 hours after insemination. Ovulation occurred within 24 hours post insemination in 92.1% of the animals and was delayed in 7.9% of the cases, with no differences between first-service and repeat-breeder heifers, indicating that the subfertility of the repeat-breeder animals was not due to delayed ovulation. The duration of the delay was at most 12 hours since all the animals had ovulated by 36 hours post insemination. Conception rate of the 19 animals with delayed ovulation (42.1%) was not different (P>O.O5) from that of the 223 heifers that ovulated on time (44.8%). In a second experiment, no differences were detected between 15 heifers with delayed ovulation and 15 animals that ovulated on time with respect to their progesterone concentrations during the first 8 days post insemination, indicating that delayed ovulation is not associated with delayed luteinization or subnormal early luteal function. In the third experiment, the conception rate of 126 repeat-breeder heifers that were treated with hCG at the time of insemination was 26.7%; the conception rate of 101 repeat-breeder heifers that were inseminated twice, at 12 and 24 hours after the onset of es&us, was 34.6%; and the conception rate of 105 repeat-breeder heifers which were not treated with hCG and which were inseminated only once was 30.5% (P>O.OS) It is concluded that delayed ovulation is not an important cause of infertility and does not constitute an impo~nt com~nent of the repeat-breeding s~drome in Holstein heifers. Key words: delayed ovulation, repeat breeder, heifer, progesterone,
infertility
INTRODUCTION In Bos taurus females ovulation normally takes place between 26 and 36 hours after the onset of estrus, with few individu~s ovulating outside this range (1). It is generally considered that animals that have not ovulated by 36 hours after the onset of estrus, or by 24 hours after insemination, have delayed ovulation (l-4). Although some authors have reported very low incidences of delayed ovulation in dairy cattle (l), others have found incidences ranging from 18 % (35) to more than 30% (2,6). Some authors have reported low conception rates in cows with delayed ovulation (2,5), and many consider delayed ovulation as a significant factor in the repeatbreeding syndrome (2,4,5,7,8). It has been suggested that in animals with delayed ovulation, the application of artificial insemination 12 hours after the onset of es&us
Copyright 0 1993 Bu~e~orth-Heinemann
1074
Theriogenology
could result in a large interval between in~m~ation and ovulation, resulting in sperm aging and a lack of fertilizing ability (2,5). Other authors have suggested that the hormonal asynchrony associated with delayed ovulation results in delayed or subnormal luteal function (g-12), which can in turn result in abnormal embryo development (10) or in alterations of the uterine environment that lead to early embryonic death (9,11,-13). To improve fertility in repeat-breeder cows through the correction of delayed ovulation or subnormal luteal function, some authors have resorted to the administration of ovulation-inducing hormones such as gonadotropin releasing hormone (GnRH; 4,7,8,12,14,15) or human chorionic gonadotropin (hCG; 4,14,16). Others have reported the use of double insemination in repeat-breeder animals (12,17) or in animals with’diagnosed delayed ovulation (2,5). The degree of success with these approaches has been variable, and no consensus exists on their effectiveness. All the information presented above has been obtained from experiments with adult cows. There is almost no info~ation about the incidence. effects on fertility and treatment of delayed ovulation in dairy heifers. There is only’one experiment which indirectly suggests that delayed ovulation is present in repeat-breeder heifers, since the heifers had a preovulatory release of LH that was delayed with respect to the onset of estrus (18). Results from adult cows should not be extrapolated to heifers, since lactation imposes a burden on the energy balance of the cow that affects the neuroendocrine control of ovarian activity (19-22), and the heifer is not exposed to such burden. As an example of the potential impact of this difference between cows and heifers, it can be mentioned that other ovulation disorders such as ovarian cysts are characteristic of lactating cows (23) and are strongly associated with ketosis (24,25), a metabolic disorder of cows in strong negative energy balance due to lactation. The objectives of this study were to determine the incidence of delayed ovulation and its importance as part of the repeat-brewing syndrome in Holstein heifers, to compare the progesterone profdes of heifers with delayed or normal ovulation during the first 7 days post-insemination, and to evaluate the use of hCG or reinsemination to improve fertility in repeat-breeder heifers. MATERIALS AND METHODS This study was carried out on a large dairy cattle farm located in the Mexican state of Hidalgo, on the central plateau of the country. The animals were kept stabled and were fed according to NRC requirements (26), using mainly alfalfa hay, corn silage and concentrates. Experiment I We used 108 repeat-breeder and 134 first-service Holstein heifers. To be classified as a repeat breeder, the heifer had to have been unsuccessfully in~minated at Least 3 times, should have estrous cycles of normal length (19 to 22 days), and should not have genital abnormalities. During the study, estrus detection was carried out by continuous observation of homosexual activity 24 hours a day, and artificial insemination was carried out 12 hours after the onset of standing e&us. All animals were palpated per rectum at the time of artificial insemination to determine the location of the preovulatory follicle. They were palpated again 24 hours after in~mination (36 hours after estrns onset) to
Theriogenology
1075
determine if ovulation had taken place. Heifers that had not ovulated at that time were classified as delayed ovulators and continued to be palpated every 12 hours until the occurrence of ovulation was determined. A comparison was made between the incidence of delayed ovulation in first-service heifers and in repeat-breeder animals. Also, in order to determine the effects of delayed ovulation on fertility, the conception rate of the animals with delayed ovulation was compared with that of the animals that ovulated at a normal time. Both comparisons were done using Chi-square analysis. Experiment II To determine if the animals with delayed ovulation had an endocrine or follicular disturbance that would result in abnormal function of the corpus luteum during the first 8 days post insemination, blood was collected every day for 8 days from 15 animals in which ovulation had not taken place by 24 hours after insemination and 15 animals in which it had occurred on time. The blood was collected into heparinized tubes and was centrifuged within 15 minutes to separate the plasma, which was kept frozen until being assayed for progesterone using a solid-phase radioimmunoassay (27). The concentrations of progesterone were compared by split-plot analysis of variance for repeated measures, with the type of ovulation (normal or delayed) as the whole plot factor, the day post insemination (1 to 8 days) as sub-plot factor, and the subject nested within type of ovulation as replication. Experiment III Three groups of repeat-breeder heifers were randomly formed to determine if either hCG administration or reinsemination imnroved fertilitv in such animals. The hCG group consisted of 126 heifers which were inseminated’12 hours after the onset of estrus and which were intramuscularly injected with 5,000 IU hCG (Gonaforte, Parfarm, Mexico) at the time of insemination. The reinseminated group consisted of 101 heifers that were inseminated twice, 12 and 24 hours after the onset of estrus. The control group consisted of 105 heifers not treated with hCG and inseminated only once, 12 hours after the onset of estrus and. The conception rates of the 3 groups were compared by X’ test. RESULTS Experiment I All the heifers had a large preovulatory follicle at the time of artificial insemination. As expected, the conception rate at the service under study was lower IP
Theriogenology
1076
Table 1.
Incidence of delayed ovulationa in repeat-breeder heifers
and first-service Holstein
Type of heifer
Time of ovulation
Repeat-breede? n=lOS
First-servicec n=134
Within 24 hours post insemination 24 to 36 hours post insemination More than 36 hours post insemination
10
(9.3%)
9
(6.7%)
0
(0.0%)
0
(0.0%)
Row values did not differ (P > 0.05). a Estrus detection was carried out by continuous observation of homosexual activity 24 hours a day, and artificial insemination was carried out 12 hours after the onset of standing estrus. Heifers that had not ovulated by 24 hours after insemination were classitied as delayed ovulators. Normal ovulation ocurred during the first 24 hours post insemination. b Repeat-breeder heifers were animals which had been unsuccesfully inseminated at least 3 times, had estrous cycles of normal length (19 to 22 days), and did not have genital abnormalities. ’ First-service heifers were inseminated during their first estrus after reaching 15 months of age and 320 kg of body weight.
Table 2. Conception rate” of heifers with delayed and normal ovulation
Type of ovulationb
Total number of heifers
Pregnant
Nonpregnant
Conception rate
Delayed
19
8
11
42.1%
Normal
223
100
123
44.8%
Total
242
108
134
44.6%
‘Conception rates did not differ (P > 0.05) b Estrus detection was carried out by continuous observation of homosexual activity 24 hours a day, and artificial insemination was carried out 12 hours after the onset of standing estrus. Heifers that had not ovulated by 24 hours after insemination were classified as delayed ovulators. Normal ovulation ocurred during the first 24 hours post insemination.
1077
Theriogenology
Experiment II Table 3 shows that the progesterone concentrations of 15 animals with delayed ovulation and of 15 heifers with normal ovulation did not differ between the 2 groups of animals in any of the first 8 days post insemination (P> 0.05). Table 3.
Progesterone concentrations in plasma (nglml) during the first 8 days post insemination in Holstein heifers with delayed and normal ovulation Type of ovulation’
Day post insemination
Delayed (n = 15)
; 3 4 5 6 7 8
Normal (n = 15)
Mean
Mean
Pooled SEM
0.44 0.40 0.36 0.57 0.77 1.13 1.69 2.09
0.29
0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.17
0.37 0.35 0.58 0.83 1.47 1.87 2.28
Row values did not differ (p > 0.05). ’ Estrus detection was carried out by continuous observation of homosexual activity 24 hours a day, and artificial insemination was carried out 12 hours after the onset of standing es&us. Heifers that had not ovulated by 24 hours after insemination were classified as delayed ovulators. Ovulations ocurring by 24 hours post insemination were classified as normal.
Experiment III Neither insemination
hCG treatment at the time of insemination nor use of double improved the conception rate of repeat-breeder heifers (Table 4). DISCUSSION
The overall incidence of delayed ovulation found in dairy heifers in this study (8%) was much lower than the incidences reported by other authors in adult dairy cows (2,3,5,6). Furthermore, we did not find a single case of ovulation taking place more than 36 hours after artificial insemination, whereas Bostedt (2) found that 9.8% of the cows had not yet ovulated 48 hours after insemination, and Van Rensburg and De Vos (5) found that in some cows ovulation was delayed for more than 72 hours. This difference between heifers and cows may be even greater than it appears, since in the present study, estrus detection was carried out 24 hours a day, and thus artificial insemination was performed exactly 12 hours after the onset of estrus;
1078
Theriogenology
whereas in the other studies, estrus detection was carried out by the farmers following traditional practices; thus, insemination could have been performed in some cows up to 24 hours after the actual onset of estrus. It is likely that the higher incidence of delayed ovulation and the longer intervals to ovulation in adult cows as compared with heifers are due to metabolic and neuroendocrine alterations associated with lactation (19-25). Table 4.
Conception rates of repeat-breedela double insemination
Holstein heifers treated with hCG or
Treatmentb
Total number of heifers
hCG
126
33
93
26.7%
Double AI
101
35
66
34.6%
Control
105
32
73
30.5%
Pregnant
Nonpregnant
Conception rate
Conception rates did not differ (P > 0.05). a Repeat-breeder heifers were animals which had been unsuccesfully inseminated at least 3 times, had estrous cycles of normal length (19 to 22 days), and did not have genital abnormalities. b hCG = Intramuscularly injected with 5,000 IU hCG at the time of insemination, 12 hours after the onset of es&us; double AI = inseminated twice, 12 and 24 hours after the onset of estrus; control = not treated with hCG and inseminated only once, 12 hours after the onset of estrus.
The results of this experiment suggest that in heifers delayed ovulation is not associated with infertility, since the incidence of the condition was similar in firstservice and repeat-breeder animals, and the conception rate of heifers with delayed ovulation was not different from that in heifers ovulating at a normal time. In contrast, Bostedt (2) reported that the conception rate was reduced from 63.1% in cows that ovulated on time to 9.5% in cows with delayed ovulation. Van Rensburg and De Vos (5) reported 0% conception in cows with delayed ovulation that were inseminated only once. It is possible that the different effects of delayed ovulation on the fertility of heifers and cows is due to the much shorter delay that occurs in cases of delayed ovulation in heifers. Although in our present study palpations were performed only every 12 hours, and thus no precise estimate of the magnitude of the delay exists, the interval between insemination and ovulation in the delayed ovulators ranged between 24 and 36 hours, and was never longer than 36 hours. Erb et al. (9), working with adult cows, found that the conception rate of cows ovulating 2 days after the onset of estrus (42%) was similar to that of cows ovulating on time (40%), whereas the conception rate of cows in which ovulation was delayed more than 2 days was 0%. Some authors have suggested that delayed ovulation may be associated with delayed luteinization or subnormal luteal function during the early luteal phase, thus leading to early embryonic death (9-13). The results of the present study do not support this concept, since the conception rates of delayed ovulators were similar to those of animals ovulating on time. In addition, progesterone profiles during the first 8 days post insemination were essentially identical in animals with delayed ovulation
Theriogenology
and in normal ovulators. Although some authors have found a relation between early luteal function and fertility in cows (9-12), we have previously reported that progesterone profiles during the first 8 days post insemination are not different between first-service and repeat-breeder heifers or between pregnant and non-pregnant heifers (28). Also, Linares et al. (29) found no significant relationship between embryonic morphology and progesterone levels during the first 7 days post insemination in dairy heifers. Subnormal luteal function could be more common in lactating cows than in heifers, since luteal function is affected by the energy balance of the animal (19). Some authors have suggested that the administration of hCG (4) or GnRH (4,7,8,12) may improve the conception rate of repeat-breeder cows through the prevention of delayed ovulation. Lee et al. (7), however, found that the administration of GnRH significantly improved the conception rates of first-service and repeat breeder cows that were producing more than 7000 kg of milk per lactation, whereas no improvement occurred in heifers. They suggested that the effect of GnRH in cows could result from the correction of delayed ovulation, and that the lack of effect in heifers could be due to the fact that they were not lactating. The results of our present study support this concept, since the administration of hCG to repeat-breeder heifers at the time of insemination did not improve conception rates. This outcome could be expected if one considers the low incidence and short duration of delayed ovulation in repeat-breeder heifers, as well as the lack of detrimental effect of delayed ovulation on luteal function. These same factors could explain the lack of effect of reinsemination in heifers, in contrast with cows, in which Bostedt (2) reported that the conception rate of delayed ovulators improved from 9.5 % in those inseminated only once to 52.9% in animals that were reinseminated. Van Rensburg and De Vos (5) reported that conception rate in cows with delayed ovulation increased from 0% to 62.7% when reinsemination was performed. It is concluded that delayed ovulation is not a major cause of infertility in dairy heifers, and is not a significant component of the repeat-breeder syndrome in such animals. Thus, treatments or management practices oriented toward preventing delayed ovulation in heifers are not likely to be successful. REFERENCES Zemjanis, R. Repeat-breeding or conception failure in cattle. In: Morrow, D. A. (ed.), Current Therapy in Theriogenology. W. B. Saunders Co., Philadelphia, 1980. pp. 205-213. Bostedt, H. Delayed ovulation as a cause of sterility in the AI of cattle. 8th. Intl. Congress on Anim. Reprod. pp. 552-554 (1976). Leidl, W., Bostedt, H., Lamprecht, W., Prinzen, R. and Went, V. The influence of GnRH and hCG on the time of ovulation in cows artificially inseminated. Tieraerztl Umsch, x:546-555 (1979). Nakao, T., Shirakawa, J., Tsurubayashi, M., Ohboshi, K., Abe, T., Sawamukai, Y., Saga, N., Tsunoda, N. and Kawata, K. A preliminary report on the treatment of ovulation failure in cows with gonadotropin-releasing hormone analog or human chorionic gonadotropin combined with insemination. Anim. Reprod. Sci. 2:489-495 (1984).
T~ef~ogenoiogy
1080 5.
Van Rensburg, S.W.J. and De Vos, W.H. Ovulatory Onderstepoort J. Vet. Res. 29:55-78 (1962).
failure
in bovines.
6.
Hancock, J.L. The clinical analysis of reproductive @:513-517 (1948).
7.
Lee, C.N., Maurice, E., Ax, R.L., Pennington, J.A., Hoffman, W.F. and Brown, M.D. Efficacy of gonadotropin-releasing hormone administered at the time of artificial insemination of heifers and postpartum and repeat-breeder dairy cows. Am. J. Vet. Res. @:2160-2163 (1983).
8.
Lucy, M.C. and Stevenson, J.S. Gonadotropin-releasing hormone at estrus: luteinizing hormone, estradiol and progesterone during the periestrual and postinsemination periods in dairy cattle. Biol. Reprod. 3.&300-311 (1986).
9.
Erb, R-E., Garverick, H.A., Randell, RD., Brown, B.L. and Callahan, C.J, Profiles of reproductive hormones associated with fertile and nonfertile inseminations of dairy cows. Theriogenology 5:27-41 (1976).
failure in cattle. Vet Rec.
10.
Maurer, R.R. and Echternkamp, S.E. Hormonal development. The~og~nology u: 1l-22 (1982).
11.
Shelton, K., Gayerie de Abreu, M.F., Hunter, M.G., Parkinson, T. J. and Lamming, G.E. Luteal inadequacy during the early luteal phase of subfertile cows. J. Reprod. Fertil. 90: l-10 (1990).
12.
Stevenson, J.S., Call, E.P. and Scoby, gonadotropin-releasing hormone treatment Dairy Sci. a: 1766-1772 (1990).
13.
Kimura, M., Nakao, T., Moriyoshi, M. and Kawata, K. Luteal phase deficiency as a possible cause of repeat breeding in dairy cows. Br. Vet. J. ,&$:560-566 (1987).
14.
Swanson, L.V. and Young, A.J. Failure of gonadotropin-releasing hormone or human chorionic gonadotropin to enhance the fertility of repeat breeder cows when administered at the time of insemination. Theriogenology $&955-963 (1990).
15.
Bon Durant, R-H., Revah, I., Franti, C., Harman, R.J., Hird, D., Klingborg, D., McCloskey, M., Weaver, L. and Wilgenberg, B. Effect of gonadotropinreleasing hormone on fertility in repeat-breeder California dairy cows. Theriogenology %:365-374 (1991).
16.
Hansel, W., Spalding, R.W., Larson, L.L., Laster, D.B., Wagner, J.F. and Braun, R.K. Influence of human chononic gonadotropin on pregnancy rates in lactatmg dairy and beef cows. J. Dairy Sci. 59:751-759 (1976).
17.
Drost, M. A practice approach to the infertile cow. The Bovine Practitioner 12: 117-121 (1980).
18.
Gustafsson, H., Larsson, K., Kindahl, H. and Madej, A. Sequential endocrine changes and behavior during oestrus and metoestrus in repeat breeder and virgin heifers. Anim. Reprod. Sci. u:261-273 (1986).
asynchrony
and embryonic
R.K. Double insemination and of repeat-breeding dairy cattle. J.
1081
Theriogenology 19.
Villa-Godoy, A., Hughes, T.L., Emery, R.S., Chapin, L.T. and Fogwell, R.L. Association between energy balance and luteal function in lactating dairy cows. J. Dairy Sci. 21:1063-1072 (1988).
20.
Staples, C.R. and Thatcher, J.H. Relationship between ovarian activity and energy status during the early postpartum period of high producing dairy cows. J. Dairy Sci. 73:938-947 (1990).
21.
Lucy, M.C., Staples, C.R., Michel, EM. and Thatcher, W. W. Energy balance and size and number of ovarian follicles detected by ultrasonography in early postpartum dairy cows. J. Dairy Sci. z:473-482 (1991).
22.
Canfield, R.W. and Butler, W.R. Energy balance, first ovulation and the effects of naloxone on LH secretion in early postpartum dairy cows. 5. Anim. Sci. 69: 740-746 (1991).
23.
Arthur, G.H., Noakes, D.E. and Pearson, H. Veterinary Obstetrics. Bailliere Tindall, London, 1982, pp. 295-327.
24.
Andersson, L. and Emanuelson, U.: An epidemiological study hyperketonemia in Swedish dairy cows. Prev. Vet. Med. 3~449-462 (1985).
25.
Andersson, L., Gustafsson, A.H. and Emanuelson, U.: Effect of hyperketonemia and feeding on fertility in dairy cows. Theriogenology 36:521536 (1991).
26.
National Research Council. Nutrient Requirements Academy Press, Washington, 1988.
27.
Pulido, A., Zarco, L., Galina, C.S., Murcia, C., Flores, G. and Posadas, E. Progesterone metabolism during storage of blood samples from Gyr cattle: effects of anticoagulant, time and temperature of incubation. Theriogenology 3:965-975 (1991).
28.
Hernandez, C.J., Zarco, Q.L. and Lima, T.V. Niveles de progesterona plasmatica durante 10s primeros 7 dias posinseminacidn en vaquillas Holstein repetidoras y de primer servicio. Vet. Mex. a:189-192 (1992).
29.
Linares, T., Larsson, K. and Edqvist, L.-E. Plasma progesterone levels from estrus through Day 7 after AI in heifers carrying embryos with normal or deviating morphology. Theriogenology 17: 125-132 (1982).
Reproduction
of Dairy Cattle.
and of
National