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The extent and timing of prenatal loss in gilts
THERIOGENOLOGY
THE EXTENT AND TIMING OF PRENATAL LOSS IN GILTS E.Lambert,lp4
D.H. Williams,* P.B. Lynch,4 T.J. Hanrahan,4 F.H Austin.1 M P. Boland3 and J.F. Rachel
T.A. McGeady,l
Faculties of lVeterinary Medicine, *Science and 3Agriculture University College Dublin, Ireland 4Teagasc, Moorepark Research and Development Division Fermoy, Co. Cork, Ireland Received for publication: Accepted:
December
13,
1990
JUZY 29, 1991
ABSTRACT The potential litter size of gilts that is baaed on the ovulation .rate is much higher than the actual litter size, which depends on the fertilization rate and subsequent prenatal mortality. Prenatal mortality is divided into embryonic mortality (before Day 30) and fetal mortality (after Day 30). Prenatal loss includes both fertilization failure and prenatal mortality. Crossbred gilts (n =149) were bred at the first observed estrus after being exposed to the boar at 200 days of age. Time of the first insemination after estrus detection was determined by measurement of vaginal conductivity using a Walsmeta meter. A second insemination was administered either 8 or 16 hours later. Artificial insemination with fresh semen (0 to 3 days old) was used throughout the experiment. Gilts were slaughtered on Day 3 (n = 26), Day 10 (n = 42), Day 30 of gestation (n = 45) or they were allowed to farrow (n = 36). Gilts slaughtered on Day 3 were used to estimate the fertilization rate. Gilts slaughtered on Day 10 and Day 30 were used to calculate embryonic mortality, while fetal mortality was calculated from the gilts that fat-rowed. The mean (+SEM) number of corpora lutea (CL) was 13.15kO.46, 13.36kO.37 and 12.9750.39 for gilts slaughtered at Days 3, 10 and 30, respectively (p>O.O5), and the mean (*SEM) number of normal embryos recovered was 11.12*0.69, 9.46kO.55 and 9.33kO.58, respectively. Litter size at parturition was 9.10f0.54. There was a significant difference between the number of normal embryos on Day 3 and Day 30 (P=O.O5) and also between the number of normal embryos at Day 3 and the number of piglets at term. Ninety percent of the ova were recovered at Day 3. The fertilization rate was calculated either 1) assuming that unrecovered ova .had -a similar fertilization rate as the recovered ova (FRER=94.5?2.0%) or 2) assuming_ that unrecovered ova were unfertilized (FROR=84.5+2.5%): It was concludedcihat FRER was a more accurate estimation of the fertilization rate. Based on this fertilization rate, embryonic mortality between Day 3 and Day 10 was 20.8+8.3%, with an additional 12.5+7.10/o loss between Day 10 and Day 30, when all gilts were included (P = 0.308). Thus the total prenatal loss, including fertilization failure, up to Day 10 was 26.3% and to Day 30 it was 38.8%. Fetal mortality was 2.2%, giving a total prenatal mortality (excluding fertilization failure) of 35.5% and a prenatal loss of 4 1%. Most of the prenatal loss was due to embryonic mortality. In those gilts that remained pregnant most of the embryonic loss occurred before Day 10 (19.0+6.3%; P=O.O03). There was no further loss between Day 10 and 30 of pregnancy. There was a significant difference between the loss from Day 3 to Day 10 compared with the loss from Day 10 to Day 30 (P=O.O5); therefore, most of the embryonic loss in pregnant gilts occurred before Day 10. Since fetal mortality was 3.2+6.30/o, most of the prenatal loss was due to embryonic mortality. Key words: pig, fertilization, embryo mortality, litter size
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INTRODUCTION In the pig, potential litter size, as determined by the ovulation rate is much greater than the actual litter size, which depends on the level of prenatal loss (1,2). Prenatal development can be divided into three periods: the ovum, the embryo and the fetus. There are difficulties in determining the limits of these periods and in determining accurately the time of death. Therefore it is difficult to classify prenatal mortality on the basis of these periods. There is considerable variation in the application of the terms used in describing prenatal mortality (3-7). Most authors use the term embryonic mortality to describe prenatal mortality occurring at any time during the period of the ovum, the period of the embryo and the early part of the period of the fetus. Embryonic mortality, generally refers to fertility losses during the embryonic period from conception to completion of the stage of differentiation. In the pig, this is arbitrarily taken as Day 30 of gestation, although a sharp distinction cannot be made on a physiological basis. There is by no means a universal acceptance of this arbitrarily chosen 30&y limit, but for the purpose of this experiment this is the limit taken. The fertilization rate in pigs is higher than 90% (8,9). Because losses due to fertilization failure are small, in many studies the fertilization rate is assumed to be lOO%, and the level of embryonic loss is then simply calculated as the difference between the number of normal embryos and the number of corpora lutea present. Despite this, the greater part of prenatal loss is due to embryonic mortality. There is a general consensus that 30 to 35 % of fertilized ova are not represented by viable embryos at Day 25 to 30. There are conflicting data in the literature on the stage at which most embryonic loss occurs. One study (8) found little evidence of embryonic loss up to 9 days after the onset of estrus. In a different study (9), it was found that up to the sixth day all fertilized ova appeared to be developing normally; between the sixth and ninth day, however, degenerating embryos were found and accounted for 22% of the total. An even greater loss was observed in sows and gilts killed between the 13th and 18th day (28%). A high incidence of visible abnormalities during the 6 to lo-day period of pregnancy was observed (10). Approximately 20% of blastocysts collected from individual gilts may exhibit reduced viability as early as Day 6 of pregnancy (11). The time at which elongation and first attachment to the wall of the uterus occurs (Day 10 to Day 13 of gestation) appears to be the stage at which the bulk of embryonic loss occurred in another study (12). A loss of 17% of potential embryos by Day 18 was reported in a further study (13). Few degenerating embryos were recovered at any stage (Day 9 to 18), suggesting rapid dissolution once mortality occurred (13). Embryonic loss is not a feature of all gilts. Up to 20% of gilts have no embryonic loss by Day 30 of pregnancy (2). Other gilts sustain complete loss of the litter. The period from the 12th to the 13th day of gestation is critical as this is the stage when the signals for recognition and maintenance of pregnancy are produced (14). At least four viable embryos must be present at this critical stage or else the corpora lutea will regress, resulting in the termination of pregnancy (15,16). Because of a lack of consensus in the literature regarding the timing and extent of prenatal loss in the pig, there was a need to investigate it further, since this is a necessary prelude before carrying out experiments to reduce this loss. Therefore the objective of this experiment was to determine the extent of prenatal loss in gilts in our herd and to establish the stage at which most embryonic loss occurred.
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MATERIALS AND METHODS Gilt Management One hundred and forty-nine Large White and Landrace gilts from a herd operating a reciprocal backcross breeding system were used over a nine-month period from October 1988 to June 1989. Gilts were fed a cereal-soya-meat and bone meal sow diet (13.5 MJ DE&g; 17% CP). The daily allowance was 3 to 3.5 kg per gilt. Groups of 10 to 12 gilts were housed in partially slatted pens 19m2 in area. Breeding Management Gilts were first exposed to the boar at an average age of 200 days and at an average weight of 112 kg. A mature boar was put into each pen of gilts for 5 to 6 hours daily until the first gilt showed signs of estrus. Thereafter, a boar was allowed into the pen for 30 minutes per day and was observed to ensure that no gilt was mated. Checking for estrus was carried out twice daily at 0900 and 1600 hours in the presence of a boar. Gilts were bred by artificial insemination (AI) on their first observed estrus (Day 0, standing to back pressure in the presence of a boar ). Time of first insemination after estrus detection was determined by measurement of the vaginal mucus conductivity using a Walsmeta (Mark II) meter. A reading of 55 to 65 indicated the optimum time for insemination. Gilts inseminated in the morning were inseminated again that evening, while those inseminated in the evening were inseminated again the following morning. Artificial insemination with fresh semen was used throughout the experiment. Gilts were randomly assigned to the following groups at the onset of estrus: 1) slaughter on Day 3 of gestation to determine fertilization rate (n = 26); 2) slaughter on Day 10 of gestation to determine early embryonic mortality (n = 42); 3) slaughter on Day 30 of gestation to determine late embryonic mortality (n = 45); and 4) allocated to furow to determine fetal mortality (n = 36). Collection of Embryos The reproductive and were transported minutes of slaughter. that returned to estrus
tracts were removed from gilts within 20 to 30 minutes of slaughter to the laboratory on ice where embryos were recovered within 45 The number of CL was recorded for all gilts, (excluding the 16 gilts and the 29 gilts that farrowed).
Dav 3. The oviduct was detached from the uterine horn at a point just below the utero tubal junction. The oviduct was then flushed with 4 ml of phosphate buffered saline (PBS), and the upper section of the uterine horn was flushed with 20 ml of PBS. Ova were examined using a stereomicroscope and were classified as either fertilized or unfertilized, and the numbers in each category were recorded for each gilt. Dav 10. Both uterine horns were flushed completely using 40 ml of PBS per horn. The fhtshmg solution was examined with the aid of a stereomicroscope, and embryos were classified as normal or degenerated (degenerated embryos had not developed beyond the late morula/early blastocyst stage). Dav 30. An incision was made along the outer curvature of the uterine horn and the embryos were dissected out and were classified as either normal or degenerated, based on their morphology. Farrowinn aroun. Gilts allocated to farrow were kept in their original groups for 14 to 2 1 days after insemination and were then housed in individual stalls and fed 2.25 kg per
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day of the 17% crude protein sow diet until parturition. At 110 days of pregnancy, they were moved into the farrowing house. The total number of pigs born live and dead was recorded.
Statistical Analysis A one-way ANOVA was used to compare prenatal loss where the experimental unit was an individual animal. An embryo survival rate was calculated for each animal. The mean value was then calculated from these figures. Dav 3. The gilts from the Day-3 group were used to calculate a fertilization rate which could subsequently be used for gilts in the Day-10 and Day-30 groups to calculate embryonic mortality and for the farrowing group of gilts to estimate fetal mortality. However, in gilts slaughtered at Day 3, not all ova were recovered so the fertilization rate was calculated in two different ways for each gilt using the following assumptions: 1) the fertilization rate based on ova recovery (FRER), assuming that unrecovered ova had the same fertilization rate as those recovered FRER= (No. of fertilized ova recovered)/(Total no. of ova recovered). 2) The fertilization rate was based on ovulation unrecovered ova were unfertilized
rate (FROR),
assuming
that the
FROR= (No. fertilized ova recovered)/(No.of CL). Davs 10 and 30. It was assumed that all normal embryos were recovered, thus to estimate the number of ova initially fertilized, the number of CL for each gilt was multiplied by the mean fertilization rate (FRER or FROR). The proportion of embryos surviving to this stage was estimated for each animal as follows: (No. normal embryos recovered)/(No. Data was analyzed both by including pregnancy.
of CL x fertilization rate).
and excluding gilts that did not maintain their
Farrowing grout. An estimate of the number of CL for each gilt was taken as the mean number of CL for the other three groups (i.e., gilts slaughtered at Days 3, 10 and 30). This gave a mean value of 13.13 CL; hence the proportion of embryos surviving was calculated as follows: (Total no. pigs born live)/( 13.13 x fertilization rate). The total number of pigs born for each gilt included stillborn pigs, which were not included in the fetal mortality category. Data were analyzed using both fertilization rates and by including and excluding the nonpregnant gilts.
RESULTS Pregnancy Rate All 26 animals slaughtered on Day 3 had more than seven fertilized ova at slaughter, giving a pregnancy rate of 100%. The pregnancy rate of gilts slaughtered on Day 10 of
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gestation was 98%. This was because one of the 42 gilts inseminated had no embryos, but six unfertilized ova were recovered from this gilt. Another seven gilts had less than 4 embryos and were included as pregnant gilts as they had embryos present at the time of slaughter. Nineteen percent of the Day- 10 group could potentially have returned to service, based on embryo numbers present at slaughter. Of the 45 animals originally assigned to be slaughtered on Day 30, nine returned to service giving a pregnancy rate of 80%. Seven of the 36 gilts in the farrowing group returned to service. The pregnancy rate for this group was 81% (Table 1). Ovulation Rate and Embryo Survival Of the 26, 42 and 36 gilts slaughtered on Days 3, 10 or 30, respectively, the total number of ovulations was 342,557 and 467 giving a mean (+SEM) number of ovulations of 13.1520.46, 13.36kO.37 and 12.97kO.39, respectively (P>O.O5; Table 1). The recovery rates at Days 3, 10 and 30 were 90, 77 and 74%, respectively. The number of fertilized ova or normal embryos or piglets born was 11.12kO.69, 9.46iO.55, 9.33kO.58 and 9.1kO.54, respectively, for gilts at Day 3, Day 10, Day 30 of gestation and those that farrowed (P=O. 135, Table 1). However, on further analysis there was a significant difference between the mean number of normal embryos on Day 3 and Day 30 (P=O.O5) and also between the mean number of embryos on Day 3 and the mean number of piglets born in the farrowing group (P=O.O35). (Nonpregnant animals in the Day-30 and farrowing groups were not used in the calculations). There was no prenatal loss in 6, 7 and 6 gilts from the Day-3, Day-10 and Day-30 groups, respectively (the number of CL = the number of embryos) . Fertilization and Embryo Survival The fertilization rate was 94.5+2.0% based on ova recovery (FRER) and was 84.5+2.5% based on ovulation rate (FROR; Table 2). The mean percentage of embryonic survival in gilts (including pregnant and nonpregnant gilts) in the Day- 10, Day-30, and the farrowing group based on FRER was 73.7*4.9%, 61.2+5.9% and 59.1+6.0%, respectively. If nonpregnant gilts were excluded (one gilt from the Day-10 group, nine gilts from the Day-30 group and seven gilts from the farrowing group), then the percentage of embryonic survival among pregnant gilts was 75.5*4.7%, 76.5*4.6% and 73.3&4.4% in the Day-lo, Day-30, and the farrowing groups, respectively (Table 2). The corresponding values for all gilts (pregnant and nonpregnant) using FROR were 82.4k5.5, 68.5k6.6 and 66.1+6.8%. When nonpregnant gilts are excluded the level of embryonic survival in the Day-lo, Day-30 and farrowing group was 84.4k5.2, 86.6k5.1 and 82.0f4.9%, respectively (Table 2). Embryonic/Fetal Mortality Based on FRER embryonic mortality between Days 3 and 10 of pregnancy was 20.8f8.3% (P=O.Ol), and between Days 10 and 30 it was 12.5+7.19/o (P=O.O5) in all gilts. Fetal loss between Day 30 and farrowing was 2.247.4% (Table 3). When the level of embryonic loss from Day 3 to Day 10 was compared to the loss from Day 10 to Day 30 (a difference of 8.3?13%), there was no significant difference. However, the loss from Day 3 to Day 30 (a difference of 31.1+13.03%) was significantly different (P=O.O2) compared with the loss from Day 30 to parturition. This indicates that embryonic loss accounts for most prenatal mortality in gilts.
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Table 1. Pregnancy rate and embryo recovery rate (with ANOVA model standard errors) in gilts slaughtered at Days 3, 10 and 30 of gestation and piglets born in a group that were allowed to farrow Treatment
Day 3
Day 10
No. of gilts inseminated
26
42
No. of gilts yielding embryos
26
41
36
29
% pregnant
100
98
80
81
Total no. of CL
342
557
467
13.15kO.46
13.36kO.37
12.97*0.39
No. of CL mean (+SEM)
Total no. of embryos recovered
307
428
Total no. of normal embryos recovered
289
388
No. of normal embryos recovered mean (SEM)
11.12kO.69
9.46+0.55
Day 30
Farrowing
P value for overall F-test
36
336
0.767
264a’
9.33kO.58
9.10+0.54a
0.135
a = Number of piglets born. The extent of complete or partial loss was examined. Excluding the gilts with complete prenatal loss changed the pattern of partial prenatal loss in pregnant gilts (Table 3). The extent of embryonic loss between Days 3 and 10 of gestation remained unchanged (19.Ok6.3 versus 20.8+8.3%). However, there was no further loss from Day 10 to Day 30 of gestation. Fetal mortality was 3.2+6.39/o and was not significant. However, when comparing the level of loss from Day 3 to Day 10 with the loss from Day 10 to Day 30, there was a significant difference (P=O.O5), suggesting that most of the loss in pregnant gilts occurred before Day 10 of gestation. Based on FROR only 2.1+9.3% embryonic loss occurred by Day 10 of gestation in all gilts. A further 13.9+7.9% loss occurred between Day 10 and Day 30 of gestation, with fetal mortality at 2.4+8.3% (Table 3). None of these differences were significant. When nonpregnant gilts were excluded, the level of embryonic mortality in pregnant gilts from Day 3 to Day 10 was 0.0+7.1%, from Day 10 to Day 30 it was - 1.2*6.4% and fetal mortality was 3.6+7.0% (Table 3). None of these values were significantly different.
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THERIOGENOLOGY Table 2. The percentage of embryos surviving (within-group standard errors) in gilts slaughtered on Days 3, 10, 30 of gestation and at farrowing, based on expressing fertilization rate on the basis of ova recovery or ovulation rate % surviving at : Day 10 Day 30
Day 3
Farrowing
Allgilts
Pregnant gilts only
F&R FROR
94.5*2.0 26 84.5k2.5
73.7k4.9 42 82.4k5.5
61.2*5.9 45 68.5*6.6
59. E6 0 66.1+6:8
F&R
26 94.5k2.0 84.5k2.5
41 75.5k4.7 84.4k5.2
36 76.5k4.6 86.6k5.1
29 73.3k4.4 82.Ok4.9
FROR
Table 3. The percentage embryonic and fetal mortality (with ANOVA model standard errors), based on expressing fertilization rate on the basis of recovery of ova or the ovulation rate %loss from Day 3 to Day 10
% loss from Day 10 to Day 30
%loss from Day 30 to Farrow
All gilts
Pregnant gilts only
FRER FROR
20.8+8.3 2.159.3
12.557.1 13.9k7.9
2.2k7.4 2.458.3
FRER FROR
19.Ok6.3 O.Ok7.1
-1.Ok5.7 -1.2k6.4
3.2k6.3 3.6k7.0
DISCUSSION Many studies have been carried out to study embryonic mortality, but direct comparisons with such work are difficult because of the variation in use of the term embryonic mortality. In some studies this includes loss due to fertilization failure and, to a varying extent, fetal mortality (4-6,9). The level of embryonic mortality depends on whether it is a primiparous (gilt) or a multiparous (sow) animal. It is established that sows sustain a higher level of embryonic mortality than gilts (4). Not all authors distinguish between embryonic mortality in gilts and sows, so direct comparisons with embryonic mortality in gilts alone are not possible (9). Another difficulty lies in interpreting the data.
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It has already been shown that the fertilization rate in pigs is higher than 90% (8,9). To get a more complete picture of the losses at each stage (ova, embryo and fetal stages) in a group of animals kept under similar management and environmental conditions, it was deemed necessary first to establish the fertilization rate in a group of these animals (Day 3) as an estimate for the fertilization rate in other groups (Days 10 and 30 and the farrowing group). This was then used in the calculation of embryonic mortality in gilts at Days 10 and 30 of gestation and of fetal mortality. Variation in the fertilization rate was not allowed for. At Day 3 only 90% of the ova were recovered. The question then arises as to whether those ova not recovered had a similar fertilization rate to those recovered (FRER), or whether they were unfertilized (FROR). Data were analyzed using both assumptions. The 94.5% fertilization rate (FRER) is similar to other reports (8,9) while the 84.5% fertilization rate (FROR) is considerably lower. A second difficulty in our study was with gilts slaughtered at Day 10 in the definition of pregnancy. Recognition of pregnancy occurs around Day 12 (14), although it is possible that there are subtle signals before Day 12. However, the animal is considered pregnant once embryos are present in the uterus. It has been shown that gilts with less than four viable embryos at Day 12 will not maintain pregnancy (15,16) so 19% of the Day-10 group, had they not been slaughtered, presumably would have returned to es&us. This would be in line with the 20 and 19% of returns in the Day-30 and farrowing groups, respectively. This suggests that complete fertilization failure was not the cause of complete litter loss, but, in fact, viable embryos were present although in insufficient numbers at the critical stage (Day 12) to generate an adequate signal for the maintenance of pregnancy. A combination of a low fertilization rate and a high embryonic mortality rate before this critical stage would result in pregnancy failure. Another difficulty arose in gilts with complete litter loss. One gilt at Day 10 had no embryos;while nine and seven gilts returned to estrus from the Day-30 and farrowing groups, respectively. The question is whether they should be included in the estimation of embryonic/fetal mortality or should the level of embryonic/fetal mortality be estimated in pregnant gilts separately. Once again, data were analysed both ways and also using the two fertilization rates. A different picture emerges depending on the method of calculation. Fertilization Rate (FRER) and Subsequent Prenatal Loss With a fertilization rate of 94.5% there was a significant embryonic loss at Day 10 (20%), with a further loss of 12% between Day 10 to Day 30 of gestation (all gilts). As this 12% loss was not significantly different from the 20% loss, no statistical conclusion could be drawn regarding the timing of embryonic loss in all gilts. The total embryonic loss was significantly higher than the fetal loss, as expected (1,2). When nonpregnant gilts were excluded, there was a significant embryonic loss by Day 10 (19%), but in gilts that remained pregnant no more embryonic loss occurred after Day 10. By comparing the two it can be seen that approximately 50% of the loss from Day 10 to Day 30, when all gilts were included, could be attributed to complete litter loss in the gilts that returned to estrus. Fertilization Rate (FROR) and Subsequent Prenatal Loss With a fertilization rate of 84.5% most of the loss occurred at the ovum stage due to fertilization failure. This is in conflict with data in the literature since in general pigs have high fertilization rates (8,9), although certain individual animals for reasons such as incorrect timing of mating, poor semen quality, or infection may have a low fertilization
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rate. These factors can be discounted in this investigation since management factors were standardized across all treatment groups, and strict hygienic procedures were followed to minimise the risk of infection. Using FROR and including all gilts, practically no embryonic loss occurred by Day 10 (2+9%). This low level of embryonic mortality by Day 10 is in agreement with one report (8) (
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gestation. In gilts that remained pregnant, there was 19% embryonic mortality and a further 3% fetal mortality. The bulk of this prenatal loss was due to embryonic mortality, and this occurred before Day 10 of pregnancy. REFERENCES 1.
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THE EXTENT AND TIMING OF PRENATAL LOSS IN GILTS E.Lambert,lp4
D.H. Williams,* P.B. Lynch,4 T.J. Hanrahan,4 F.H Austin.1 M P. Boland3 and J.F. Rachel
T.A. McGeady,l
Faculties of lVeterinary Medicine, *Science and 3Agriculture University College Dublin, Ireland 4Teagasc, Moorepark Research and Development Division Fermoy, Co. Cork, Ireland Received for publication: Accepted:
December
13,
1990
JUZY 29, 1991
ABSTRACT The potential litter size of gilts that is baaed on the ovulation .rate is much higher than the actual litter size, which depends on the fertilization rate and subsequent prenatal mortality. Prenatal mortality is divided into embryonic mortality (before Day 30) and fetal mortality (after Day 30). Prenatal loss includes both fertilization failure and prenatal mortality. Crossbred gilts (n =149) were bred at the first observed estrus after being exposed to the boar at 200 days of age. Time of the first insemination after estrus detection was determined by measurement of vaginal conductivity using a Walsmeta meter. A second insemination was administered either 8 or 16 hours later. Artificial insemination with fresh semen (0 to 3 days old) was used throughout the experiment. Gilts were slaughtered on Day 3 (n = 26), Day 10 (n = 42), Day 30 of gestation (n = 45) or they were allowed to farrow (n = 36). Gilts slaughtered on Day 3 were used to estimate the fertilization rate. Gilts slaughtered on Day 10 and Day 30 were used to calculate embryonic mortality, while fetal mortality was calculated from the gilts that fat-rowed. The mean (+SEM) number of corpora lutea (CL) was 13.15kO.46, 13.36kO.37 and 12.9750.39 for gilts slaughtered at Days 3, 10 and 30, respectively (p>O.O5), and the mean (*SEM) number of normal embryos recovered was 11.12*0.69, 9.46kO.55 and 9.33kO.58, respectively. Litter size at parturition was 9.10f0.54. There was a significant difference between the number of normal embryos on Day 3 and Day 30 (P=O.O5) and also between the number of normal embryos at Day 3 and the number of piglets at term. Ninety percent of the ova were recovered at Day 3. The fertilization rate was calculated either 1) assuming that unrecovered ova .had -a similar fertilization rate as the recovered ova (FRER=94.5?2.0%) or 2) assuming_ that unrecovered ova were unfertilized (FROR=84.5+2.5%): It was concludedcihat FRER was a more accurate estimation of the fertilization rate. Based on this fertilization rate, embryonic mortality between Day 3 and Day 10 was 20.8+8.3%, with an additional 12.5+7.10/o loss between Day 10 and Day 30, when all gilts were included (P = 0.308). Thus the total prenatal loss, including fertilization failure, up to Day 10 was 26.3% and to Day 30 it was 38.8%. Fetal mortality was 2.2%, giving a total prenatal mortality (excluding fertilization failure) of 35.5% and a prenatal loss of 4 1%. Most of the prenatal loss was due to embryonic mortality. In those gilts that remained pregnant most of the embryonic loss occurred before Day 10 (19.0+6.3%; P=O.O03). There was no further loss between Day 10 and 30 of pregnancy. There was a significant difference between the loss from Day 3 to Day 10 compared with the loss from Day 10 to Day 30 (P=O.O5); therefore, most of the embryonic loss in pregnant gilts occurred before Day 10. Since fetal mortality was 3.2+6.30/o, most of the prenatal loss was due to embryonic mortality. Key words: pig, fertilization, embryo mortality, litter size
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INTRODUCTION In the pig, potential litter size, as determined by the ovulation rate is much greater than the actual litter size, which depends on the level of prenatal loss (1,2). Prenatal development can be divided into three periods: the ovum, the embryo and the fetus. There are difficulties in determining the limits of these periods and in determining accurately the time of death. Therefore it is difficult to classify prenatal mortality on the basis of these periods. There is considerable variation in the application of the terms used in describing prenatal mortality (3-7). Most authors use the term embryonic mortality to describe prenatal mortality occurring at any time during the period of the ovum, the period of the embryo and the early part of the period of the fetus. Embryonic mortality, generally refers to fertility losses during the embryonic period from conception to completion of the stage of differentiation. In the pig, this is arbitrarily taken as Day 30 of gestation, although a sharp distinction cannot be made on a physiological basis. There is by no means a universal acceptance of this arbitrarily chosen 30&y limit, but for the purpose of this experiment this is the limit taken. The fertilization rate in pigs is higher than 90% (8,9). Because losses due to fertilization failure are small, in many studies the fertilization rate is assumed to be lOO%, and the level of embryonic loss is then simply calculated as the difference between the number of normal embryos and the number of corpora lutea present. Despite this, the greater part of prenatal loss is due to embryonic mortality. There is a general consensus that 30 to 35 % of fertilized ova are not represented by viable embryos at Day 25 to 30. There are conflicting data in the literature on the stage at which most embryonic loss occurs. One study (8) found little evidence of embryonic loss up to 9 days after the onset of estrus. In a different study (9), it was found that up to the sixth day all fertilized ova appeared to be developing normally; between the sixth and ninth day, however, degenerating embryos were found and accounted for 22% of the total. An even greater loss was observed in sows and gilts killed between the 13th and 18th day (28%). A high incidence of visible abnormalities during the 6 to lo-day period of pregnancy was observed (10). Approximately 20% of blastocysts collected from individual gilts may exhibit reduced viability as early as Day 6 of pregnancy (11). The time at which elongation and first attachment to the wall of the uterus occurs (Day 10 to Day 13 of gestation) appears to be the stage at which the bulk of embryonic loss occurred in another study (12). A loss of 17% of potential embryos by Day 18 was reported in a further study (13). Few degenerating embryos were recovered at any stage (Day 9 to 18), suggesting rapid dissolution once mortality occurred (13). Embryonic loss is not a feature of all gilts. Up to 20% of gilts have no embryonic loss by Day 30 of pregnancy (2). Other gilts sustain complete loss of the litter. The period from the 12th to the 13th day of gestation is critical as this is the stage when the signals for recognition and maintenance of pregnancy are produced (14). At least four viable embryos must be present at this critical stage or else the corpora lutea will regress, resulting in the termination of pregnancy (15,16). Because of a lack of consensus in the literature regarding the timing and extent of prenatal loss in the pig, there was a need to investigate it further, since this is a necessary prelude before carrying out experiments to reduce this loss. Therefore the objective of this experiment was to determine the extent of prenatal loss in gilts in our herd and to establish the stage at which most embryonic loss occurred.
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MATERIALS AND METHODS Gilt Management One hundred and forty-nine Large White and Landrace gilts from a herd operating a reciprocal backcross breeding system were used over a nine-month period from October 1988 to June 1989. Gilts were fed a cereal-soya-meat and bone meal sow diet (13.5 MJ DE&g; 17% CP). The daily allowance was 3 to 3.5 kg per gilt. Groups of 10 to 12 gilts were housed in partially slatted pens 19m2 in area. Breeding Management Gilts were first exposed to the boar at an average age of 200 days and at an average weight of 112 kg. A mature boar was put into each pen of gilts for 5 to 6 hours daily until the first gilt showed signs of estrus. Thereafter, a boar was allowed into the pen for 30 minutes per day and was observed to ensure that no gilt was mated. Checking for estrus was carried out twice daily at 0900 and 1600 hours in the presence of a boar. Gilts were bred by artificial insemination (AI) on their first observed estrus (Day 0, standing to back pressure in the presence of a boar ). Time of first insemination after estrus detection was determined by measurement of the vaginal mucus conductivity using a Walsmeta (Mark II) meter. A reading of 55 to 65 indicated the optimum time for insemination. Gilts inseminated in the morning were inseminated again that evening, while those inseminated in the evening were inseminated again the following morning. Artificial insemination with fresh semen was used throughout the experiment. Gilts were randomly assigned to the following groups at the onset of estrus: 1) slaughter on Day 3 of gestation to determine fertilization rate (n = 26); 2) slaughter on Day 10 of gestation to determine early embryonic mortality (n = 42); 3) slaughter on Day 30 of gestation to determine late embryonic mortality (n = 45); and 4) allocated to furow to determine fetal mortality (n = 36). Collection of Embryos The reproductive and were transported minutes of slaughter. that returned to estrus
tracts were removed from gilts within 20 to 30 minutes of slaughter to the laboratory on ice where embryos were recovered within 45 The number of CL was recorded for all gilts, (excluding the 16 gilts and the 29 gilts that farrowed).
Dav 3. The oviduct was detached from the uterine horn at a point just below the utero tubal junction. The oviduct was then flushed with 4 ml of phosphate buffered saline (PBS), and the upper section of the uterine horn was flushed with 20 ml of PBS. Ova were examined using a stereomicroscope and were classified as either fertilized or unfertilized, and the numbers in each category were recorded for each gilt. Dav 10. Both uterine horns were flushed completely using 40 ml of PBS per horn. The fhtshmg solution was examined with the aid of a stereomicroscope, and embryos were classified as normal or degenerated (degenerated embryos had not developed beyond the late morula/early blastocyst stage). Dav 30. An incision was made along the outer curvature of the uterine horn and the embryos were dissected out and were classified as either normal or degenerated, based on their morphology. Farrowinn aroun. Gilts allocated to farrow were kept in their original groups for 14 to 2 1 days after insemination and were then housed in individual stalls and fed 2.25 kg per
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day of the 17% crude protein sow diet until parturition. At 110 days of pregnancy, they were moved into the farrowing house. The total number of pigs born live and dead was recorded.
Statistical Analysis A one-way ANOVA was used to compare prenatal loss where the experimental unit was an individual animal. An embryo survival rate was calculated for each animal. The mean value was then calculated from these figures. Dav 3. The gilts from the Day-3 group were used to calculate a fertilization rate which could subsequently be used for gilts in the Day-10 and Day-30 groups to calculate embryonic mortality and for the farrowing group of gilts to estimate fetal mortality. However, in gilts slaughtered at Day 3, not all ova were recovered so the fertilization rate was calculated in two different ways for each gilt using the following assumptions: 1) the fertilization rate based on ova recovery (FRER), assuming that unrecovered ova had the same fertilization rate as those recovered FRER= (No. of fertilized ova recovered)/(Total no. of ova recovered). 2) The fertilization rate was based on ovulation unrecovered ova were unfertilized
rate (FROR),
assuming
that the
FROR= (No. fertilized ova recovered)/(No.of CL). Davs 10 and 30. It was assumed that all normal embryos were recovered, thus to estimate the number of ova initially fertilized, the number of CL for each gilt was multiplied by the mean fertilization rate (FRER or FROR). The proportion of embryos surviving to this stage was estimated for each animal as follows: (No. normal embryos recovered)/(No. Data was analyzed both by including pregnancy.
of CL x fertilization rate).
and excluding gilts that did not maintain their
Farrowing grout. An estimate of the number of CL for each gilt was taken as the mean number of CL for the other three groups (i.e., gilts slaughtered at Days 3, 10 and 30). This gave a mean value of 13.13 CL; hence the proportion of embryos surviving was calculated as follows: (Total no. pigs born live)/( 13.13 x fertilization rate). The total number of pigs born for each gilt included stillborn pigs, which were not included in the fetal mortality category. Data were analyzed using both fertilization rates and by including and excluding the nonpregnant gilts.
RESULTS Pregnancy Rate All 26 animals slaughtered on Day 3 had more than seven fertilized ova at slaughter, giving a pregnancy rate of 100%. The pregnancy rate of gilts slaughtered on Day 10 of
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gestation was 98%. This was because one of the 42 gilts inseminated had no embryos, but six unfertilized ova were recovered from this gilt. Another seven gilts had less than 4 embryos and were included as pregnant gilts as they had embryos present at the time of slaughter. Nineteen percent of the Day- 10 group could potentially have returned to service, based on embryo numbers present at slaughter. Of the 45 animals originally assigned to be slaughtered on Day 30, nine returned to service giving a pregnancy rate of 80%. Seven of the 36 gilts in the farrowing group returned to service. The pregnancy rate for this group was 81% (Table 1). Ovulation Rate and Embryo Survival Of the 26, 42 and 36 gilts slaughtered on Days 3, 10 or 30, respectively, the total number of ovulations was 342,557 and 467 giving a mean (+SEM) number of ovulations of 13.1520.46, 13.36kO.37 and 12.97kO.39, respectively (P>O.O5; Table 1). The recovery rates at Days 3, 10 and 30 were 90, 77 and 74%, respectively. The number of fertilized ova or normal embryos or piglets born was 11.12kO.69, 9.46iO.55, 9.33kO.58 and 9.1kO.54, respectively, for gilts at Day 3, Day 10, Day 30 of gestation and those that farrowed (P=O. 135, Table 1). However, on further analysis there was a significant difference between the mean number of normal embryos on Day 3 and Day 30 (P=O.O5) and also between the mean number of embryos on Day 3 and the mean number of piglets born in the farrowing group (P=O.O35). (Nonpregnant animals in the Day-30 and farrowing groups were not used in the calculations). There was no prenatal loss in 6, 7 and 6 gilts from the Day-3, Day-10 and Day-30 groups, respectively (the number of CL = the number of embryos) . Fertilization and Embryo Survival The fertilization rate was 94.5+2.0% based on ova recovery (FRER) and was 84.5+2.5% based on ovulation rate (FROR; Table 2). The mean percentage of embryonic survival in gilts (including pregnant and nonpregnant gilts) in the Day- 10, Day-30, and the farrowing group based on FRER was 73.7*4.9%, 61.2+5.9% and 59.1+6.0%, respectively. If nonpregnant gilts were excluded (one gilt from the Day-10 group, nine gilts from the Day-30 group and seven gilts from the farrowing group), then the percentage of embryonic survival among pregnant gilts was 75.5*4.7%, 76.5*4.6% and 73.3&4.4% in the Day-lo, Day-30, and the farrowing groups, respectively (Table 2). The corresponding values for all gilts (pregnant and nonpregnant) using FROR were 82.4k5.5, 68.5k6.6 and 66.1+6.8%. When nonpregnant gilts are excluded the level of embryonic survival in the Day-lo, Day-30 and farrowing group was 84.4k5.2, 86.6k5.1 and 82.0f4.9%, respectively (Table 2). Embryonic/Fetal Mortality Based on FRER embryonic mortality between Days 3 and 10 of pregnancy was 20.8f8.3% (P=O.Ol), and between Days 10 and 30 it was 12.5+7.19/o (P=O.O5) in all gilts. Fetal loss between Day 30 and farrowing was 2.247.4% (Table 3). When the level of embryonic loss from Day 3 to Day 10 was compared to the loss from Day 10 to Day 30 (a difference of 8.3?13%), there was no significant difference. However, the loss from Day 3 to Day 30 (a difference of 31.1+13.03%) was significantly different (P=O.O2) compared with the loss from Day 30 to parturition. This indicates that embryonic loss accounts for most prenatal mortality in gilts.
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Table 1. Pregnancy rate and embryo recovery rate (with ANOVA model standard errors) in gilts slaughtered at Days 3, 10 and 30 of gestation and piglets born in a group that were allowed to farrow Treatment
Day 3
Day 10
No. of gilts inseminated
26
42
No. of gilts yielding embryos
26
41
36
29
% pregnant
100
98
80
81
Total no. of CL
342
557
467
13.15kO.46
13.36kO.37
12.97*0.39
No. of CL mean (+SEM)
Total no. of embryos recovered
307
428
Total no. of normal embryos recovered
289
388
No. of normal embryos recovered mean (SEM)
11.12kO.69
9.46+0.55
Day 30
Farrowing
P value for overall F-test
36
336
0.767
264a’
9.33kO.58
9.10+0.54a
0.135
a = Number of piglets born. The extent of complete or partial loss was examined. Excluding the gilts with complete prenatal loss changed the pattern of partial prenatal loss in pregnant gilts (Table 3). The extent of embryonic loss between Days 3 and 10 of gestation remained unchanged (19.Ok6.3 versus 20.8+8.3%). However, there was no further loss from Day 10 to Day 30 of gestation. Fetal mortality was 3.2+6.39/o and was not significant. However, when comparing the level of loss from Day 3 to Day 10 with the loss from Day 10 to Day 30, there was a significant difference (P=O.O5), suggesting that most of the loss in pregnant gilts occurred before Day 10 of gestation. Based on FROR only 2.1+9.3% embryonic loss occurred by Day 10 of gestation in all gilts. A further 13.9+7.9% loss occurred between Day 10 and Day 30 of gestation, with fetal mortality at 2.4+8.3% (Table 3). None of these differences were significant. When nonpregnant gilts were excluded, the level of embryonic mortality in pregnant gilts from Day 3 to Day 10 was 0.0+7.1%, from Day 10 to Day 30 it was - 1.2*6.4% and fetal mortality was 3.6+7.0% (Table 3). None of these values were significantly different.
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THERIOGENOLOGY Table 2. The percentage of embryos surviving (within-group standard errors) in gilts slaughtered on Days 3, 10, 30 of gestation and at farrowing, based on expressing fertilization rate on the basis of ova recovery or ovulation rate % surviving at : Day 10 Day 30
Day 3
Farrowing
Allgilts
Pregnant gilts only
F&R FROR
94.5*2.0 26 84.5k2.5
73.7k4.9 42 82.4k5.5
61.2*5.9 45 68.5*6.6
59. E6 0 66.1+6:8
F&R
26 94.5k2.0 84.5k2.5
41 75.5k4.7 84.4k5.2
36 76.5k4.6 86.6k5.1
29 73.3k4.4 82.Ok4.9
FROR
Table 3. The percentage embryonic and fetal mortality (with ANOVA model standard errors), based on expressing fertilization rate on the basis of recovery of ova or the ovulation rate %loss from Day 3 to Day 10
% loss from Day 10 to Day 30
%loss from Day 30 to Farrow
All gilts
Pregnant gilts only
FRER FROR
20.8+8.3 2.159.3
12.557.1 13.9k7.9
2.2k7.4 2.458.3
FRER FROR
19.Ok6.3 O.Ok7.1
-1.Ok5.7 -1.2k6.4
3.2k6.3 3.6k7.0
DISCUSSION Many studies have been carried out to study embryonic mortality, but direct comparisons with such work are difficult because of the variation in use of the term embryonic mortality. In some studies this includes loss due to fertilization failure and, to a varying extent, fetal mortality (4-6,9). The level of embryonic mortality depends on whether it is a primiparous (gilt) or a multiparous (sow) animal. It is established that sows sustain a higher level of embryonic mortality than gilts (4). Not all authors distinguish between embryonic mortality in gilts and sows, so direct comparisons with embryonic mortality in gilts alone are not possible (9). Another difficulty lies in interpreting the data.
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It has already been shown that the fertilization rate in pigs is higher than 90% (8,9). To get a more complete picture of the losses at each stage (ova, embryo and fetal stages) in a group of animals kept under similar management and environmental conditions, it was deemed necessary first to establish the fertilization rate in a group of these animals (Day 3) as an estimate for the fertilization rate in other groups (Days 10 and 30 and the farrowing group). This was then used in the calculation of embryonic mortality in gilts at Days 10 and 30 of gestation and of fetal mortality. Variation in the fertilization rate was not allowed for. At Day 3 only 90% of the ova were recovered. The question then arises as to whether those ova not recovered had a similar fertilization rate to those recovered (FRER), or whether they were unfertilized (FROR). Data were analyzed using both assumptions. The 94.5% fertilization rate (FRER) is similar to other reports (8,9) while the 84.5% fertilization rate (FROR) is considerably lower. A second difficulty in our study was with gilts slaughtered at Day 10 in the definition of pregnancy. Recognition of pregnancy occurs around Day 12 (14), although it is possible that there are subtle signals before Day 12. However, the animal is considered pregnant once embryos are present in the uterus. It has been shown that gilts with less than four viable embryos at Day 12 will not maintain pregnancy (15,16) so 19% of the Day-10 group, had they not been slaughtered, presumably would have returned to es&us. This would be in line with the 20 and 19% of returns in the Day-30 and farrowing groups, respectively. This suggests that complete fertilization failure was not the cause of complete litter loss, but, in fact, viable embryos were present although in insufficient numbers at the critical stage (Day 12) to generate an adequate signal for the maintenance of pregnancy. A combination of a low fertilization rate and a high embryonic mortality rate before this critical stage would result in pregnancy failure. Another difficulty arose in gilts with complete litter loss. One gilt at Day 10 had no embryos;while nine and seven gilts returned to estrus from the Day-30 and farrowing groups, respectively. The question is whether they should be included in the estimation of embryonic/fetal mortality or should the level of embryonic/fetal mortality be estimated in pregnant gilts separately. Once again, data were analysed both ways and also using the two fertilization rates. A different picture emerges depending on the method of calculation. Fertilization Rate (FRER) and Subsequent Prenatal Loss With a fertilization rate of 94.5% there was a significant embryonic loss at Day 10 (20%), with a further loss of 12% between Day 10 to Day 30 of gestation (all gilts). As this 12% loss was not significantly different from the 20% loss, no statistical conclusion could be drawn regarding the timing of embryonic loss in all gilts. The total embryonic loss was significantly higher than the fetal loss, as expected (1,2). When nonpregnant gilts were excluded, there was a significant embryonic loss by Day 10 (19%), but in gilts that remained pregnant no more embryonic loss occurred after Day 10. By comparing the two it can be seen that approximately 50% of the loss from Day 10 to Day 30, when all gilts were included, could be attributed to complete litter loss in the gilts that returned to estrus. Fertilization Rate (FROR) and Subsequent Prenatal Loss With a fertilization rate of 84.5% most of the loss occurred at the ovum stage due to fertilization failure. This is in conflict with data in the literature since in general pigs have high fertilization rates (8,9), although certain individual animals for reasons such as incorrect timing of mating, poor semen quality, or infection may have a low fertilization
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rate. These factors can be discounted in this investigation since management factors were standardized across all treatment groups, and strict hygienic procedures were followed to minimise the risk of infection. Using FROR and including all gilts, practically no embryonic loss occurred by Day 10 (2+9%). This low level of embryonic mortality by Day 10 is in agreement with one report (8) (
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gestation. In gilts that remained pregnant, there was 19% embryonic mortality and a further 3% fetal mortality. The bulk of this prenatal loss was due to embryonic mortality, and this occurred before Day 10 of pregnancy. REFERENCES 1.
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