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Correlation between LH response to challenges with GNRH and naloxone during lactation, and LH secretion and follicular development after weaning in the sows
Animal Reproduction Science 56 Ž1999. 143–152
Correlation between LH response to challenges with GNRH and naloxone during lactation, and LH secretion and follicular development after weaning in the sows Fabio De Rensis
a,)
, George R. Foxcroft
b
a
Istituto di Clinica Ostetrica Veterinaria, UniÕersita` degli Studi di Parma, Õia del Taglio 8, 43100, Parma, Italy b Department of Agricultural, Food and Nutritional Science, 310-C Agriculturer Forestry Building, UniÕersity of Alberta, Edmonton, Alberta, Canada T6G 2P5 Accepted 18 May 1999
Abstract The aim of this study was of establishing a correlation between endogenous LH secretion and the magnitude of the LH response to challenges with GnRH and the opioid antagonist naloxone during lactation, and between these characteristics and LH secretion and follicular development after weaning. Sows Ž n s 9. were sampled for 6 h at day 2 post-partum, for 12 h on day 26 of lactation and for 6 h immediately after weaning at day 27 of lactation. Four hours after the beginning of sampling at day 26 of lactation all sows were injected with 2 mgrkg i.v. of naloxone hydrochloride and 5 h later with 100 mgrsow of GnRH. Follicular development was studied in all sows at slaughter the day after weaning. There was an effect of time Žsampling period; P - 0.001. on mean plasma LH, with an increase Ž P - 0.05. in LH the day after weaning compared to mean LH concentrations during lactation. Naloxone and GnRH treatment both increased Ž P - 0.05. mean LH concentrations. A positive relationship Ž r s 0.58, P - 0.01. between mean plasma LH after GnRH and after weaning was established. Although there were differences Ž P - 0.001. between sows in follicular fluid volume, there were no correlations between mean follicular fluid volume and mean LH concentrations after GnRH or after weaning. These data indicate that the LH response to GnRH during late lactation could be useful predictor of LH activity after weaning. However, none of the measures of endogenous or induced LH secretion were associated with differences in ovarian follicular size after weaning. Direct evidence is therefore still needed for
)
Corresponding author. Tel.: q39-0521-902659; fax: q39-0521-902662; e-mail: [email protected] 0378-4320r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 3 8 - X
144
F. De Rensis, G.R. Foxcroftr Animal Reproduction Science 56 (1999) 143–152
afunctional link between differences in LH in lactation and differences in fertility after weaning. q 1999 Elsevier Science B.V. All rights reserved. Keywords: LH; Lactation; Pig-endocrinology; GnRH; Opioids
1. Introduction Shaw and Foxcroft Ž1985. reported a reciprocal relationship between mean LH concentrations in late lactation and the length of the weaning to estrus interval and suggested that the LH status of the sow during lactation might be causally related to the resumption of ovarian activity and to fertility after weaning. This suggestion was supported by the subsequent data from the same laboratory ŽFoxcroft et al., 1987. and by the data of Tokach et al. Ž1992.. In other studies in which differences in LH secretion occurred in response to differences in nutritional management during lactation, there were differential effects of these treatments on the weaning-to-estrus interval, ovulation rate and embryonic survival, which might also be functionally related to the pattern of LH secretion in lactation ŽZak et al., 1997.. However, in this study marked differences in the pattern of LH secretion during lactation were not reflected in differences on the LH response to weaning. Further evidence is therefore needed to establish whether differences in the pattern of LH secretion during lactation in the sow are functionally related to differences in LH secretion, and in ovarian development and fertility, after weaning. Associations between gonadotrophin status and fertility have been established in a number of species. For example, a positive correlation between peripheral concentrations of FSH in the lamb and genetic merit for litter size in the mature ewe has been reported ŽRicordeu, 1982, Ricordeu et al., 1994.. The other gonadotrophin that can be considered is LH. However, since endogenous LH release is pulsatile, and accurate characterization of LH secretion requires frequent sampling, it is a poor candidate for use as a practical indicator of potential fertility, whereas LH release after GnRH injection is more easily quantified. Interestingly therefore in sheep, various associations have been reported amongst endogenous LH concentrations, the GnRH-induced increase in plasma LH and prolificacy ŽThimonier et al., 1972; Carr and Land, 1975; Land et al., 1982; Evans et al., 1994.. Studies of the prolific Finnish Landrace ewe and its crosses, in comparison with lambs from less prolific breeds, also suggested a correlation between LH responses to GnRH and the index of prolificacy ŽLand and Carr, 1979.. While the later study of Hanrahan Ž1982. did not support this conclusion, Haley et al. Ž1989. suggest that there is a different response between low- and high-line ewes to GnRH stimulation at puberty or at the beginning of the breeding season. Although similar experimental approaches have been taken to establish differences in gonadotrophin status in prolific Meishan compared to European breeds of pigs ŽTilton et al., 1994., comparable studies are lacking in lactating and weaned sows of different fertility. However, bolus injections of GnRH significantly increase LH secretion during lactation in the pig ŽBevers et al., 1981; Rojanasthien et al., 1987; De Rensis et al., 1991; Sesti and Britt, 1994. and gonadotrophin responses to GnRH injection have been
F. De Rensis, G.R. Foxcroftr Animal Reproduction Science 56 (1999) 143–152
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used to characterize reproductive status in prepubertal gilts ŽDeligeorgis et al., 1985.. The injection of opioid antagonists Ži.e. naloxone. also increases LH secretion during lactation ŽBarb et al., 1986; Armstrong et al., 1988; De Rensis et al., 1998; Okrasa and Kalamarz, 1996. and may indicate the potential pattern of LH secretion that will occur when the inhibitory effects of suckling are removed at weaning. The aim of this study was therefore to evaluate the practicality of establishing a correlation between endogenous LH secretion and the magnitude of the LH response to challenges with GnRH and naloxone during lactation, and between these characteristics and LH secretion and follicular development after weaning.
2. Materials and methods 2.1. Animals and blood collection A total of 10 multiparous Camborough sows ŽPIC, Acme, AB, Canada. from the University of Alberta herd were used. Sows were housed in conventional farrowing crates and water was provided ad libitum throughout lactation. A standard lactating sow ration was provided twice daily at 0800 and 1600 h on the basis of body weight and lactational requirements for each sow. Daily food intake and body weight loss during lactation in each sow were determined. The average weight of the sows one day after farrowing was 175 " 1.2 kg and the average litter size during the experiment was 9.7 " 0.7 piglets. All animals were cannulated non-surgically using ear vein catheters 22–24 h after the end of farrowing. Blood samples were then collected for 6 h at 15 min intervals on day 2 post-partum and catheters removed immediately after the end of this initial sampling period. Seven days before weaning at day 27 of lactation all sows were provided surgically with indwelling jugular catheters under general anesthesia as described previously ŽCosgrove et al., 1993.. Blood samples were again collected at 15 min intervals for a 12 h period on day 26 of lactation. Four hours after the beginning of sampling all sows received 2 mgrkg i.v. of naloxone hydrochloride ŽSigma. and 5 h later, 100 mgrsow i.v. of GnRH ŽSigma.. Finally, starting 6 h after weaning on day 27 of lactation, sows were sampled for a further 6-h period. All animal procedures were carried out with prior approval of the University Animal Policy and Welfare Committee and according to CCAC guidelines. 2.2. OÕarian deÕelopment Follicular development was studied in the left ovary of all sows after slaughter at a commercial abattoir the day after weaning; the other ovary was used for mRNA extraction for analyses not reported in this paper. Follicular development was assessed by measuring the volume of follicular fluid recovered from the 10 largest follicles on the dissected ovary.
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F. De Rensis, G.R. Foxcroftr Animal Reproduction Science 56 (1999) 143–152
2.3. Hormone assays Plasma LH was quantified in all samples using the double antibody RIA described by De Rensis et al. Ž1993b.. Mean inter- and intra-assay c.v. were 12.7 and 4.1%, respectively, and the sensitivity of the assay, defined as 90% of total binding, was 0.12 ngrml. 2.4. Statistical analysis The jugular cannulae of one sow failed to remain patent throughout the period of sampling at day 26 and data from this animal were not included in the analyses. LH data from the 6-h sampling windows on day 2 post-partum and after weaning were characterized as means of a single period of 6 h Žperiods 1 and 6, respectively.. Data from the 12-h sampling window on day 26 of lactation was characterized as means representing a 4-h period before naloxone injections Žperiod 2., a 2-h period after naloxone injections Žperiod 3., and two 3-h periods before Žperiod 4. and after Žperiod 5. GnRH injections. Consideration of the plasma LH profiles in this study indicated that the adoption of computerized pulse analysis programs was inappropriate because in many cases discrete pulsatile secretion was not evident at times when plasma LH concentrations were apparently changing. Therefore LH profiles were analysed using the sliding window technique described by Shaw and Foxcroft Ž1985., to provide minimum, mean and maximum characteristics of LH secretion and thus a complete and more appropriate method of analysis ŽFoxcroft et al., 1988.. Preliminary analysis of the data determined from the sliding windows analysis indicated that treatment effects on minimum, maximum and mean LH characteristics were similar; therefore only the data for mean LH are presented in the results. Comparison of means for periods 1, 2 and 6 were analysed separately using a repeated measures analysis of variance within the PROC GLM procedure of the SAS statistical package ŽSAS Institute, 1988. to establish changes in endogenous LH secretion during lactation and after weaning. In the presence of significant period effects, specific comparisons between these means were made using Student– Newman–Keuls multiple range test. The LH response to naloxone during sampling on day 26 of lactation was analysed using a priori comparisons between means for periods 2 and 3 by the same statistical procedures. Having established no differences between period 2 and 4, and thus the absence of any carry-over effect of naloxone treatment on LH secretion, the response to the GnRH challenge Žperiod 5. was tested using repeat measures analysis between period 4 and 5. Differences in mean follicular volume were examined by PROC ANOVA. Regression analysis was used to determine relationships among follicular volume and mean plasma LH concentrations and among different LH means. 3. Results Fig. 1 shows individual patterns of LH secretion in four representative sows. Endogenous LH secretion was affected by day of sampling Ž P - 0.001., and was
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Fig. 1. Individual plasma LH profiles in four sows at day 1 and day 26 of lactation and on the day of weaning. On day 26 of lactation challenges with i.v. bolus injections of naloxone and GnRH occurred 3 and 9 h after the start of sampling.
reflected in higher Ž P - 0.05. mean LH concentrations on the day after weaning Žperiod 6. compared to mean LH concentrations during lactation Žperiods 1, 2 and 4. ŽFig. 2.. There were no significant correlations between LH concentrations in periods 1 and 2, and LH concentrations after weaning. Naloxone Ž P - 0.05. and GnRH Ž P - 0.05.
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Fig. 2. Mean ŽqS.E.M.. plasma LH from all sows Ž ns9. at day 2 after farrowing Žperiod 1. at day 26 of lactation Žperiods 2 and 4. and 6 h after weaning Žperiod 6.. a,bs Means with different superscripts differ Ž P - 0.05..
treatment increased mean LH concentrations ŽFig. 3.. However, there was no relationship between the LH response to naloxone and to GnRH treatments. A positive
Fig. 3. ŽPanel A. Mean ŽqS.E.M.. plasma LH from all sows Ž ns9. at day 26 of lactation pre- and after naloxone injection Žperiods 2 and 3.. ŽPanel B. Mean ŽqS.E.M.. plasma LH from all sows Ž ns9. at day 26 of lactation pre- and after GnRH injection Žperiods 4 and 5.. a,bs Means with different superscripts differ Ž P - 0.05..
F. De Rensis, G.R. Foxcroftr Animal Reproduction Science 56 (1999) 143–152
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Fig. 4. Correlation Ž r s 0.58, P - 0.01. between mean plasma LH after GnRH ŽPeriod 5. and after weaning ŽPeriod 6..
relationship Ž r s 0.58, P - 0.01. between mean plasma LH after GnRH Žperiod 5. and after weaning Žperiod 6. was established ŽFig. 4.. The mean Ž"S.E.M.. follicular fluid volumes for the 10 largest follicles dissected from the nine sows were 66.1 " 7.2, 317.8 " 44.6, 186.5 " 15.0, 67.5 " 13.4, 48.5 " 4.3, 68.5 " 7.1, 57.1 " 5.5, 55.2 " 3.2, 12.7 " 2.1 ml, respectively, and although there were differences Ž P - 0.001. among sows in mean follicular fluid volume, there were no correlations between mean follicular fluid volume and any of the measures of LH secretion. 4. Discussion The lack of any correlation between LH concentrations during lactation and immediately after weaning in the present study is consistent with the data of Zak et al. Ž1997. in the same population of sows and our data provide no evidence that the endogenous pattern of LH secretion in early or late lactation can be used as a reliable predictor of LH activity after weaning, even when sow management is standardized. Thus, although earlier studies established significant differences between sows in LH secretion immediately after farrowing ŽDe Rensis et al., 1993b., it appears that other Žmetabolic and environmental. factors determine the eventual pattern of LH secretion in established lactation and after weaning. Haley et al. Ž1989. classified two lines of lambs as high or low responders on the basis of the response to parenteral GnRH. They observed that the LH response to GnRH stimulation in their male progeny was positively correlated with body weight and mean testis diameter, suggesting that selection for improved reproductive performance on the basis of the LH response to a GnRH challenge is possible. These observations have been recently confirmed in the adult ewe by Evans et al. Ž1994.. In the present experiment a positive correlation between mean plasma LH concentrations after GnRH treatment in late lactation and after weaning was established. Although this might suggest that the use of a GnRH challenge before weaning could therefore potentially be used as a
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predictor of LH response to weaning, neither Shaw and Foxcroft Ž1985. nor Foxcroft et al. Ž1987. were able to correlate LH secretory characteristics after weaning with measures of ovarian activity or subsequent fertility. The opioids are involved in the mechanisms mediating the inhibitory effect of suckling stimulus in established lactation on LH secretion ŽBarb et al., 1986; Armstrong et al., 1988; De Rensis et al., 1993a, 1998; Okrasa and Kalamarz, 1996.. In the present experiment naloxone treatment increased LH concentration, but a more robust LH response was observed after GnRH administration. In part these data confirm and extend the result of Barb et al. Ž1986. and indicate that in pigs, naloxone alone may not be able to totally disinhibit the suckling-induced block of LH secretion and that naloxone challenges to lactating sows may not provide a reliable estimate of potential LH secretory activity. Even though the ability to predict the pattern of LH secretion after weaning on the basis of LH responses to a GnRH challenge in late lactation may not help in predicting subsequent fertility, it is still possible that a measure of uninhibited LH secretion in lactation may show a better correlation to subsequent ovarian activity and fertility than an estimate of endogenous LH secretion. Given the existence of very low episodic LH frequencies in lactation, there are considerable technical difficulties in accurately characterizing endogenous LH secretion. However, Foxcroft et al. Ž1987. observed that mean plasma LH in the preweaning period was positively correlated to several characteristics of ovarian development 48 h after weaning, and Prunier et al. Ž1993. reported that delayed estrus was associated with a low secretion of oestradiol by the ovaries the day after weaning. Although in the present work we observed large variability in the follicular development among sows, there were no correlations between any measure of LH secretion during lactation and follicular development after weaning. The reasons for this discrepancy between studies is uncertain, but these data suggest that more direct evidence is needed to support the suggestion that the resumption of follicular development after weaning in the sow is functionally related to the pattern of endogenous LH secretion or to size of the releasable pool of LH in lactation.
Acknowledgements This research was supported by grants from C.N.R., Italy, Žcontribution no. 95.04411.CT04, posiz. 115.30478. n 132405., by MURST 60% and by contributions and grant no. 121.08575, prot. 048290 and from the Natural Sciences and Engineering Research council of Canada. For the experimental support we acknowledge D. Irwin, T. Wiesak, S. Charlton and E. Clowes.
References Armstrong, J.D., Kraeling, R.R., Britt, J.H., 1988. Effects of naloxone or transient weaning on secretion of LH and prolactin in lactating sows. J. Reprod. Fertil. 83, 301–308. Barb, C.R., Kraeling, R.R., Rampacek, G.B., Whisnant, C.S., 1986. Opioid inhibition of luteinizing hormone secretion in the post-partum lactating sow. Biol. Reprod. 35, 368–371.
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Bevers, M.M., Willemse, M.H., Kruip, T.A.M., Van de Wiel, D.F.M., 1981. Prolactin levels and the LH response to synthetic LHRH in lactating sow. Anim. Reprod. Sci. 4, 155–163. Carr, W.R., Land, R.B., 1975. Plasma LH levels and testis diameter of ram lambs of different breeds. J. Reprod. Fertil. 42, 325–333. Cosgrove, J.R., Urbanski, H.F., Foxcroft, G.R., 1993. Maturational changes in gonadotrophin secretion: the LH response to realimentation and a nocturnal increment in LH secretion of feed-restricted prepubertal gilts. J. Reprod. Fertil. 98, 293–300. Deligeorgis, S.G., English, P.R., Lodge, G.A., 1985. Interrelationships between growth, gonadotrophin secretion and sexual maturation in gilts reared in different litter sizes. Anim. Prod. 41, 393–401. De Rensis, F., Hunter, M.G., Grant, S.A., Lancaster, R.T., Foxcroft, G.R., 1991. Effect of oestrogen administration on endogenous and LHRH-induced LH secretion and follicular development in lactating sow. Biol. Reprod. 44, 975–982. De Rensis, F., Cosgrove, J.R., Foxcroft, G.R., 1993a. LH and prolactin response to naloxone vary with stage of lactation in the sow. Biol. Reprod. 48, 970–976. De Rensis, F., Hunter, M.G., Foxcroft, G.R., 1993b. Suckling-induced inhibition of LH secretion and follicular development in the early post-partum sow. Biol. Reprod. 48, 964–969. De Rensis, F., Cosgrove, J.R., Foxcroft, G.R., 1998. Ontogeny of the opioidergic regulation of LH and prolactin secretion in lactating sow: I. Failure of naloxone to antagonize suckling-induced changes in LH and prolactin secretion in early lactation, irrespective of pattern of administration. J. Reprod. Fertil. 112, 79–85. Evans, N.P., McNeilly, J.R., Webb, R., 1994. Alterations in endogenous gonadotropin secretion and pituitary responsiveness to gonadotropin-releasing hormone in adult ewes, following indirect selection in prepubertal male lambs. Biol. Reprod. 51, 913–919. Foxcroft, G.R., Shaw, H.J., Hunter, M.G., Booth, P.J., Lancaster, R.T., 1987. Relationships between luteinizing hormone, follicle-stimulating hormone and prolactin secretion and ovarian follicular development in the weaned sow. Biol. Reprod. 36, 175–191. Foxcroft, G.R., Haresign, N.B., Haynes, N.B., Lamming, G.E., Peters, A.R., 1988. Gonadotropins — domestic animals. Acta Endocrinol. 288, 41–50. Haley, C.S., Lee, G.J., Fordyce, M., Baxter, G., Land, R.B., Webb, R., 1989. Study of LH response to GnRH in the young female as a criterion of genetic merit for female reproduction in the sheep. J. Reprod. Fertil. 86, 119–133. Hanrahan, J.P., 1982. Selection for increased ovulation rate, litter size, and embryo survival. Proc. 2nd World Congr. Genet. Appl. Livest. Prod. 5, 294–309. Land, R.B., Carr, W.R., 1979. Reproduction in domestic mammals. In: Shire, J.G.M. ŽEd.., Genetic Variation in Hormone Systems. CRC Press, London, pp. 89–112. Land, R.B., Gauld, I.K., Lee, G.J., Webb, R., 1982. Further possibilities for manipulating the reproductive process. In: Barker, J.S.F., Hammond, K., McClimtock ŽEds.., Future Developments in Genetic Improvement of Animals. Academic Press, Sydney, pp. 59–87. Okrasa, S., Kalamarz, H., 1996. Involvement of opioid system in the control of LH secretion in sows. Reprod. Domest. Anim. 31, 575–583. Prunier, A., Dourmad, J.Y., Etienne, M., 1993. Feeding level, metabolic parameters and reproductive performance of primiparous sows. Livest. Prod. Sci. 37, 185–196. Ricordeu, G., 1982. Selection for reduced seasonality and post-partum anoestrus. Proc. 2nd World Congr. Genet. Appl. Livest. Prod. 5, 338–347. Ricordeu, G., Blanc, M.R., Bodin, L., 1994. Teneurs plasmatiques en FSH et LH des agneaux males et femelles issus de belivers Lacauneprolifiques et non prolifiques et non prolifiques. Gen. Sel. Evol. 16, 195–210. Rojanasthien, S.A.M., Lundeheim, N., Einarsson, S., 1987. Luteinizing hormone response to different doses of synthetic gonadotrophin releasing hormone during early and late lactation in primiparous sows. Anim. Reprod. Sci. 13, 299–307. SAS Institute, 1988. Statistical Analysis System. SAS Institute, Cary, NC, USA. Sesti, L.A.C., Britt, J.H., 1994. Secretion of gonadotropins and estimated releasable pools of gonadotropin-releasing hormone and gonadotropins during establishment of suckling-induced inhibition of gonadotropin secretion in the sow. Biol. Reprod. 50, 1078–1086.
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Shaw, H.J., Foxcroft, G.R., 1985. Relationship between LH, FSH, and prolactin secretion and reproductive activity in the weaned sow. J. Reprod. Fertil. 75, 17–28. Thimonier, J., Pelletier, J., Land, R.B., 1972. The concentration of plasma LH in male and female lambs of high and low prolificacy breed types. J. Reprod. Fertil. 31, 498–499. Tilton, J.E., Biggs, C., Hunter, M.G., Foxcroft, G.R., 1994. Gonadotropin secretion in ovariectomized Chinese Meishan and hybrid Large White gilts: responses to challenges with estradiol benzoate, gonadotropin releasing hormone or porcine follicular fluid. Biol. Reprod. 51, 963–970. Tokach, M.D., Pettigrew, J.E., Dial, G.D., Wheaton, J.E., Crooker, B.A., Johnston, L.J., 1992. Characterization of LH secretion in the primiparous lactating sow: relationship to blood metabolites and return to estrus interval. J. Anim. Sci. 70, 2195–2201. Zak, L.J., Cosgrove, J.R., Aherne, F.X., Foxcroft, G.R., 1997. Pattern of feed intake and associated metabolic and endocrine changes differentially affect postweaning fertility in primiparous lactating sow. J. Anim. Sci. 75, 208–216.
Correlation between LH response to challenges with GNRH and naloxone during lactation, and LH secretion and follicular development after weaning in the sows Fabio De Rensis
a,)
, George R. Foxcroft
b
a
Istituto di Clinica Ostetrica Veterinaria, UniÕersita` degli Studi di Parma, Õia del Taglio 8, 43100, Parma, Italy b Department of Agricultural, Food and Nutritional Science, 310-C Agriculturer Forestry Building, UniÕersity of Alberta, Edmonton, Alberta, Canada T6G 2P5 Accepted 18 May 1999
Abstract The aim of this study was of establishing a correlation between endogenous LH secretion and the magnitude of the LH response to challenges with GnRH and the opioid antagonist naloxone during lactation, and between these characteristics and LH secretion and follicular development after weaning. Sows Ž n s 9. were sampled for 6 h at day 2 post-partum, for 12 h on day 26 of lactation and for 6 h immediately after weaning at day 27 of lactation. Four hours after the beginning of sampling at day 26 of lactation all sows were injected with 2 mgrkg i.v. of naloxone hydrochloride and 5 h later with 100 mgrsow of GnRH. Follicular development was studied in all sows at slaughter the day after weaning. There was an effect of time Žsampling period; P - 0.001. on mean plasma LH, with an increase Ž P - 0.05. in LH the day after weaning compared to mean LH concentrations during lactation. Naloxone and GnRH treatment both increased Ž P - 0.05. mean LH concentrations. A positive relationship Ž r s 0.58, P - 0.01. between mean plasma LH after GnRH and after weaning was established. Although there were differences Ž P - 0.001. between sows in follicular fluid volume, there were no correlations between mean follicular fluid volume and mean LH concentrations after GnRH or after weaning. These data indicate that the LH response to GnRH during late lactation could be useful predictor of LH activity after weaning. However, none of the measures of endogenous or induced LH secretion were associated with differences in ovarian follicular size after weaning. Direct evidence is therefore still needed for
)
Corresponding author. Tel.: q39-0521-902659; fax: q39-0521-902662; e-mail: [email protected] 0378-4320r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 3 8 - X
144
F. De Rensis, G.R. Foxcroftr Animal Reproduction Science 56 (1999) 143–152
afunctional link between differences in LH in lactation and differences in fertility after weaning. q 1999 Elsevier Science B.V. All rights reserved. Keywords: LH; Lactation; Pig-endocrinology; GnRH; Opioids
1. Introduction Shaw and Foxcroft Ž1985. reported a reciprocal relationship between mean LH concentrations in late lactation and the length of the weaning to estrus interval and suggested that the LH status of the sow during lactation might be causally related to the resumption of ovarian activity and to fertility after weaning. This suggestion was supported by the subsequent data from the same laboratory ŽFoxcroft et al., 1987. and by the data of Tokach et al. Ž1992.. In other studies in which differences in LH secretion occurred in response to differences in nutritional management during lactation, there were differential effects of these treatments on the weaning-to-estrus interval, ovulation rate and embryonic survival, which might also be functionally related to the pattern of LH secretion in lactation ŽZak et al., 1997.. However, in this study marked differences in the pattern of LH secretion during lactation were not reflected in differences on the LH response to weaning. Further evidence is therefore needed to establish whether differences in the pattern of LH secretion during lactation in the sow are functionally related to differences in LH secretion, and in ovarian development and fertility, after weaning. Associations between gonadotrophin status and fertility have been established in a number of species. For example, a positive correlation between peripheral concentrations of FSH in the lamb and genetic merit for litter size in the mature ewe has been reported ŽRicordeu, 1982, Ricordeu et al., 1994.. The other gonadotrophin that can be considered is LH. However, since endogenous LH release is pulsatile, and accurate characterization of LH secretion requires frequent sampling, it is a poor candidate for use as a practical indicator of potential fertility, whereas LH release after GnRH injection is more easily quantified. Interestingly therefore in sheep, various associations have been reported amongst endogenous LH concentrations, the GnRH-induced increase in plasma LH and prolificacy ŽThimonier et al., 1972; Carr and Land, 1975; Land et al., 1982; Evans et al., 1994.. Studies of the prolific Finnish Landrace ewe and its crosses, in comparison with lambs from less prolific breeds, also suggested a correlation between LH responses to GnRH and the index of prolificacy ŽLand and Carr, 1979.. While the later study of Hanrahan Ž1982. did not support this conclusion, Haley et al. Ž1989. suggest that there is a different response between low- and high-line ewes to GnRH stimulation at puberty or at the beginning of the breeding season. Although similar experimental approaches have been taken to establish differences in gonadotrophin status in prolific Meishan compared to European breeds of pigs ŽTilton et al., 1994., comparable studies are lacking in lactating and weaned sows of different fertility. However, bolus injections of GnRH significantly increase LH secretion during lactation in the pig ŽBevers et al., 1981; Rojanasthien et al., 1987; De Rensis et al., 1991; Sesti and Britt, 1994. and gonadotrophin responses to GnRH injection have been
F. De Rensis, G.R. Foxcroftr Animal Reproduction Science 56 (1999) 143–152
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used to characterize reproductive status in prepubertal gilts ŽDeligeorgis et al., 1985.. The injection of opioid antagonists Ži.e. naloxone. also increases LH secretion during lactation ŽBarb et al., 1986; Armstrong et al., 1988; De Rensis et al., 1998; Okrasa and Kalamarz, 1996. and may indicate the potential pattern of LH secretion that will occur when the inhibitory effects of suckling are removed at weaning. The aim of this study was therefore to evaluate the practicality of establishing a correlation between endogenous LH secretion and the magnitude of the LH response to challenges with GnRH and naloxone during lactation, and between these characteristics and LH secretion and follicular development after weaning.
2. Materials and methods 2.1. Animals and blood collection A total of 10 multiparous Camborough sows ŽPIC, Acme, AB, Canada. from the University of Alberta herd were used. Sows were housed in conventional farrowing crates and water was provided ad libitum throughout lactation. A standard lactating sow ration was provided twice daily at 0800 and 1600 h on the basis of body weight and lactational requirements for each sow. Daily food intake and body weight loss during lactation in each sow were determined. The average weight of the sows one day after farrowing was 175 " 1.2 kg and the average litter size during the experiment was 9.7 " 0.7 piglets. All animals were cannulated non-surgically using ear vein catheters 22–24 h after the end of farrowing. Blood samples were then collected for 6 h at 15 min intervals on day 2 post-partum and catheters removed immediately after the end of this initial sampling period. Seven days before weaning at day 27 of lactation all sows were provided surgically with indwelling jugular catheters under general anesthesia as described previously ŽCosgrove et al., 1993.. Blood samples were again collected at 15 min intervals for a 12 h period on day 26 of lactation. Four hours after the beginning of sampling all sows received 2 mgrkg i.v. of naloxone hydrochloride ŽSigma. and 5 h later, 100 mgrsow i.v. of GnRH ŽSigma.. Finally, starting 6 h after weaning on day 27 of lactation, sows were sampled for a further 6-h period. All animal procedures were carried out with prior approval of the University Animal Policy and Welfare Committee and according to CCAC guidelines. 2.2. OÕarian deÕelopment Follicular development was studied in the left ovary of all sows after slaughter at a commercial abattoir the day after weaning; the other ovary was used for mRNA extraction for analyses not reported in this paper. Follicular development was assessed by measuring the volume of follicular fluid recovered from the 10 largest follicles on the dissected ovary.
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2.3. Hormone assays Plasma LH was quantified in all samples using the double antibody RIA described by De Rensis et al. Ž1993b.. Mean inter- and intra-assay c.v. were 12.7 and 4.1%, respectively, and the sensitivity of the assay, defined as 90% of total binding, was 0.12 ngrml. 2.4. Statistical analysis The jugular cannulae of one sow failed to remain patent throughout the period of sampling at day 26 and data from this animal were not included in the analyses. LH data from the 6-h sampling windows on day 2 post-partum and after weaning were characterized as means of a single period of 6 h Žperiods 1 and 6, respectively.. Data from the 12-h sampling window on day 26 of lactation was characterized as means representing a 4-h period before naloxone injections Žperiod 2., a 2-h period after naloxone injections Žperiod 3., and two 3-h periods before Žperiod 4. and after Žperiod 5. GnRH injections. Consideration of the plasma LH profiles in this study indicated that the adoption of computerized pulse analysis programs was inappropriate because in many cases discrete pulsatile secretion was not evident at times when plasma LH concentrations were apparently changing. Therefore LH profiles were analysed using the sliding window technique described by Shaw and Foxcroft Ž1985., to provide minimum, mean and maximum characteristics of LH secretion and thus a complete and more appropriate method of analysis ŽFoxcroft et al., 1988.. Preliminary analysis of the data determined from the sliding windows analysis indicated that treatment effects on minimum, maximum and mean LH characteristics were similar; therefore only the data for mean LH are presented in the results. Comparison of means for periods 1, 2 and 6 were analysed separately using a repeated measures analysis of variance within the PROC GLM procedure of the SAS statistical package ŽSAS Institute, 1988. to establish changes in endogenous LH secretion during lactation and after weaning. In the presence of significant period effects, specific comparisons between these means were made using Student– Newman–Keuls multiple range test. The LH response to naloxone during sampling on day 26 of lactation was analysed using a priori comparisons between means for periods 2 and 3 by the same statistical procedures. Having established no differences between period 2 and 4, and thus the absence of any carry-over effect of naloxone treatment on LH secretion, the response to the GnRH challenge Žperiod 5. was tested using repeat measures analysis between period 4 and 5. Differences in mean follicular volume were examined by PROC ANOVA. Regression analysis was used to determine relationships among follicular volume and mean plasma LH concentrations and among different LH means. 3. Results Fig. 1 shows individual patterns of LH secretion in four representative sows. Endogenous LH secretion was affected by day of sampling Ž P - 0.001., and was
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Fig. 1. Individual plasma LH profiles in four sows at day 1 and day 26 of lactation and on the day of weaning. On day 26 of lactation challenges with i.v. bolus injections of naloxone and GnRH occurred 3 and 9 h after the start of sampling.
reflected in higher Ž P - 0.05. mean LH concentrations on the day after weaning Žperiod 6. compared to mean LH concentrations during lactation Žperiods 1, 2 and 4. ŽFig. 2.. There were no significant correlations between LH concentrations in periods 1 and 2, and LH concentrations after weaning. Naloxone Ž P - 0.05. and GnRH Ž P - 0.05.
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Fig. 2. Mean ŽqS.E.M.. plasma LH from all sows Ž ns9. at day 2 after farrowing Žperiod 1. at day 26 of lactation Žperiods 2 and 4. and 6 h after weaning Žperiod 6.. a,bs Means with different superscripts differ Ž P - 0.05..
treatment increased mean LH concentrations ŽFig. 3.. However, there was no relationship between the LH response to naloxone and to GnRH treatments. A positive
Fig. 3. ŽPanel A. Mean ŽqS.E.M.. plasma LH from all sows Ž ns9. at day 26 of lactation pre- and after naloxone injection Žperiods 2 and 3.. ŽPanel B. Mean ŽqS.E.M.. plasma LH from all sows Ž ns9. at day 26 of lactation pre- and after GnRH injection Žperiods 4 and 5.. a,bs Means with different superscripts differ Ž P - 0.05..
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Fig. 4. Correlation Ž r s 0.58, P - 0.01. between mean plasma LH after GnRH ŽPeriod 5. and after weaning ŽPeriod 6..
relationship Ž r s 0.58, P - 0.01. between mean plasma LH after GnRH Žperiod 5. and after weaning Žperiod 6. was established ŽFig. 4.. The mean Ž"S.E.M.. follicular fluid volumes for the 10 largest follicles dissected from the nine sows were 66.1 " 7.2, 317.8 " 44.6, 186.5 " 15.0, 67.5 " 13.4, 48.5 " 4.3, 68.5 " 7.1, 57.1 " 5.5, 55.2 " 3.2, 12.7 " 2.1 ml, respectively, and although there were differences Ž P - 0.001. among sows in mean follicular fluid volume, there were no correlations between mean follicular fluid volume and any of the measures of LH secretion. 4. Discussion The lack of any correlation between LH concentrations during lactation and immediately after weaning in the present study is consistent with the data of Zak et al. Ž1997. in the same population of sows and our data provide no evidence that the endogenous pattern of LH secretion in early or late lactation can be used as a reliable predictor of LH activity after weaning, even when sow management is standardized. Thus, although earlier studies established significant differences between sows in LH secretion immediately after farrowing ŽDe Rensis et al., 1993b., it appears that other Žmetabolic and environmental. factors determine the eventual pattern of LH secretion in established lactation and after weaning. Haley et al. Ž1989. classified two lines of lambs as high or low responders on the basis of the response to parenteral GnRH. They observed that the LH response to GnRH stimulation in their male progeny was positively correlated with body weight and mean testis diameter, suggesting that selection for improved reproductive performance on the basis of the LH response to a GnRH challenge is possible. These observations have been recently confirmed in the adult ewe by Evans et al. Ž1994.. In the present experiment a positive correlation between mean plasma LH concentrations after GnRH treatment in late lactation and after weaning was established. Although this might suggest that the use of a GnRH challenge before weaning could therefore potentially be used as a
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predictor of LH response to weaning, neither Shaw and Foxcroft Ž1985. nor Foxcroft et al. Ž1987. were able to correlate LH secretory characteristics after weaning with measures of ovarian activity or subsequent fertility. The opioids are involved in the mechanisms mediating the inhibitory effect of suckling stimulus in established lactation on LH secretion ŽBarb et al., 1986; Armstrong et al., 1988; De Rensis et al., 1993a, 1998; Okrasa and Kalamarz, 1996.. In the present experiment naloxone treatment increased LH concentration, but a more robust LH response was observed after GnRH administration. In part these data confirm and extend the result of Barb et al. Ž1986. and indicate that in pigs, naloxone alone may not be able to totally disinhibit the suckling-induced block of LH secretion and that naloxone challenges to lactating sows may not provide a reliable estimate of potential LH secretory activity. Even though the ability to predict the pattern of LH secretion after weaning on the basis of LH responses to a GnRH challenge in late lactation may not help in predicting subsequent fertility, it is still possible that a measure of uninhibited LH secretion in lactation may show a better correlation to subsequent ovarian activity and fertility than an estimate of endogenous LH secretion. Given the existence of very low episodic LH frequencies in lactation, there are considerable technical difficulties in accurately characterizing endogenous LH secretion. However, Foxcroft et al. Ž1987. observed that mean plasma LH in the preweaning period was positively correlated to several characteristics of ovarian development 48 h after weaning, and Prunier et al. Ž1993. reported that delayed estrus was associated with a low secretion of oestradiol by the ovaries the day after weaning. Although in the present work we observed large variability in the follicular development among sows, there were no correlations between any measure of LH secretion during lactation and follicular development after weaning. The reasons for this discrepancy between studies is uncertain, but these data suggest that more direct evidence is needed to support the suggestion that the resumption of follicular development after weaning in the sow is functionally related to the pattern of endogenous LH secretion or to size of the releasable pool of LH in lactation.
Acknowledgements This research was supported by grants from C.N.R., Italy, Žcontribution no. 95.04411.CT04, posiz. 115.30478. n 132405., by MURST 60% and by contributions and grant no. 121.08575, prot. 048290 and from the Natural Sciences and Engineering Research council of Canada. For the experimental support we acknowledge D. Irwin, T. Wiesak, S. Charlton and E. Clowes.
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