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Effect of hCG administration at the onset of oestrus on early embryo survival and development in Meishan gilts
ANIMAL REPRODUCTION SCIENCE ELSEVIER
Animal Reproduction Science 38 (1995) 231-238
Effect of hCG administration at the onset of oestrus on early embryo survival and development in Meishan gilts M.G. Hunter*, H.M. Picton Department of Physiology and Environmental Science, University of Nottingham, Sutton Bonington Campus, Sutton Bonington Leicestershire LE12 5RD, UK Accepted 12 October 1994
Abstract
A study was designed to advance the time of the ovulatory luteinizing hormone (LH) surge in Meishan gilts by human chorionic gonadotrophin (hCG) administration at the onset of oestrus and assess the effect on embryo survival and development. Twelve Meishan gilts were observed six times daily for oestrous behaviour and bred at 24, 36 and 48 h after observed oestrous onset. Six of those gilts were administered an ovulatory dose of hCG (500 IU) at observed oestrous onset. Blood samples were collected at oestrous onset (Day - 2 ) and on Days 0, 2, 6, 9, 13, 16, 20, 23, 27 and 30 of gestation. All gilts were slaughtered on Day 30 of gestation and embryo survival and conceptus development assessed. Ovulation rate did not differ between control and hCG treated gilts ( 18.5 and 17.7 respectively; P > 0.1 ) while the number of live conceptuses per gilt ( 17.2 and 12.8 respectively; P<0.08) and embryo survival rate (92.1 and 75.8% respectively; P<0.1) both tended to be reduced by the hCG treatment. Placental weight ( 17.2 and 23.1 g; P < 0.01 ) was significantly increased in hCG treated gilts, while embryo weight (1.2 and 1.4 g; P < 0.06) and placental length (42.8 and 47.2 cm; P < 0.07) both tended to be increased in hCG treated gilts. Crown rump length (P> 0.1 ) and allantoic fluid volume (P> 0,1 ) did not differ between the treatment groups. Serum progesterone concentrations did not differ with treatment overall (P> 0.1 ) but were significantly elevated ( P < 0.05) at 48 h postoestrus in the hCG treated gilts compared to control gilts. Overall, these results indicate that advancing the time of the LH surge to oestrous onset, as in European breeds, compromised embryo survival and suggests that the longer time interval between oestrous onset and ovulation is important for the high rate of embryo survival in the Meishan pig. Keywords: Pig; Human chorionic gonadotrophin, hCG; Oestrus; Pregnancy; Embryology
* Corresponding author. 0378-4320/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI0378-4320(94)01365-9
232
M. G. Hunter, H.34. Picton / Animal Reproduction Science 38 (1995) 231-238
1. Introduction
The Chinese Meishan pig is characterized by its prolificacy compared to European breeds, producing an average of three to four more piglets per litter (Bolet et al., 1986; Bidanel et al., 1989; Haley and Lee, 1993 ). Although Meishan sows and gilts that have experienced an extended number of oestrous cycles have a higher ovulation rate than their European counterparts (Haley and Lee, 1993; Christenson et al., 1993; Hunter et al., 1993 ), this is not the major cause of their prolificacy. This prolificacy is considered to result from an increased level of embryo survival, regardless of ovulation rate (Bazer et al., 1988; Haley and Lee, 1990). This increased survival has been reported to occur as early as Day 12 of gestation (Bazer et al., 1988 ) and is clearly evident by Day 20 of gestation (Haley and Lee, 1993). Studies of the timing of peri-ovulatory events have shown that the interval between the onset of behavioural oestrus and ovulation is longer in Meishan than in European breeds, at approximately 49 h as opposed to 34 h respectively (Terqui et al., 1990; Wilmut et al., 1992; Faillace et al., 1994). Administration of human chorionic gonadotrophin (hCG) to Meishan gilts at the onset of oestrus significantly shortened the time to ovulation (Faillace et al., 1994), suggesting that the luteinizing hormone (LH) surge may normally occur later in the oestrous period in Meishan animals. Indeed, endocrine studies of the peri-ovulatory period confirmed that Meishan gilts exhibit behavioural oestrus significantly earlier than Large White females relative to the oestradiol and LH peaks (Hunter et al., 1993 ). Although preliminary investigations by Ellendorf et al. ( 1988 ) suggested that the LH surge is higher in Meishan pigs, this was not supported by the more substantial studies of Hunter et al. (1993) who found no difference between the breeds. The aim of the present study, therefore, was to investigate the importance of the later occurrence of the LH surge during the oestrous period on embryo survival in Meishan animals. The timing of the endogenous LH surge in Meishan gilts was altered by hCG administration at the onset of oestrus, thus making the timing similar to that in European breeds. Embryo survival and development on Day 30 of gestation as well as circulating progesterone concentrations were assessed in control and hCG treated animals.
2. Materials and methods 2.1. A n i m a l s
Twelve Meishan gilts exhibiting regular oestrous cycles were used in this study. Each of the gilts had experienced at least eight oestrous cycles. Gilts were penned
M.G. Hunter, H.M. Picton/Animal Reproduction Science 38 (1995) 231-238
233
randomly in two groups in a ventilated building with natural daylength and temperature, fed once daily and had unlimited access to drinking water. 2.2. Treatment
Oestrous detection, using vasectomized boars, '-was carried out six times daily from at least 2 days prior to anticipated oestrous onset. At the onset of oestrus, six of the 12 gilts were administered an intramuscular dose of 500 IU of hCG (Sigma Chemical Co., Dorset, UK). All gilts were bred by a fertile boar at 24, 36 and 48 h after the onset of oestrus. Blood samples (8 ml) were taken by venepuncture from the anterior vena cava of each gilt within 4 h of the onset ofoestrus (Day - 2 of gestation), at 48 h post-oestrus (Day 0 of gestation) and twice weekly (Days 2, 6, 9, 13, 16, 20, 23, 27, 30) until slaughter on Day 30. Samples were left at room temperature until a clot had formed and centrifuged at 1500Xg for 10 min; serum was aspirated and stored at - 2 0 °C until radioimmunoassay. 2.3. Conceptus dissection and measurement
The entire reproductive tract was recovered following slaughter and individual conceptuses were recovered. Allantoic fluid volume was measured and a 5 ml aliquot stored at - 20 oC. Conceptus development was measured as described by Knight et al. ( 1977 ) and included crown-rump length, embryo weight, placental length and weight and uterine weight and length. The ovaries were also dissected to determine ovulation rate. 2.4. Radioimmunoassay
The concentrations of progesterone in serum and allantoic fluid were measured by the extracted radioimmunoassay described by Hunter et al. (1986). Mean extraction efficiency was 88.8%, limit of sensitivity was 9.3 pg per tube and interand intra-assay coefficients of variation were 16.9 and 17.0% respectively. 2.5. Statistical analysis
All data were analysed by analysis of variance using the GENSTAT statistical package (GENSTAT 5 Committee, 1989). The pooled variance was used to calculate the standard error of the difference (SED) and comparisons were made using a model that included treatment, and employed gilt as the block structure. Plasma concentrations of progesterone were analysed by a multiple regression model which included treatment, day of gestation and their interaction, and employed gilt as the block structure.
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3. Results 3.1. Whole litter measurements The whole litter characteristics as measured at Day 30 of gestation are summarized in Table 1. The ovulation rate was similar between the control and hCG treated animals (P> 0.1 ), as were uterine length and weight (P> 0.1 ). However, the number of live embryos (P< 0.08 ) and percentage embryo survival (P< 0.1 ) both tended to be reduced in the hCG treated animals compared to controls. 3.2. Conceptus characteristics Individual conceptus characteristics which are summarized in Table 2 showed no differences ( P > 0.1 ) in crown-rump length or allantoic fluid volume between control and hCG treated animals. There was, however, a consistent trend for embryo weight (P<0.06), placental length (P<0.07) and placental weight ( P < 0.01 ) to be greater in the hCG treated animals than in the controls. Neither uterine weight ( P > 0.1 ) or uterine length (P> 0.1 ) were different between the groups. Table 1 Whole litter characteristics in control (n = 6 ) and hCG treated (n = 6 ) Meishan gilts
Ovulation rate (CL) Live embryos (no.) Embryo survival (%) Uterine length (cm) Uterine weight (kg)
Control
hCG treated
SED
18.5 17.2 92.1 286.0 1.12
17.7 12.8" 75.8** 281.0 1.17
2.68 2.25 8.89 29.3 0.15
*P<0.08;**P<0.1. SED, standard error of difference. Table 2 Individual conceptus characteristics in control (n = 6) and hCG treated (n = 6) Meishan gilts
Crown-rump (mm) Embryo weight (g) Placental length (cm) Placental weight (g) Allantoic fluid (ml) *P< 0.07; **P< 0.01. SED0 standard error of difference.
Control
hCG treated
SED
24.1 1.20* 42.8* 17.2"* 144.2
25.0 1.40 47.2 23.1 149.3
0,6 0.10 2.1 1.9 18.1
M.G. Hunter, H.M. Picton /Animal Reproduction Science 38 (1995) 231-238
235
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Fig. l. Serum progesterone concentrations (mean _+SEM ) in control ( O; n = 6 ) and h CG treated ( ®; n = 6 ) Meishan gilts from the onset ofoestrus (Day - 2 ) until Day 30 of gestation. *P<0.05.
3.3. Progesterone concentrations The concentration of progesterone in allantoic fluid was similar between the control and hCG treated groups ( 1.08 and 1.12 ng m l - ~respectively, SED = 0.09; P > 0.1 ), as was total allantoic fluid progesterone per conceptus ( 154 and 172 ng respectively, SED = 21.3; P > 0.1 ). Serum progesterone concentrations until Day 30 of gestation are shown in Fig. 1. Overall, there was no significant difference between the two treatment groups ( P > 0.1 ), but when individual days were analysed separately the concentration of progesterone was significantly higher ( P < 0.05) in the hCG treated group on Day 0.
4. Discussion
The administration of hCG at the onset of oestrus did not affect ovulation rate, which implies that the ovulatory follicular population had been selected by the time of oestrous onset and responded with ovulation to the hCG administration. This supports the findings of Grant et al. ( 1989 ) who observed that the ovulatory population of follicles in Large White hybrid gilts could readily be distinguished on Day 20 of the oestrous cycle, i.e. prior to oestrous onset. Anderson and Melampy (1972) and Anderson (1987) reported that hCG administration to European gilts at the time of onset of behavioural oestrus did not alter ovulation rate. The elevated serum progesterone in the hCG treated group on Day 0 of gesta-
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M.G. Hunter, H.M. Picton / Animal Reproduction Science 38 (1995)231-238
tion is not unexpected since the treatment would have induced ovulation earlier than in control animals. At the onset of oestrus (Day - 2 ) serum progesterone was not significantly different between the two groups of gilts. The subsequent elevation in the hCG treated group confirms that ovulation occurred earlier in these animals compared to the control group as had been observed in a previous study (Faillace et al., 1994), and after this time progesterone concentrations were similar in the two groups. It is interesting that there was a trend for both the number of live conceptuses and percentage survival to be reduced by the hCG treatment, suggesting that the earlier induction of ovulation may have had a negative effect on embryo survival. This contrasts with results from Lambert et al. ( 1991 ) who found no change in embryonic survival in Large White gilts administered hCG at the onset of oestrus. It should be noted, however, that the survival rate in the control group of Large White gilts was 79%, considerably lower than the Meishan control gilts in the present study. Furthermore, in European breeds the first signs ofbehavioural oestrus are usually approximately coincident with the preovulatory surges of gonadotrophins (Tilton et al., 1982 ) and thus the administration ofhCG at this time would result in a combined L H / h C G peak at a similar time to the endogenous LH surge. The high level of embryo mortality in European breeds is considered to result from asynchronous development of litter mates during the early stages of pregnancy, which leads to poorly coordinated biochemical interactions between the conceptuses and the maternal systems (Pope, 1988; Pope et al., 1990). The smaller, less developed embryos are lost because they become out of phase with the uterine environment. This variation in embryo development is thought to result from heterogeneous follicle development and oocyte maturation (Hunter and Wiesak, 1990; Pope et al., 1990) and a skewed ovulation with a few oocytes shed later in the process of ovulation (Pope et al., 1988 ). In the prolific Meishan pig, however, ovulation occurs over a shorter time duration (Terqui et al., 1990) although no difference in heterogeneity was found compared to European breeds (Biggs et al., 1993 ). Meishan follicles have a higher concentration of oestradiol in follicular fluid and elevated follicular aromatase activity compared to their European counterparts (Biggs et al., 1993; Hunter et al., 1994). Furthermore, within 8 h of predicted ovulation, more oocytes from Meishan animals were in the advanced stages of meiotic maturation than those from Large White hybrid animals (Faillace and Hunter, 1994) and collectively these results suggest enhanced maturation of Meishan follicles and oocytes prior to ovulation. The administration of hCG at the onset of oestrus in the present study would induce ovulation before the follicles had undergone their final maturation, and this would also apply to the oocytes. The tendency for the resultant reduction in embryo survival indicates that the final maturational processes are essential for the high embryo survival in the Meishan pig. Placental weight was significantly increased in hCG treated animals, and placental length and fetal weight both tended to be greater. Placental dimensions are probably the most important conceptus measurements at this stage since it is dif-
M.G. Hunter, H.M. Picton / Animal Reproduction Science 38 (1995) 231-238
237
ficult to negatively or positively influence embryo size at this stage (Knight et al., 1977 ). This increase presumably occurred due to more space becoming available in the uterus due to the loss of embryos in the hCG treated animals, since uterine weight and length were unaffected by treatment. In conclusion, this study has shown that advancing the time of the LH surge in Meishan gilts to the onset of oestrus did not affect ovulation rate but tended to reduce the number of live embryos and hence percentage survival. This was accompanied by an increase in placental weight and length. These results indicate that the extended oestrus-to-ovulation interval and enhanced follicle/oocyte maturation are critical for the prolificacy of the Meishan pig.
Acknowledgements
We acknowledge the AFRC for financial support. We thank L.S. Faillace and K. Jennings and members of the Joint Animal Breeding Unit for technical assistance.
References Anderson, L.L., 1987. Reproductive cycles--pigs. In: E.S.E. Hafez (Editor), Reproduction in Farm Animals. Butterworths, London, pp. 324-344. Anderson, L.L. and Melampy, R.M., 1972. Factors affecting ovulation rate in the pig. In: D.J.A. Cole (Editor), Pig Production. Butterworths, London, pp. 329-366. Bazer, F.W., Thatcher, W.W., Martinat-Botte, F. and Terqui, M., 1988. Conceptus development in Large White and prolific Chinese Meishan pigs. J. Repro& Fertil., 84:37-42. Bidanel, J.P., Caritaz, J.C. and Legault, C., 1989. Estimation of cross-breeding parameters between Large White and Meishan porcine breeds, i. Reproductive performance. Genet. Sel. Evol., 21: 507-526. Biggs, C., Tilton J.E., Craigon, J.C., Foxcroft, G.R., Ashworth, C.A. and Hunter, M.G., 1993. Comparison of follicular heterogeneity and ovarian characteristics in Meishan and Large- White hybrid pigs. J. Reprod. Fertil., 97: 263-269. Bolet, G., Martinat-Botte, F., Locatelli, A., Gruand, J., Terqui, M. and Berthelot, F., 1986. Components of prolificacy in hyperprolific Large White sows compared with the Meishan and Large White breeds. Genet. Sel. Evol., 18: 333-342. Christenson, R.K., Vallet, J.L., Leymaster, K.A. and Young, L.D., 1993. Uterine function in Meishan pigs. J. Reprod. Fertil. Suppl., 48: 279-289. Ellendorf, F., Martinat-Botte, F., Signoret, J.P. and Terqui, M., 1988. Duration of oestrus and preovulatory LH in the high fertility Meishan pig. Proceedings 1lth World Congress Animal Reproduction and A.I., June 1988, Dublin, Vol. 2, Abstract 24. Faillace, L.S. and Hunter, M.G., 1994. Follicle development and oocyte maturation during the immediate preovulatory period in Meishan and White hybrid gilts. J. Reprod. Fertil., 101 : 139-144. Faillace, L.S., Biggs, C. and Hunter, M.G., 1994. Factors affecting age at onset of puberty, ovulation rate and time of ovulation in Chinese Meishan gilts. J. Reprod. Fertil., 100: 353-357. GENSTAT 5 Committee, 1989. GENSTAT 5 Reference Manual. Clarendon Press, Oxford. Grant, S.A., Hunter, M.G. and Foxcroft, G.R., 1989. Morphological and biochemical characteristics during ovarian follicular development in the pig. J. Reprod. Fertil., 86:171 - 183. Haley, C.S. and Lee, G.J., 1990. Genetic components of litter size in Meishan and Large White pigs
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and their crosses. In: W.G. Hill, R. Thompson and J.A. Woolliams (Editors), Proceedings of the 4th World Congress on Genetics Applied to Livestock Production XV, Edinburgh, pp. 458-461. Haley, C.S. and Lee, G.J., 1993. Genetic basis of prolificacy in Meishan pigs. J. Reprod. Fertil. Suppl., 48: 247-259. Hunter, M.G. and Wiesak, T., 1990. Evidence for and implications of follicular heterogeneity in pigs. J. Reprod. Fertil. Suppl., 40:163-171. Hunter, M.G., Southee, J.A., McLeod, B.J. and Haresign, W., 1986. Progesterone pretreatment has a direct effect on GnRH induced preovulatory follicles to determine their ability to develop into normal corpora lutea in anoestrous ewes. J. Reprod. Fertil., 76: 349-363. Hunter, M.G., Biggs, C. and Faillace, L.S., 1993. Endocrine and follicular studies in Meishan pigs. J. Reprod. Fertil. Suppl., 48:261-270. Hunter, M.G., Biggs, C., Pickard, A.R. and Faillace, L.S., 1994. Differences in follicular aromatase activity between Meishan and Large-White hybrid gilts. J. Reprod. Fertil., 101:139-144. Knight, J.W., Bazer, F.W., Thatcher, W.W., Franke, D.E. and Wallace, H.D., 1977. Conceptus development in intact and unilaterally hysterectomized-ovariectomizedgilts: interrelationships among hormonal status, placental development, fetal fluids and fetal growth. J. Anim. Sci., 44: 620-637. Lambert, E., Williams, D.H., Lynch, P.B., Hanrahan, T.J., McGeady, T.A., Austin, F.H., Boland, M.P. and Roche, J.F., 1991. The effects of administration of gonadotrophins and estrogens on prenatal survival in gilts. Theriogenology, 36: 77-85. Pope, W.F., 1988. Uterine asynchrony: a cause of embryonic loss. Biol. Repro&, 39: 999-1003. Pope, W.F., Wilde, M.H. and Xie, S., 1988. Effect of electrocautery of nonovulated Day 1 folliles on subsequent morphological variation among Day 11 embryos. Biol. Reprod., 39: 882-887. Pope, W.F., Xie, S., Broermann, D.M. and Nephew, K.P., 1990. Causes and consequences of early embryonic diversity in pigs. J. Reprod. Fertil. Suppl., 40:251-260. Terqui, M., Bazer, F.W. and Marinat-Botte, F., 1990. Mechanisms involved in the high prolificacy of the Meishan breed. In: N. Molenat and C. Legault (Editors), Symposium sur le Porc Chinois, Toulouse. Inst. Nat. Recherche Agron., Jouy-en-Josas, France, pp. 17-32. Tilton, J.E., Foxcroft, G.R., Ziecik, A.J., Coombs, S.L. and Williams, G.L., 1982. Time of the preovulatory LH surge in the gilt and sow in relation to the onset ofbehavioural estrus. Theriogenology, 18: 227-236. Wilmut, I., Ritchie, W.A., Haley, C.S., Ashwortb, C.J. and Aitken, R.P., 1992. A comparison of rate and uniformity of embryo development in Meishan and Large White pigs. J. Reprod. Fertil., 95: 24-56.
Animal Reproduction Science 38 (1995) 231-238
Effect of hCG administration at the onset of oestrus on early embryo survival and development in Meishan gilts M.G. Hunter*, H.M. Picton Department of Physiology and Environmental Science, University of Nottingham, Sutton Bonington Campus, Sutton Bonington Leicestershire LE12 5RD, UK Accepted 12 October 1994
Abstract
A study was designed to advance the time of the ovulatory luteinizing hormone (LH) surge in Meishan gilts by human chorionic gonadotrophin (hCG) administration at the onset of oestrus and assess the effect on embryo survival and development. Twelve Meishan gilts were observed six times daily for oestrous behaviour and bred at 24, 36 and 48 h after observed oestrous onset. Six of those gilts were administered an ovulatory dose of hCG (500 IU) at observed oestrous onset. Blood samples were collected at oestrous onset (Day - 2 ) and on Days 0, 2, 6, 9, 13, 16, 20, 23, 27 and 30 of gestation. All gilts were slaughtered on Day 30 of gestation and embryo survival and conceptus development assessed. Ovulation rate did not differ between control and hCG treated gilts ( 18.5 and 17.7 respectively; P > 0.1 ) while the number of live conceptuses per gilt ( 17.2 and 12.8 respectively; P<0.08) and embryo survival rate (92.1 and 75.8% respectively; P<0.1) both tended to be reduced by the hCG treatment. Placental weight ( 17.2 and 23.1 g; P < 0.01 ) was significantly increased in hCG treated gilts, while embryo weight (1.2 and 1.4 g; P < 0.06) and placental length (42.8 and 47.2 cm; P < 0.07) both tended to be increased in hCG treated gilts. Crown rump length (P> 0.1 ) and allantoic fluid volume (P> 0,1 ) did not differ between the treatment groups. Serum progesterone concentrations did not differ with treatment overall (P> 0.1 ) but were significantly elevated ( P < 0.05) at 48 h postoestrus in the hCG treated gilts compared to control gilts. Overall, these results indicate that advancing the time of the LH surge to oestrous onset, as in European breeds, compromised embryo survival and suggests that the longer time interval between oestrous onset and ovulation is important for the high rate of embryo survival in the Meishan pig. Keywords: Pig; Human chorionic gonadotrophin, hCG; Oestrus; Pregnancy; Embryology
* Corresponding author. 0378-4320/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI0378-4320(94)01365-9
232
M. G. Hunter, H.34. Picton / Animal Reproduction Science 38 (1995) 231-238
1. Introduction
The Chinese Meishan pig is characterized by its prolificacy compared to European breeds, producing an average of three to four more piglets per litter (Bolet et al., 1986; Bidanel et al., 1989; Haley and Lee, 1993 ). Although Meishan sows and gilts that have experienced an extended number of oestrous cycles have a higher ovulation rate than their European counterparts (Haley and Lee, 1993; Christenson et al., 1993; Hunter et al., 1993 ), this is not the major cause of their prolificacy. This prolificacy is considered to result from an increased level of embryo survival, regardless of ovulation rate (Bazer et al., 1988; Haley and Lee, 1990). This increased survival has been reported to occur as early as Day 12 of gestation (Bazer et al., 1988 ) and is clearly evident by Day 20 of gestation (Haley and Lee, 1993). Studies of the timing of peri-ovulatory events have shown that the interval between the onset of behavioural oestrus and ovulation is longer in Meishan than in European breeds, at approximately 49 h as opposed to 34 h respectively (Terqui et al., 1990; Wilmut et al., 1992; Faillace et al., 1994). Administration of human chorionic gonadotrophin (hCG) to Meishan gilts at the onset of oestrus significantly shortened the time to ovulation (Faillace et al., 1994), suggesting that the luteinizing hormone (LH) surge may normally occur later in the oestrous period in Meishan animals. Indeed, endocrine studies of the peri-ovulatory period confirmed that Meishan gilts exhibit behavioural oestrus significantly earlier than Large White females relative to the oestradiol and LH peaks (Hunter et al., 1993 ). Although preliminary investigations by Ellendorf et al. ( 1988 ) suggested that the LH surge is higher in Meishan pigs, this was not supported by the more substantial studies of Hunter et al. (1993) who found no difference between the breeds. The aim of the present study, therefore, was to investigate the importance of the later occurrence of the LH surge during the oestrous period on embryo survival in Meishan animals. The timing of the endogenous LH surge in Meishan gilts was altered by hCG administration at the onset of oestrus, thus making the timing similar to that in European breeds. Embryo survival and development on Day 30 of gestation as well as circulating progesterone concentrations were assessed in control and hCG treated animals.
2. Materials and methods 2.1. A n i m a l s
Twelve Meishan gilts exhibiting regular oestrous cycles were used in this study. Each of the gilts had experienced at least eight oestrous cycles. Gilts were penned
M.G. Hunter, H.M. Picton/Animal Reproduction Science 38 (1995) 231-238
233
randomly in two groups in a ventilated building with natural daylength and temperature, fed once daily and had unlimited access to drinking water. 2.2. Treatment
Oestrous detection, using vasectomized boars, '-was carried out six times daily from at least 2 days prior to anticipated oestrous onset. At the onset of oestrus, six of the 12 gilts were administered an intramuscular dose of 500 IU of hCG (Sigma Chemical Co., Dorset, UK). All gilts were bred by a fertile boar at 24, 36 and 48 h after the onset of oestrus. Blood samples (8 ml) were taken by venepuncture from the anterior vena cava of each gilt within 4 h of the onset ofoestrus (Day - 2 of gestation), at 48 h post-oestrus (Day 0 of gestation) and twice weekly (Days 2, 6, 9, 13, 16, 20, 23, 27, 30) until slaughter on Day 30. Samples were left at room temperature until a clot had formed and centrifuged at 1500Xg for 10 min; serum was aspirated and stored at - 2 0 °C until radioimmunoassay. 2.3. Conceptus dissection and measurement
The entire reproductive tract was recovered following slaughter and individual conceptuses were recovered. Allantoic fluid volume was measured and a 5 ml aliquot stored at - 20 oC. Conceptus development was measured as described by Knight et al. ( 1977 ) and included crown-rump length, embryo weight, placental length and weight and uterine weight and length. The ovaries were also dissected to determine ovulation rate. 2.4. Radioimmunoassay
The concentrations of progesterone in serum and allantoic fluid were measured by the extracted radioimmunoassay described by Hunter et al. (1986). Mean extraction efficiency was 88.8%, limit of sensitivity was 9.3 pg per tube and interand intra-assay coefficients of variation were 16.9 and 17.0% respectively. 2.5. Statistical analysis
All data were analysed by analysis of variance using the GENSTAT statistical package (GENSTAT 5 Committee, 1989). The pooled variance was used to calculate the standard error of the difference (SED) and comparisons were made using a model that included treatment, and employed gilt as the block structure. Plasma concentrations of progesterone were analysed by a multiple regression model which included treatment, day of gestation and their interaction, and employed gilt as the block structure.
234
M.G. Hunter, H.M. Picton / Animal Reproduction Science 38 (1995) 231-238
3. Results 3.1. Whole litter measurements The whole litter characteristics as measured at Day 30 of gestation are summarized in Table 1. The ovulation rate was similar between the control and hCG treated animals (P> 0.1 ), as were uterine length and weight (P> 0.1 ). However, the number of live embryos (P< 0.08 ) and percentage embryo survival (P< 0.1 ) both tended to be reduced in the hCG treated animals compared to controls. 3.2. Conceptus characteristics Individual conceptus characteristics which are summarized in Table 2 showed no differences ( P > 0.1 ) in crown-rump length or allantoic fluid volume between control and hCG treated animals. There was, however, a consistent trend for embryo weight (P<0.06), placental length (P<0.07) and placental weight ( P < 0.01 ) to be greater in the hCG treated animals than in the controls. Neither uterine weight ( P > 0.1 ) or uterine length (P> 0.1 ) were different between the groups. Table 1 Whole litter characteristics in control (n = 6 ) and hCG treated (n = 6 ) Meishan gilts
Ovulation rate (CL) Live embryos (no.) Embryo survival (%) Uterine length (cm) Uterine weight (kg)
Control
hCG treated
SED
18.5 17.2 92.1 286.0 1.12
17.7 12.8" 75.8** 281.0 1.17
2.68 2.25 8.89 29.3 0.15
*P<0.08;**P<0.1. SED, standard error of difference. Table 2 Individual conceptus characteristics in control (n = 6) and hCG treated (n = 6) Meishan gilts
Crown-rump (mm) Embryo weight (g) Placental length (cm) Placental weight (g) Allantoic fluid (ml) *P< 0.07; **P< 0.01. SED0 standard error of difference.
Control
hCG treated
SED
24.1 1.20* 42.8* 17.2"* 144.2
25.0 1.40 47.2 23.1 149.3
0,6 0.10 2.1 1.9 18.1
M.G. Hunter, H.M. Picton /Animal Reproduction Science 38 (1995) 231-238
235
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. 15
. 20
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Gestation
Fig. l. Serum progesterone concentrations (mean _+SEM ) in control ( O; n = 6 ) and h CG treated ( ®; n = 6 ) Meishan gilts from the onset ofoestrus (Day - 2 ) until Day 30 of gestation. *P<0.05.
3.3. Progesterone concentrations The concentration of progesterone in allantoic fluid was similar between the control and hCG treated groups ( 1.08 and 1.12 ng m l - ~respectively, SED = 0.09; P > 0.1 ), as was total allantoic fluid progesterone per conceptus ( 154 and 172 ng respectively, SED = 21.3; P > 0.1 ). Serum progesterone concentrations until Day 30 of gestation are shown in Fig. 1. Overall, there was no significant difference between the two treatment groups ( P > 0.1 ), but when individual days were analysed separately the concentration of progesterone was significantly higher ( P < 0.05) in the hCG treated group on Day 0.
4. Discussion
The administration of hCG at the onset of oestrus did not affect ovulation rate, which implies that the ovulatory follicular population had been selected by the time of oestrous onset and responded with ovulation to the hCG administration. This supports the findings of Grant et al. ( 1989 ) who observed that the ovulatory population of follicles in Large White hybrid gilts could readily be distinguished on Day 20 of the oestrous cycle, i.e. prior to oestrous onset. Anderson and Melampy (1972) and Anderson (1987) reported that hCG administration to European gilts at the time of onset of behavioural oestrus did not alter ovulation rate. The elevated serum progesterone in the hCG treated group on Day 0 of gesta-
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tion is not unexpected since the treatment would have induced ovulation earlier than in control animals. At the onset of oestrus (Day - 2 ) serum progesterone was not significantly different between the two groups of gilts. The subsequent elevation in the hCG treated group confirms that ovulation occurred earlier in these animals compared to the control group as had been observed in a previous study (Faillace et al., 1994), and after this time progesterone concentrations were similar in the two groups. It is interesting that there was a trend for both the number of live conceptuses and percentage survival to be reduced by the hCG treatment, suggesting that the earlier induction of ovulation may have had a negative effect on embryo survival. This contrasts with results from Lambert et al. ( 1991 ) who found no change in embryonic survival in Large White gilts administered hCG at the onset of oestrus. It should be noted, however, that the survival rate in the control group of Large White gilts was 79%, considerably lower than the Meishan control gilts in the present study. Furthermore, in European breeds the first signs ofbehavioural oestrus are usually approximately coincident with the preovulatory surges of gonadotrophins (Tilton et al., 1982 ) and thus the administration ofhCG at this time would result in a combined L H / h C G peak at a similar time to the endogenous LH surge. The high level of embryo mortality in European breeds is considered to result from asynchronous development of litter mates during the early stages of pregnancy, which leads to poorly coordinated biochemical interactions between the conceptuses and the maternal systems (Pope, 1988; Pope et al., 1990). The smaller, less developed embryos are lost because they become out of phase with the uterine environment. This variation in embryo development is thought to result from heterogeneous follicle development and oocyte maturation (Hunter and Wiesak, 1990; Pope et al., 1990) and a skewed ovulation with a few oocytes shed later in the process of ovulation (Pope et al., 1988 ). In the prolific Meishan pig, however, ovulation occurs over a shorter time duration (Terqui et al., 1990) although no difference in heterogeneity was found compared to European breeds (Biggs et al., 1993 ). Meishan follicles have a higher concentration of oestradiol in follicular fluid and elevated follicular aromatase activity compared to their European counterparts (Biggs et al., 1993; Hunter et al., 1994). Furthermore, within 8 h of predicted ovulation, more oocytes from Meishan animals were in the advanced stages of meiotic maturation than those from Large White hybrid animals (Faillace and Hunter, 1994) and collectively these results suggest enhanced maturation of Meishan follicles and oocytes prior to ovulation. The administration of hCG at the onset of oestrus in the present study would induce ovulation before the follicles had undergone their final maturation, and this would also apply to the oocytes. The tendency for the resultant reduction in embryo survival indicates that the final maturational processes are essential for the high embryo survival in the Meishan pig. Placental weight was significantly increased in hCG treated animals, and placental length and fetal weight both tended to be greater. Placental dimensions are probably the most important conceptus measurements at this stage since it is dif-
M.G. Hunter, H.M. Picton / Animal Reproduction Science 38 (1995) 231-238
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ficult to negatively or positively influence embryo size at this stage (Knight et al., 1977 ). This increase presumably occurred due to more space becoming available in the uterus due to the loss of embryos in the hCG treated animals, since uterine weight and length were unaffected by treatment. In conclusion, this study has shown that advancing the time of the LH surge in Meishan gilts to the onset of oestrus did not affect ovulation rate but tended to reduce the number of live embryos and hence percentage survival. This was accompanied by an increase in placental weight and length. These results indicate that the extended oestrus-to-ovulation interval and enhanced follicle/oocyte maturation are critical for the prolificacy of the Meishan pig.
Acknowledgements
We acknowledge the AFRC for financial support. We thank L.S. Faillace and K. Jennings and members of the Joint Animal Breeding Unit for technical assistance.
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