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Embryo yield and plasma progesterone profiles in superovulated dairy cows and heifers
Animal Reproduction Science 69 (2002) 1–8
Embryo yield and plasma progesterone profiles in superovulated dairy cows and heifers J. Chagas e Silva a , L. Lopes da Costa b,∗ , J. Robalo Silva b b
a Divisão de Selecção e Reprodução Animal, Rua Elias Garcia 30, 2704-507 Venda Nova, Portugal Faculdade de Medicina Veterinária, CIISA, Rua Prof. Cid dos Santos, Polo Universitário, Alto da Ajuda, 1300-477, Lisboa, Portugal
Received 12 December 2000; received in revised form 26 July 2001; accepted 22 August 2001
Abstract This study was conducted to compare the superovulatory (SOV) response of dairy cows (n = 172) and heifers (n = 172), with two SOV treatments started at the mid-luteal-phase of the estrus cycle. Donors were randomly treated either with equine chorionic gonadotrophin (eCG) plus neutra-eCG serum (eCG + N group, n = 167) or follicle stimulating gonadotrophin (FSH-P group, n = 177). No significant differences were observed among groups in the percentage of superovulatory responsive donors (SR donors; corpora lutea (CL) ≥2), the mean number of total ova, fertilized ova and viable embryos recovered. Cows yielded significantly less total ova and less fertilized ova (P < 0.05) and tended to yield less viable embryos (P < 0.06) than heifers. Plasma progesterone (P4) concentrations (n = 135 donors) on the day of PGF2␣ (PGF) injection and on the day of SOV estrus were significantly higher (P < 0.01) in eCG + N than in FSH-P donors and, the increase between those 2 days was also significantly higher (P < 0.05) in group eCG + N than in group FSH-P, suggesting a higher luteotrophic effect of eCG than FSH-P. SR donors had P4 levels significantly higher (P < 0.001) than non-SR donors only on day 5 after the SOV estrus and on the day of embryo recovery. Plasma P4 concentrations at 5 days after the SOV estrus and at embryo recovery correlated significantly (r = 0.76, P < 0.001). Heifers had significantly higher P4 levels than cows at gonadotrophin injection (P < 0.01), PGF injection (P < 0.001), 5 days (P < 0.01) and 7 days (P < 0.001) after the SOV estrus. At day 7 after the SOV estrus, P4 concentrations per ova recovered were significantly higher in heifers than in cows (P < 0.01). The increase of plasma P4 per ova recovered, between days 5 and 7 after the SOV estrus, was significantly (P < 0.01) higher in heifers than in cows. Also, the increase of plasma P4 between injections of gonadotrophin and PGF was significantly higher (P < 0.05) in heifers than in cows.
∗ Corresponding author. Tel.: +351-21-3652825; fax: +351-21-3652827. E-mail address: [email protected] (L. Lopes da Costa).
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 7 2 - 5
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J. Chagas e Silva et al. / Animal Reproduction Science 69 (2002) 1–8
These results suggest that heifers have higher plasma P4 concentrations at diestrus (either before or after the SOV treatment) and this is associated with a higher embryo yield and quality, as compared to lactating cows. These higher plasma P4 concentrations reflect not only differences in ovulation rate as well as the competence of the corpus luteum, which is potentialized by gonadotrophin stimulation. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Cattle-reproductive technology; Superovulation; Gonadotrophins; Plasma progesterone
1. Introduction Despite the increasing use of ovum pick-up and in vitro embryo production, superovulation is still widely used in cattle embryo transfer programs. Equine chorionic gonadotrophin (eCG) and follicle stimulating gonadotrophin (FSH) preparations have long been used in superovulatory (SOV) treatments, the latter being preferred by many researchers and practitioners due to a better embryo yield (Elsden et al., 1978; Goulding et al., 1991). Concentrations of progesterone (P4) in blood or milk have been used for identification of potential causes of poor response and oocyte/embryo quality or to exclude potentially poor donors (Allen and Foote, 1988; Callesen et al., 1988). Although correlation between P4 concentrations and the outcome of the SOV treatment continues to be a matter of controversy (reviewed by Britt and Holt, 1988), it is accepted that abnormal P4 profiles are negatively related to the SOV response, embryo yield and quality (Callesen et al., 1986, 1988; Greve et al., 1995), and that there is a threshold in P4 concentration at several points of the SOV treatment, below which the SOV response is impaired (Goto et al., 1987; Allen and Foote, 1988; Herrier et al., 1990). SOV induced abnormal LH and P4 profiles have been identified and related to low oocyte competence (Callesen et al., 1986) and embryo yield and quality (Callesen et al., 1988). However, comparisons of plasma P4 profiles among donors submitted to eCG and FSH treatments are scarce (Yadav et al., 1986; Nibart et al., 1988) and comparisons of P4 profiles between donor cows and heifers are also apparently lacking. Therefore, it would be interesting to evaluate whether possible differences in embryo yield and quality between eCG and FSH treatments and between lactating cows and heifers are associated to differences in plasma P4 profiles. The objectives of this study were to (a) compare the embryo yield and quality of dairy cows and heifers treated either with eCG or FSH and (b) compare plasma P4 profiles of eCG and FSH treated donors and of cows and heifers, and evaluate whether differences in embryo yield and quality are associated to differences and/or abnormalities in P4 profiles.
2. Materials and methods The study was conducted in 28 dairy herds, in Holstein–Friesian donors. Overall, 344 SOV treatments were attempted, of which 172 were in lactating cows (mean±S.D.: 3.2±2.1 lactations, range: 1–9) and 172 in heifers.
J. Chagas e Silva et al. / Animal Reproduction Science 69 (2002) 1–8
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Prior to the SOV treatment, all females were cycling normally and were healthy and gynecologically normal, as judged by rectal examination. All donor cows were submitted to the SOV treatment after 50 days postpartum. At 9–11 days after a reference estrus (day 0), donors were assigned at random to one of two groups: (a) group eCG + N (n = 167) received an i.m. injection of eCG (2000–2500 IU, folligon), followed by an i.m. injection of PGF (15 mg, prosolvin) 48 and 60 h later and, an i.v. injection of 5 ml of an anti-eCG monoclonal serum (neutra-PMSG) 12 h after the onset of the ensuing estrus. All three drugs were purchased from Intervet International, Boxmeer, The Netherlands; (b) group FSH-P (n = 177) received a total dose of 32–36 mg (Armour standard units) of FSH (Schering Corporation, Kenilworth, NJ, USA) distributed in eight i.m. injections in decreasing dose, at 12 h intervals. An i.m. injection of PGF (15 mg, prosolvin) was administered at the time of the seventh and of the eighth FSH injections. At 12 and 24 h after the onset of the SOV estrus, donors were artificially inseminated with one straw of frozen–thawed semen from one of several dairy bulls with proven fertility. On days 6.5–8 after the SOV estrus (day 0 of SOV cycle), ova were recovered by standard non-surgical procedures. Recovered ova were evaluated for stage of development and quality according to IETS guidelines (IETS, 1998). Embryos of stages 4–7 and qualities 1–2 were classified as viable. At the time of uterine flushing, the donor’s ovaries were palpated per rectum and the number of corpora lutea (CL) assessed. Donors (CL ≥2) were considered to be SOV responsive donors (SR donors). Non-SR donors (CL 0–1) also included those that did not show estrus and were therefore, not submitted to AI. In 135 donors from a single herd, plasma P4 was measured by a validated solid-phase radioimmunoassay, without extraction, using commercial kits (Coat-A-Count, Diagnostic Product Corporation, Los Angeles, CA, USA). Blood samples were collected from the coccygeal vessels into heparinized tubes, immediately centrifuged and plasma stored frozen until assay. Samples were assayed in duplicate. Intra-assay and inter-assay coefficients of variation of sessions run at the time of the study were 8.9 and 9.6%, respectively. Sampling days were the following: reference estrus, 7 days after reference estrus, first gonadotrophin injection, first PGF injection, SOV estrus or 48 h after PGF injection in donors that did not show estrus behavior, 5 days after SOV estrus and 7 days after SOV estrus. In donors where plasma P4 was measured, cows and heifers were evenly distributed among eCG + N and FSH-P treatments. Also, the day when gonadotrophin treatment started (days 9–11) was evenly distributed among treatments. Ovarian palpation tended to underestimate the number of ovulations, mainly in donors displaying many CL per ovary, therefore, ovarian palpation results were only used to distinguish non-SR (CL 0–1) from SR donors (CL ≥2) and, the magnitude of the SOV response was depicted from the total ova recovered (all flushings were performed by the same two technicians and no variation among technicians on the estimated efficiency of flushing was observed). Differences among groups (age, treatment) were analyzed by χ 2 -tests, ANOVA and LSD, where appropriate. Total ova recovered were used as a covariate in analysis (ANCOVA) of effects of treatment and cow/heifer status of donor on plasma P4 concentrations. Regression analysis between P4 concentrations on sampling days and total ova, fertilized ova and viable embryos recovered used the linear equation y = ax + b.
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Table 1 SR and ova recovered for different groups Group
n
Ova recovered in SR donors—mean ± S.E.M.
SR n (%)
Total
Fertilized
Viable
Cow Heifer P valuea
172 172
140 (81.4) 151 (87.8) NSb
7.9 ± 0.5 9.9 ± 0.6 0.05
6.0 ± 0.4 7.6 ± 0.5 0.05
5.2 ± 0.4 6.4 ± 0.5 0.06
eCG + N FSH-P P value
167 177
152 (91.0) 139 (78.5) NS
8.5 ± 0.5 9.5 ± 0.5 NS
6.3 ± 0.4 7.4 ± 0.6 NS
5.2 ± 0.4 6.5 ± 0.5 NS
a b
P values for ANOVA, except column SR for χ 2 -test. NS: non-significant.
3. Results Table 1 presents the number of donors with SOV response and the mean number of total ova, fertilized ova and viable embryos recovered. No significant differences were observed among treatments in the percentage of responsive donors, the mean number of total ova, fertilized ova and viable embryos recovered per SR donor. Also, the mean number of viable embryos per treated donor was not significantly different between treatments (4.8 ± 0.4 versus 5.0 ± 0.5, P > 0.05, for eCG + N and FSH-P, respectively). Donor cows yielded significantly (P < 0.05) less total ova and less fertilized ova and tended (P < 0.06) to yield less viable embryos than donor heifers. Table 2 presents the mean plasma P4 values for eCG + N and FSH-P donors, for cows and heifers and, for SR and non-SR donors. Donors treated with eCG + N had Table 2 Mean plasma P4 values for donors in different groupsa Group
n
Plasma P4 (ng/ml)—mean ± S.E.M. Sampling days d7
Gon
PGF
Estrus
D5
D7
eCG + N∗ FSH-P∗ P value
30 80
2.9 ± 0.2 2.9 ± 0.1 NSb
4.2 ± 0.2 4.1 ± 0.1 NS
7.4 ± 0.5 6.0 ± 0.3 0.01
0.5 ± 0.04 0.4 ± 0.03 0.05
7.4 ± 0.8 9.6 ± 0.9 NS
16.6 ± 1.8 18.9 ± 1.5 NS
Cow∗ Heifer∗ P value
40 70
2.6 ± 0.2 3.0 ± 0.1 NS
3.7 ± 0.2 4.4 ± 0.1 0.01
5.3 ± 0.4 7.0 ± 0.3 0.001
0.5 ± 0.04 0.4 ± 0.03 NS
6.8 ± 0.8 10.2 ± 0.9 0.01
11.3 ± 1.4 22.3 ± 1.5 0.001
SR Non-SR P value
110 25
2.9 ± 0.1 2.8 ± 0.2 NS
4.1 ± 0.1 4.2 ± 0.3 NS
6.4 ± 0.2 6.4 ± 0.4 NS
0.4 ± 0.02 0.4 ± 0.09 NS
9.0 ± 0.7 0.4 ± 0.2 0.001
18.3 ± 1.2 1.1 ± 0.3 0.001
a
d7: day 7 after the reference estrus; Gon: first gonadotrophin injection; PGF: PGF injection; Estrus: SOV estrus; D5 and D7: days after the SOV estrus (D0). b NS: non-significant. ∗ Only SR donors considered. P values for ANOVA.
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Table 3 Plasma P4 concentrations per recovered ova in superovulated dairy cows and heifers P4 per recovered ova
At day 5 after the SOV estrus At day 7 after the SOV estrus Increase day 5–7 Increase gonadotrophin–PGF∗ Increase day 7–gonadotrophin∗∗
Plasma P4 (ng/ml)—mean ± S.E.M. Cows (n = 40)
Heifers (n = 70)
0.97 ± 0.19 1.63 ± 0.23 0.70 ± 0.12 1.89 ± 0.33 1.11 ± 0.32
1.06 ± 0.08 2.83 ± 0.27 1.77 ± 0.24 2.81 ± 0.26 1.38 ± 0.13
P value
NSa 0.01 0.01 0.05 NS
a
NS: non-significant. Between first gonadotrophin injection and PGF injection. ∗∗ Between 7 days after the reference estrus and PGF injection. P values for ANOVA. ∗
significantly higher P4 levels at PGF injection (P< 0.01) and at the SOV estrus (P < 0.05) than FSH-P treated donors. Plasma P4 significantly increased from gonadotrophin injection to PGF injection (4.1 ± 0.1 to 6.4 ± 0.2, P < 0.001, n = 110) and this increase was significantly higher (P < 0.05) in eCG + N than in FSH-P donors. SR donors had significantly higher (P < 0.001) P4 concentrations at 5 and 7 days after the SOV estrus than non-SR donors. The P4 levels at 7 and 5 days after the SOV estrus were significantly (P < 0.001) correlated with the number of total ova (r = 0.65 and r = 0.32), of fertilized ova (r = 0.54 and 0.31) and viable embryos (r = 0.49 and 0.32) recovered. Plasma P4 levels at 5 and 7 days after the SOV estrus also correlated significantly (r = 0.76; P < 0.001). Cows had significantly lower P4 levels at all diestrus sampling days (except at day 7 after the reference estrus) than heifers (Table 2). Analysis of variance of P4 concentrations, using CL number (depicted from the total ova recovered) as a covariate, showed significant differences between cows and heifers at 5 and 7 days after the SOV estrus (P < 0.05 and < 0.001, respectively). Table 3 presents the results of analysis of variance of P4 concentrations and of increase of P4 concentrations per ova recovered, between cows and heifers, at diestrus sampling days. The increase on P4 concentrations between injections of gonadotrophin and of PGF and, between days 5 and 7 after the SOV estrus was significantly higher in heifers than in cows (P < 0.05 and < 0.01, respectively).
4. Discussion The differences in embryo yield and quality observed among eCG + N and FSH-P treatments were not significant. Other papers (Elsden et al., 1978; Goulding et al., 1991) reported a better yield of transferable embryos by FSH preparations in comparison with eCG. The negative effect of eCG on embryo quality is thought to arise from its long half-life (Schams et al., 1978) that induces persistently high estrogen levels after ovulation or from its high LH activity that stimulates early luteinization of growing follicles, disturbing intra-follicular steroidogenesis and oocyte maturation (Callesen et al., 1986, 1988; Greve et al., 1995).
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A negative effect of LH activity of FSH preparations on embryo quality has also been reported (Donaldson and Ward, 1986). In this study, eCG + N induced significantly higher plasma P4 levels at PGF injection and at the ensuing SOV estrus than FSH-P and, the increase in P4 values between gonadotrophin injection and PGF injection was also significantly higher in eCG + N treated donors than in FSH-P donors. As no differences in CL number were likely to occur among treatment groups at gonadotrophin injection (with the possible exception of a very few double ovulations) and as P4 concentrations are kept at a plateau level during mid-diestrus, this suggests a higher luteotrophic effect of eCG compared to FSH-P, a result also observed by Nibart et al. (1988). It is accepted that eCG is a more crude preparation with high LH activity (Schams et al., 1978), compared to FSH-P (Donaldson, 1986). However, the apparent higher luteotrophic effect of eCG compared to FSH-P had no significant effects on embryo yield and quality. It remains to be elucidated whether the source of extra P4 comes from the CL or from premature follicle luteinization (Tamboura et al., 1985). In the present study, non-SR donors only had P4 levels significantly lower than SR donors on days 5 and 7 after the SOV estrus. Plasma P4 on these days was significantly, positively and increasingly correlated with viable embryos, fertilized ova and total ova recovered. This agrees with previous reports (Yadav et al., 1986; Goto et al., 1987; Allen and Foote, 1988; Nibart et al., 1988; Herrier et al., 1990; Mehmood et al., 1990; Wubishet et al., 1991). Measuring plasma P4 on day 5 after the SOV estrus onwards could identify donors with poor or no response. The lower SOV response, fertilization rate and viable embryo yield of lactating cows in relation to heifers was associated to significantly lower P4 concentrations during diestrus, either before or after the SOV treatment. These differences in P4 concentrations at diestrus can be atributted to differences in CL competence (unstimulated cycle) and to CL competence and ovulation rate (SOV cycle). The P4 concentrations per ova recovered at day 7 after the SOV estrus were significantly higher in heifers than in cows and, the increase in P4 concentrations per ova recovered after the SOV estrus (between days 5 and 7) was also significantly higher in heifers than in cows. This also suggests that after gonadotrophin stimulation, CL competence is more stimulated in heifers compared to cows. Whether P4 concentrations are associated or causal to differences in embryo yield and quality cannot be depicted from the study. In beef cows, Lerner et al. (1986) observed that total ova, fertilized ova and transferable embryos recovered, increased up to 5.6 years of age and then decreased, particularly in very old donors (up to 17 years). In our study, mean lactation number was 3 and therefore, age related changes in ovarian function might have had little influence. Also, uterine abnormalities probably did not account for the differences observed since all donors were healthy and gynecologically sound. Comparing heifers and old non-lactating dairy cows displaying contrasting SOV response, Desaulniers et al. (1995) observed a disturbed pattern of follicular development and of endocrine events in cows, which could lead to follicles of low steroidogenic capability and to low competence oocytes. These disturbances were associated with significantly lower P4 concentrations during the SOV treatment, in cows compared to heifers. The difference observed between heifers and lactating cows in P4 concentrations, SOV response and embryo yield might be related to the nutritional and
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metabolic status of donors before or at the SOV treatment (Butler, 2000). Further work is needed to test this hypothesis.
Acknowledgements The authors thank Dr. António Horta for his help in the statistical analysis.
References Allen, S.E., Foote, R.H., 1988. An enzyme-linked immunoassay of milk progesterone as a diagnostic aid in embryo transfer programs. Theriogenology 29, 893–903. Britt, J.H., Holt, L.C., 1988. Endocrinological screening of embryo donors and embryo transfer recipients: a review of research with cattle. Theriogenology 29, 189–202. Butler, W.R., 2000. Nutritional interactions with reproductive performance in dairy cattle. Anim. Reprod. Sci. 60-61, 449–457. Callesen, H., Greve, T., Hyttel, P., 1986. Preovulatory endocrinology and oocyte maturation in superovulated cattle. Theriogenology 25, 71–86. Callesen, H., Greve, T., Hyttel, P., 1988. Preovulatory evaluation of the superovulatory response in donor cattle. Theriogenology 30, 477–488. Desaulniers, D.M., Lussier, J.G., Goff, A.K., Bousquet, D., Guilbault, L.A., 1995. Follicular development and reproductive endocrinology during and after superovulation in heifers and mature cows displaying contrasting superovulatory responses. Theriogenology 12, 117–131. Donaldson, L.E., 1986. FSH-P variation. Theriogenology 33, 215 (abstract). Donaldson, L.E., Ward, D.N., 1986. Effects of luteinizing hormone on embryo production in superovulated cows. Vet. Record. 119, 625–626. Elsden, R.P., Nelson, L.D., Seidel, G.E., 1978. Superovulating cows with follicle stimulating hormone and pregnant mare’s serum gonadotrophin. Theriogenology 9, 17–26. Goto, K., Nakanishi, Y., Ohkutsu, S., Ogawa, K., Tasaki, M., Ohta, M., Inohae, S., Tateyama, S., Kawabata, T., 1987. Plasma progesterone profiles and embryo quality in superovulated Japanese black cattle. Theriogenology 27, 819–826. Goulding, D., Williams, D.H., Roche, J.F., Boland, M.P., 1991. Superovulation in heifers using either pregnant mare’s serum gonadotrophin or follicle stimulating hormone during the mid-luteal stage of the estrous cycle. Theriogenology 36, 949–958. Greve, T., Callesen, H., Hyttel, P., Hoier, R., Assey, R., 1995. The effects of exogenous gonadotropins on oocyte and embryo quality in cattle. Theriogenology 43, 41–50. Herrier, A., Elsaesser, F., Niemann, H., 1990. Rapid milk progesterone assay as a tool for the selection of potential donor cows prior to superovulation. Theriogenology 33, 415–422. IETS, 1998. Manual of the international embryo transfer society, Stringfellow, D.A. Seidel, S.M. (Eds.), Third Edition, International Embryos Transfer Society, Inc., Savoy, Illinois, USA. Lerner, S.P., Thayne, W.V., Baker, R.D., Henschen, T., Meredith, S., Inskeep, E.K., Dailey, R.A., Lewis, P.E., Butcher, R.L., 1986. Age, dose of FSH and other factors affecting superovulation in Holstein cows. J. Anim. Sci. 63, 176–183. Mehmood, A., Anwar, M., Ullah, N., Baig, S.M., Wright Jr., R.W., 1990. Pattern of sex steroids secretion and their relationship with embryo yield in jersey cows superovulated with PMSG. Theriogenology 35, 513–520. Nibart, N., Slimane, N., Herrera, R., Jeanguyot, N., Mechekour, F., Humblot, P., Thibier, M., 1988. Variations des concentrations plasmatiques des hormones gonadotropes (FSH, LH) et stéroides (oestradiol-17, progestérone) après différents traitments de superovulation chez la vache. Élevage et Insemination 226, 11–30.
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Schams, S., Menzer, Ch., Schallenberger, E., Hoffman, B., Hahn, J., Hahn, R., 1978. Some studies on pregnant mare serum gonadotrophin (PMSG) and on endocrine responses after application for superovulation in cattle. In: Sreenan, J.M. (Ed.), Control of Reproduction in the Cow. Martinus Nijhoff, The Hague, The Netherlands, 122–143. Tamboura, D., Chupin, D., Saumande, J., 1985. Superovulation in cows: A relationship between progesterone secretion before ovulation and the quality of embryos. Anim. Reprod. Sci. 8, 327–334. Wubishet, A., Kesler, D.J., Graves, C.N., Spahr, S.L., Favero, R.J., 1991. Preovulatory LH profiles of superovulated cows and progesterone concentrations at embryo recovery. Theriogenology 35, 451–457. Yadav, M.C., Walton, J.S., Leslie, K.E., 1986. Plasma concentrations of luteinizing hormone and progesterone during superovulation of dairy cows using follicle stimulating hormone or pregnant mare serum gonadotrophin. Theriogenology 26, 523–540.
Embryo yield and plasma progesterone profiles in superovulated dairy cows and heifers J. Chagas e Silva a , L. Lopes da Costa b,∗ , J. Robalo Silva b b
a Divisão de Selecção e Reprodução Animal, Rua Elias Garcia 30, 2704-507 Venda Nova, Portugal Faculdade de Medicina Veterinária, CIISA, Rua Prof. Cid dos Santos, Polo Universitário, Alto da Ajuda, 1300-477, Lisboa, Portugal
Received 12 December 2000; received in revised form 26 July 2001; accepted 22 August 2001
Abstract This study was conducted to compare the superovulatory (SOV) response of dairy cows (n = 172) and heifers (n = 172), with two SOV treatments started at the mid-luteal-phase of the estrus cycle. Donors were randomly treated either with equine chorionic gonadotrophin (eCG) plus neutra-eCG serum (eCG + N group, n = 167) or follicle stimulating gonadotrophin (FSH-P group, n = 177). No significant differences were observed among groups in the percentage of superovulatory responsive donors (SR donors; corpora lutea (CL) ≥2), the mean number of total ova, fertilized ova and viable embryos recovered. Cows yielded significantly less total ova and less fertilized ova (P < 0.05) and tended to yield less viable embryos (P < 0.06) than heifers. Plasma progesterone (P4) concentrations (n = 135 donors) on the day of PGF2␣ (PGF) injection and on the day of SOV estrus were significantly higher (P < 0.01) in eCG + N than in FSH-P donors and, the increase between those 2 days was also significantly higher (P < 0.05) in group eCG + N than in group FSH-P, suggesting a higher luteotrophic effect of eCG than FSH-P. SR donors had P4 levels significantly higher (P < 0.001) than non-SR donors only on day 5 after the SOV estrus and on the day of embryo recovery. Plasma P4 concentrations at 5 days after the SOV estrus and at embryo recovery correlated significantly (r = 0.76, P < 0.001). Heifers had significantly higher P4 levels than cows at gonadotrophin injection (P < 0.01), PGF injection (P < 0.001), 5 days (P < 0.01) and 7 days (P < 0.001) after the SOV estrus. At day 7 after the SOV estrus, P4 concentrations per ova recovered were significantly higher in heifers than in cows (P < 0.01). The increase of plasma P4 per ova recovered, between days 5 and 7 after the SOV estrus, was significantly (P < 0.01) higher in heifers than in cows. Also, the increase of plasma P4 between injections of gonadotrophin and PGF was significantly higher (P < 0.05) in heifers than in cows.
∗ Corresponding author. Tel.: +351-21-3652825; fax: +351-21-3652827. E-mail address: [email protected] (L. Lopes da Costa).
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 7 2 - 5
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J. Chagas e Silva et al. / Animal Reproduction Science 69 (2002) 1–8
These results suggest that heifers have higher plasma P4 concentrations at diestrus (either before or after the SOV treatment) and this is associated with a higher embryo yield and quality, as compared to lactating cows. These higher plasma P4 concentrations reflect not only differences in ovulation rate as well as the competence of the corpus luteum, which is potentialized by gonadotrophin stimulation. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Cattle-reproductive technology; Superovulation; Gonadotrophins; Plasma progesterone
1. Introduction Despite the increasing use of ovum pick-up and in vitro embryo production, superovulation is still widely used in cattle embryo transfer programs. Equine chorionic gonadotrophin (eCG) and follicle stimulating gonadotrophin (FSH) preparations have long been used in superovulatory (SOV) treatments, the latter being preferred by many researchers and practitioners due to a better embryo yield (Elsden et al., 1978; Goulding et al., 1991). Concentrations of progesterone (P4) in blood or milk have been used for identification of potential causes of poor response and oocyte/embryo quality or to exclude potentially poor donors (Allen and Foote, 1988; Callesen et al., 1988). Although correlation between P4 concentrations and the outcome of the SOV treatment continues to be a matter of controversy (reviewed by Britt and Holt, 1988), it is accepted that abnormal P4 profiles are negatively related to the SOV response, embryo yield and quality (Callesen et al., 1986, 1988; Greve et al., 1995), and that there is a threshold in P4 concentration at several points of the SOV treatment, below which the SOV response is impaired (Goto et al., 1987; Allen and Foote, 1988; Herrier et al., 1990). SOV induced abnormal LH and P4 profiles have been identified and related to low oocyte competence (Callesen et al., 1986) and embryo yield and quality (Callesen et al., 1988). However, comparisons of plasma P4 profiles among donors submitted to eCG and FSH treatments are scarce (Yadav et al., 1986; Nibart et al., 1988) and comparisons of P4 profiles between donor cows and heifers are also apparently lacking. Therefore, it would be interesting to evaluate whether possible differences in embryo yield and quality between eCG and FSH treatments and between lactating cows and heifers are associated to differences in plasma P4 profiles. The objectives of this study were to (a) compare the embryo yield and quality of dairy cows and heifers treated either with eCG or FSH and (b) compare plasma P4 profiles of eCG and FSH treated donors and of cows and heifers, and evaluate whether differences in embryo yield and quality are associated to differences and/or abnormalities in P4 profiles.
2. Materials and methods The study was conducted in 28 dairy herds, in Holstein–Friesian donors. Overall, 344 SOV treatments were attempted, of which 172 were in lactating cows (mean±S.D.: 3.2±2.1 lactations, range: 1–9) and 172 in heifers.
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Prior to the SOV treatment, all females were cycling normally and were healthy and gynecologically normal, as judged by rectal examination. All donor cows were submitted to the SOV treatment after 50 days postpartum. At 9–11 days after a reference estrus (day 0), donors were assigned at random to one of two groups: (a) group eCG + N (n = 167) received an i.m. injection of eCG (2000–2500 IU, folligon), followed by an i.m. injection of PGF (15 mg, prosolvin) 48 and 60 h later and, an i.v. injection of 5 ml of an anti-eCG monoclonal serum (neutra-PMSG) 12 h after the onset of the ensuing estrus. All three drugs were purchased from Intervet International, Boxmeer, The Netherlands; (b) group FSH-P (n = 177) received a total dose of 32–36 mg (Armour standard units) of FSH (Schering Corporation, Kenilworth, NJ, USA) distributed in eight i.m. injections in decreasing dose, at 12 h intervals. An i.m. injection of PGF (15 mg, prosolvin) was administered at the time of the seventh and of the eighth FSH injections. At 12 and 24 h after the onset of the SOV estrus, donors were artificially inseminated with one straw of frozen–thawed semen from one of several dairy bulls with proven fertility. On days 6.5–8 after the SOV estrus (day 0 of SOV cycle), ova were recovered by standard non-surgical procedures. Recovered ova were evaluated for stage of development and quality according to IETS guidelines (IETS, 1998). Embryos of stages 4–7 and qualities 1–2 were classified as viable. At the time of uterine flushing, the donor’s ovaries were palpated per rectum and the number of corpora lutea (CL) assessed. Donors (CL ≥2) were considered to be SOV responsive donors (SR donors). Non-SR donors (CL 0–1) also included those that did not show estrus and were therefore, not submitted to AI. In 135 donors from a single herd, plasma P4 was measured by a validated solid-phase radioimmunoassay, without extraction, using commercial kits (Coat-A-Count, Diagnostic Product Corporation, Los Angeles, CA, USA). Blood samples were collected from the coccygeal vessels into heparinized tubes, immediately centrifuged and plasma stored frozen until assay. Samples were assayed in duplicate. Intra-assay and inter-assay coefficients of variation of sessions run at the time of the study were 8.9 and 9.6%, respectively. Sampling days were the following: reference estrus, 7 days after reference estrus, first gonadotrophin injection, first PGF injection, SOV estrus or 48 h after PGF injection in donors that did not show estrus behavior, 5 days after SOV estrus and 7 days after SOV estrus. In donors where plasma P4 was measured, cows and heifers were evenly distributed among eCG + N and FSH-P treatments. Also, the day when gonadotrophin treatment started (days 9–11) was evenly distributed among treatments. Ovarian palpation tended to underestimate the number of ovulations, mainly in donors displaying many CL per ovary, therefore, ovarian palpation results were only used to distinguish non-SR (CL 0–1) from SR donors (CL ≥2) and, the magnitude of the SOV response was depicted from the total ova recovered (all flushings were performed by the same two technicians and no variation among technicians on the estimated efficiency of flushing was observed). Differences among groups (age, treatment) were analyzed by χ 2 -tests, ANOVA and LSD, where appropriate. Total ova recovered were used as a covariate in analysis (ANCOVA) of effects of treatment and cow/heifer status of donor on plasma P4 concentrations. Regression analysis between P4 concentrations on sampling days and total ova, fertilized ova and viable embryos recovered used the linear equation y = ax + b.
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Table 1 SR and ova recovered for different groups Group
n
Ova recovered in SR donors—mean ± S.E.M.
SR n (%)
Total
Fertilized
Viable
Cow Heifer P valuea
172 172
140 (81.4) 151 (87.8) NSb
7.9 ± 0.5 9.9 ± 0.6 0.05
6.0 ± 0.4 7.6 ± 0.5 0.05
5.2 ± 0.4 6.4 ± 0.5 0.06
eCG + N FSH-P P value
167 177
152 (91.0) 139 (78.5) NS
8.5 ± 0.5 9.5 ± 0.5 NS
6.3 ± 0.4 7.4 ± 0.6 NS
5.2 ± 0.4 6.5 ± 0.5 NS
a b
P values for ANOVA, except column SR for χ 2 -test. NS: non-significant.
3. Results Table 1 presents the number of donors with SOV response and the mean number of total ova, fertilized ova and viable embryos recovered. No significant differences were observed among treatments in the percentage of responsive donors, the mean number of total ova, fertilized ova and viable embryos recovered per SR donor. Also, the mean number of viable embryos per treated donor was not significantly different between treatments (4.8 ± 0.4 versus 5.0 ± 0.5, P > 0.05, for eCG + N and FSH-P, respectively). Donor cows yielded significantly (P < 0.05) less total ova and less fertilized ova and tended (P < 0.06) to yield less viable embryos than donor heifers. Table 2 presents the mean plasma P4 values for eCG + N and FSH-P donors, for cows and heifers and, for SR and non-SR donors. Donors treated with eCG + N had Table 2 Mean plasma P4 values for donors in different groupsa Group
n
Plasma P4 (ng/ml)—mean ± S.E.M. Sampling days d7
Gon
PGF
Estrus
D5
D7
eCG + N∗ FSH-P∗ P value
30 80
2.9 ± 0.2 2.9 ± 0.1 NSb
4.2 ± 0.2 4.1 ± 0.1 NS
7.4 ± 0.5 6.0 ± 0.3 0.01
0.5 ± 0.04 0.4 ± 0.03 0.05
7.4 ± 0.8 9.6 ± 0.9 NS
16.6 ± 1.8 18.9 ± 1.5 NS
Cow∗ Heifer∗ P value
40 70
2.6 ± 0.2 3.0 ± 0.1 NS
3.7 ± 0.2 4.4 ± 0.1 0.01
5.3 ± 0.4 7.0 ± 0.3 0.001
0.5 ± 0.04 0.4 ± 0.03 NS
6.8 ± 0.8 10.2 ± 0.9 0.01
11.3 ± 1.4 22.3 ± 1.5 0.001
SR Non-SR P value
110 25
2.9 ± 0.1 2.8 ± 0.2 NS
4.1 ± 0.1 4.2 ± 0.3 NS
6.4 ± 0.2 6.4 ± 0.4 NS
0.4 ± 0.02 0.4 ± 0.09 NS
9.0 ± 0.7 0.4 ± 0.2 0.001
18.3 ± 1.2 1.1 ± 0.3 0.001
a
d7: day 7 after the reference estrus; Gon: first gonadotrophin injection; PGF: PGF injection; Estrus: SOV estrus; D5 and D7: days after the SOV estrus (D0). b NS: non-significant. ∗ Only SR donors considered. P values for ANOVA.
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Table 3 Plasma P4 concentrations per recovered ova in superovulated dairy cows and heifers P4 per recovered ova
At day 5 after the SOV estrus At day 7 after the SOV estrus Increase day 5–7 Increase gonadotrophin–PGF∗ Increase day 7–gonadotrophin∗∗
Plasma P4 (ng/ml)—mean ± S.E.M. Cows (n = 40)
Heifers (n = 70)
0.97 ± 0.19 1.63 ± 0.23 0.70 ± 0.12 1.89 ± 0.33 1.11 ± 0.32
1.06 ± 0.08 2.83 ± 0.27 1.77 ± 0.24 2.81 ± 0.26 1.38 ± 0.13
P value
NSa 0.01 0.01 0.05 NS
a
NS: non-significant. Between first gonadotrophin injection and PGF injection. ∗∗ Between 7 days after the reference estrus and PGF injection. P values for ANOVA. ∗
significantly higher P4 levels at PGF injection (P< 0.01) and at the SOV estrus (P < 0.05) than FSH-P treated donors. Plasma P4 significantly increased from gonadotrophin injection to PGF injection (4.1 ± 0.1 to 6.4 ± 0.2, P < 0.001, n = 110) and this increase was significantly higher (P < 0.05) in eCG + N than in FSH-P donors. SR donors had significantly higher (P < 0.001) P4 concentrations at 5 and 7 days after the SOV estrus than non-SR donors. The P4 levels at 7 and 5 days after the SOV estrus were significantly (P < 0.001) correlated with the number of total ova (r = 0.65 and r = 0.32), of fertilized ova (r = 0.54 and 0.31) and viable embryos (r = 0.49 and 0.32) recovered. Plasma P4 levels at 5 and 7 days after the SOV estrus also correlated significantly (r = 0.76; P < 0.001). Cows had significantly lower P4 levels at all diestrus sampling days (except at day 7 after the reference estrus) than heifers (Table 2). Analysis of variance of P4 concentrations, using CL number (depicted from the total ova recovered) as a covariate, showed significant differences between cows and heifers at 5 and 7 days after the SOV estrus (P < 0.05 and < 0.001, respectively). Table 3 presents the results of analysis of variance of P4 concentrations and of increase of P4 concentrations per ova recovered, between cows and heifers, at diestrus sampling days. The increase on P4 concentrations between injections of gonadotrophin and of PGF and, between days 5 and 7 after the SOV estrus was significantly higher in heifers than in cows (P < 0.05 and < 0.01, respectively).
4. Discussion The differences in embryo yield and quality observed among eCG + N and FSH-P treatments were not significant. Other papers (Elsden et al., 1978; Goulding et al., 1991) reported a better yield of transferable embryos by FSH preparations in comparison with eCG. The negative effect of eCG on embryo quality is thought to arise from its long half-life (Schams et al., 1978) that induces persistently high estrogen levels after ovulation or from its high LH activity that stimulates early luteinization of growing follicles, disturbing intra-follicular steroidogenesis and oocyte maturation (Callesen et al., 1986, 1988; Greve et al., 1995).
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A negative effect of LH activity of FSH preparations on embryo quality has also been reported (Donaldson and Ward, 1986). In this study, eCG + N induced significantly higher plasma P4 levels at PGF injection and at the ensuing SOV estrus than FSH-P and, the increase in P4 values between gonadotrophin injection and PGF injection was also significantly higher in eCG + N treated donors than in FSH-P donors. As no differences in CL number were likely to occur among treatment groups at gonadotrophin injection (with the possible exception of a very few double ovulations) and as P4 concentrations are kept at a plateau level during mid-diestrus, this suggests a higher luteotrophic effect of eCG compared to FSH-P, a result also observed by Nibart et al. (1988). It is accepted that eCG is a more crude preparation with high LH activity (Schams et al., 1978), compared to FSH-P (Donaldson, 1986). However, the apparent higher luteotrophic effect of eCG compared to FSH-P had no significant effects on embryo yield and quality. It remains to be elucidated whether the source of extra P4 comes from the CL or from premature follicle luteinization (Tamboura et al., 1985). In the present study, non-SR donors only had P4 levels significantly lower than SR donors on days 5 and 7 after the SOV estrus. Plasma P4 on these days was significantly, positively and increasingly correlated with viable embryos, fertilized ova and total ova recovered. This agrees with previous reports (Yadav et al., 1986; Goto et al., 1987; Allen and Foote, 1988; Nibart et al., 1988; Herrier et al., 1990; Mehmood et al., 1990; Wubishet et al., 1991). Measuring plasma P4 on day 5 after the SOV estrus onwards could identify donors with poor or no response. The lower SOV response, fertilization rate and viable embryo yield of lactating cows in relation to heifers was associated to significantly lower P4 concentrations during diestrus, either before or after the SOV treatment. These differences in P4 concentrations at diestrus can be atributted to differences in CL competence (unstimulated cycle) and to CL competence and ovulation rate (SOV cycle). The P4 concentrations per ova recovered at day 7 after the SOV estrus were significantly higher in heifers than in cows and, the increase in P4 concentrations per ova recovered after the SOV estrus (between days 5 and 7) was also significantly higher in heifers than in cows. This also suggests that after gonadotrophin stimulation, CL competence is more stimulated in heifers compared to cows. Whether P4 concentrations are associated or causal to differences in embryo yield and quality cannot be depicted from the study. In beef cows, Lerner et al. (1986) observed that total ova, fertilized ova and transferable embryos recovered, increased up to 5.6 years of age and then decreased, particularly in very old donors (up to 17 years). In our study, mean lactation number was 3 and therefore, age related changes in ovarian function might have had little influence. Also, uterine abnormalities probably did not account for the differences observed since all donors were healthy and gynecologically sound. Comparing heifers and old non-lactating dairy cows displaying contrasting SOV response, Desaulniers et al. (1995) observed a disturbed pattern of follicular development and of endocrine events in cows, which could lead to follicles of low steroidogenic capability and to low competence oocytes. These disturbances were associated with significantly lower P4 concentrations during the SOV treatment, in cows compared to heifers. The difference observed between heifers and lactating cows in P4 concentrations, SOV response and embryo yield might be related to the nutritional and
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metabolic status of donors before or at the SOV treatment (Butler, 2000). Further work is needed to test this hypothesis.
Acknowledgements The authors thank Dr. António Horta for his help in the statistical analysis.
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