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Safety of altrenogest in pregnant mares and on health and development of offspring
SAFETY OF ALTRENOGEST IN PREGNANT MARES AND ON HEALTH AND DEVELOPMENT OF OFFSPRING C.F. Shoemaker, MS; E.L. Squires PhD; and R.K. Shideler, DVM
SUMMARY Fifty-one light-horse mares were utilized to evaluate the safety of an oral progestin, altrenogest, administered throughout gestation on: gestation length, embryonic and fetal loss, periparturient events, health and development of offspring, and future reproductive capabilities of the mares. Pregnancies were established by inseminating mares with 250 x 106 progressively motile spermatozoa from the same stallion every other day throughout estrus or by non-surgical transfer of embryos. Mare s were randomly assigned to 1 of 2 treatments upon confirmation of pregnancy on day 20: 1) controls, 2 ml of neobee oil orally per 44.5 kg of body weight; and 2) treated, 2 ml of altrenogest dissolved in neobee oil at a concentration of 2.2 mg/ml orally per 44.5 kg of body weight. Treamaents were administered dally from day 20 to 320 of gestation. There were no significant differences between treatment groups for duration of gestation, placental weight, time to placental expulsion and incidence of retained placental membranes. Number of female foals born from altrenogest treated mares (14 of 23) was greater (P<.05) than the number from untreated control mares (4 of 16). Female foals born from altrenogest treated mares had larger clitori (P<.05) than those from control mares. Times to sternal recumbency, standing and nursing were similar for the 2 groups (P>.05). Body weight and height at withers, heart girth circumference and length and width of cannon were measured at time of birth and at 2, 4, 6, 8, 12 and 16 weeks of age. Measurements did not differ (P>05) between treated and control foals for any development parameters. Authors' address: Animal Reproduction Laboratory, Colorado State University, Fort Collins, CO 80523. V o l u m e 9, N u m b e r 2, 1989
Beginning on day 20 postpartum, mares were teased dally. During estrus, mares were inseminated every other day with 250 x 10 6 motile spermatozoa. Teasing and/or insemination was continued for 2 cycles or until mares were 35 days pregnant. The number of mares pregnant after 1 cycle and after 2 cycles of insemination was similar (P>.05) for treated and control mares. Nineteen of 21 treated mares and 15 of 16 control mares were pregnant after 2 cycles of insemination. Number of cycles per pregnancy was similar (P>.05) for treated and control mares (1.37 vs 1.13) as was number of days mares exhibited estrus (6.30 vs 6.13). Number of inseminations per cycle did not differ (P>.05) between treated and control mares (2.92 vs 3.00). In summary, there was no effect of treatment with altrenogest from day 20 to 320 of gestation on periparturient events, viability and growth of offspring and subsequent reproductive performance of mares.
INTRODUCTION Pregnancy maintenance in mares is largely dependent on adequate production and secretion of progesterone. Initially, the primary corpus luteum and subsequently secondary corpora lutea are solely responsible for production of progesterone. Based on measurements of progesterone in the uterine vein 13 and successful maintenance of pregnancy after ovariectomy,s it would appear that the placenta becomes a source of progesterone at approximately 60 to 80 days of gestation. Inadequacies in production or secretion of progesterone due to failure of luteal function has been suggested as a cause of early embryonic death (EED) and termination of pregnancy. I Pregnant mares are frequently given injections of progesterone in an attempt to prevent abortion.2.6 Mares ovariectomized on day 25 of gestation maintained preg69
nancies when injected intramuscularly with 100 mg of progesterone in oil daily.5Pregnancy was terminated 2-6 days after treatment ended. Both injectable progesterone and a syntheticprogestin, altrenogest, effectivelymaintainedpregnancy in mares ovariectomized on day 34 or 35 of gestation.u Intramuscular injections of 1000 mg of progesterone in propylene glycol every 4 days were required to maintain endogenous levels sufficient to maintain pregnancy. Unfortunately this is an impractical and inconvenient way of administering progesterone to mares, primarily due to local inflammatory reactions at the injection site. Oral administration of a progestin would seem to be more desirable. Altrenogest, an orally active progestin, has been used effectively for suppression of eslrous behavior 14,~5,asand pregnancy maintenance. ~ Use of ovariectomized progesterone treated mares as recipients for embryo transfer has recently been reported. 3,4Thirty-five day pregnancy rates for ovariectomized progestin-treated recipients (28 of 40, 70%) were similar to those of synchronized intact mares (16 of 20, 80%). s Despite the apparent widespread use of altrenogest for pregnancy maintenance, adequate studies have not been performed to determine safety with long term supplementation. This experiment was designed to determine effect of altrenogest therapy throughout pregnancy on gestation length, pregnancy loss, periparturient events, further reproductive capabilities of mares, and overall health and development of their offspring.
MATERIALS AND METHODS General Fifty-one light-horse mares were utilized. Mares selected were 3 to 15 years of age, and weighed between 386 and 500 kg. Pregnancies were established by artificially inseminating mares with 250 x 106 progressively motile spermatozoa every other day throughout estrus (N=24) or by non-surgical transfer of embryos (N=15)? 6 The same stallion sired all foals and donor mares were from the same herd. Mares were cont'umed pregnant on day 15 and 20 post-ovulation via ultrasonography9and randomly assigned to 1 of 2 groups: 1) controls, 2 ml of neobee oil • (carder for altrenogest) orally per 44.5 kg body weight; 2) treated, 2 ml of altrenogest b dissolved in neobee oil at a concentration of 2.2 mg/ml orally per 44.5 body weight: this was twice the recommended dose for estrous suppression. Treatments were administered daily from day 20 to 320 of gestation. Originally, 27 treated and 24 control mares were assigned to this study using a table of random numbers. 7 However, the number of animals available for evaluation changed over the course of the study. Early embryonic death in 5 control and 2 altrenogest treated mares occurred between 30 and 60 days. Two control mares were excluded from the trial, due to maintenance of twin pregnancies and 1 control aNeobee Oil, Hoechst-Roussel Agri-Vet., Somerville, NJ. bReguMate, Hoechst-Roussel Agri-Vet., Somerville, N,J.
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mare died of an undetermined cause. Abortions occurred in 2 altrenogest treated mares on days 165 and 250 of gestation. One abortion was apparently due to a traumatic kick, while the cause of the second was unknown. After these losses, total number of mares per treatment group was 23 treated and 16 control.. All mares were maintained in a single pasture and fed 12 kg of alfalfa and 2 kg of grain mixture dally. Water and trace mineralized salt were available ad libitum. Mares were weighed monthly and dosages were adjusted. Ultrasonography was used to assess viability of the conceptus on day 25, 30, 35, 40, 50, 60, 80, 100 and 120 of gestation. Palpation per rectum was performed monthly thereafter to assess pregnancy status.
Periparturient events Beginning approximately 1 month prior to scheduled foaling, each mare was observed for signs of impending parturition. Those mares estimated to be within one week of foaling were placed in 4.9 x 3.7m foaling stall and closely observed. After parturition, mares and foals were examined and initial assessments made regarding general health. Assessment of foal health was determined by: 1) hair coat - normal or short; 2) attitude - aggressive, slow, dull/sleepy or comatose; 3) maturity - mature (gestation length > 320 days and normal foal), premature (gestational length <320 days) or dismature (gestational length >320 days and abnormal foal). Data collected included sex of foal, time from birth to: 1) sternal recumbency, 2) standing, 3) nursing, and 4) placental expulsion. Weight of placenta and incidence of membrane retainment were also recorded. Placental membranes were classified as retained if not expelled within 3 hours after foaling. Body weight and height at withers, bean girth circumference and length and width of cannon bone were also measured at time of birth and at 2,4,6,8, 12 and 16 weeks of age. Progestin-treated and control mares were compared to determine if treatment had an effect on duration of pregnancy. Clitori of fdlies were measured at birth and subsequent measurements were taken at 1, 4, 6, 10, 12, 15, 18 and 21 months of age. The width and length (mm) of each clitoris was measured twice by 2 technicians. The average clitoral area (mm2) was used for statistical analysis. Comparisons were made between recipient mares and natural mothers to determine effects of embryo transfer on gestational length, sex ratio and placental weight. Time from birth to: 1) sternal recumbency, 2) standing, and 3) nursing; and birth weight and height, bean girth circumference and length and width of cannon bone were also compared between foals from embryo recipients and natural dams. An outbreak of foal septicemia occurred at the Equine Reproduction Laboratory in some foals in both groups and 6 foals died as a result of identified microorganisms and were not available for progressive measurements. In addition, 1 control mare delivered a stillborn foal. Foal losses EQUINE VETERINARY SCIENCE
were similar (P>.05) between treated (N=4) and control groups (N=3). Number of live foals per group after these los ses was 19 treated foals and 13 control foals. Because of the time involved with foal observation and neonatal care, some data points were not collected. Reproductive S o u n d n e s s
Mares and foals were maintained in 3 pastures and grouped according to age of foal. Beginning 20 days postpartum, mares were teased daily until they had been inseminated 2 cycles or were 35 days pregnant. Mares were artificiallyinseminated with 250 x 106progressively motile spermatozoa from 1 stallion beginning on day 2 or 3 of estrus and continuing every other day until cessation of estrus. Data collected were: 1) number of cycles per pregnancy; 2) number of inseminations per cycle; 3) duration of estrus for cycle I and 2; 4) pregnancy rate per cycle; and 5) overall pregnancy rate for treated and control mares. Statistical Analysis
Chi-square analyses 12were used to compare: 1) number of foals from treated and control mares; 2) number of male and female foals from treated and control mares and embryo transferdams versus natural mothers; and 3) number of treated and control mares pregnant per cycle and after 2 cycles. Quantitative data were analyzed by two-sample ttests to compare differences between treatment groups. RESULTS AND DISCUSSION
in utero. Differences were statistically significant(P<.05) at all ages except I and 4 months. Presented in Table 2 are times to sternal recumbency, standing and nursing for foals in the two treatment groups. Table 2. Effects of administering altrenogest during pregnancy on per±parturient events (mean ¢S.E.) Parameters Stermal recumbancy (rain)
Groupa Altrenogest Control
6.4+3.5 5,0±1.1
Birth to stand (rain)
Birth to nurse (min)
44.1:1~..8 38.3±.2.8
77.1¢4.9 75.6±4.3
• Number of observations per mean ranged from 11 to 12 for the treated group and N=9 for controls.
Treatment of mares with altrenogest had no effect on any of these parameters. Health of foals at birth did not differ (P>.05) due to treatment. Since some pregnancies were established by embryo transfer (7 treated, 8 controls) and others by artificial insemination (16 treated, 8 controls), comparisons were made between foals from natural mothers and those from embryo Cansfer recipients. No differences were found between groups for gestational length and sex ratio. Placental weight, time to sternal recumbency, time to standing and time to nursing (Table 3) were also not affected (P>.05) by type of dam.Measurements taken at time of birth are presented in Table 4.
Duration of gestation was similar (P>.05) between treated and control mares (324.7 vs 326.9 days). Treatment of mares with altrenogest during gestation had no effect on placental weight, expulsion of placenta or incidence of retained placental membranes (Table 1). The number of female foals born from mares treated with altrenogest (14 of 23) was greater (P<.05) than the number from untreated control mares (4 out of 16). The reason for the apparent alteration of sex ratio was most likely due to small numbers and chance alone, since treat-
3.7+.19
7.6+3.8
44.0-J:1.9 79.0±4.0
Embryo transfe# 325.2.d:2.4 3.7+22
3.9i.77
39.0±3.6
Table 1. Effect of administering altrenogest during pregnancy on per±parturient events (mean ±S.E.)
aNumberof observationsper mean ranged from 10 to 24. bNumberof observationsper mean ranged from 10 to 15.
Parameters Group a
Placental weight (kg)
Placental explusion
(min)
Inddence retained placentab
Altrenogest
3.6±.22
28.4±5.9
0
Control
3.8+.15
27.1+4.9
1
aNumber observationsper mean ranged from 17 to 23 for the treated group and 13 to 16 for controls. bRetained if not expelledwithin 3 hours
ment started after pregnancy had been established. Females from treated animals had larger clitori at each measurement period indicating that altrenogest had some virilizing effect Volume 9, Number 2, 1989
Table 3. Effect of type of dam on periparturient events (mean ±S.E.) Parameters Type of dam
Gestation length (days)
Natural" 325.4+1.4
Placental Sternal weight recumbency (kg) (min)
Birth to stand (min)
Birth to nurse (min)
738+5.2
There was no effect of type of dam on birth weight or height, heart girth and cannon length, however, cannon width was smaller (P<.05) in embryo transfer foals versus natural mothers. The significance of this finding was unknown. Presented in table 5 are measurements taken on foals in treated and control groups at birth and at weeks 2, 4, 6, 8, 12, and 16. Weight changes over the time period were similar (P>.05) between treated and control animals. Similarly, changes in height at withers and heart girth measurements were not different (P>.05) between treated and controls at each of the measurement periods. In addition, width and length of cannon increased similarly (P>.05) in both groups 71
Table 4. Effect of type of dam on birth weight and height, heart girth and length and width of cannon (mean:t:S.E.) Parameters Birth weight (kg)
Type o f dam
Birth height (cm)
Heart girth (cm)
Cannon length (cm)
Cannon width (cm)
Natural a 38.2+1,5 92.8+_1.4 75.2+1.1 19.9+.28 Embryo transfed' 38.2+1.4 92.1+1.2 74.7+.92 19.95:3.0
2.9%,04 2.7d+.03
"Numberof observationsrangedfrom 23 to 24. bNumberof observationswas N=13, ='~P<.05
REFERENCES
throughout the measurement period. Thus it appeared, based on data coUected at time of birth, and measurements collected over a 16-week period, that there was no effect of treatment of pregnant mares with altrenogest on periparturient events or viability and growth of offspring. As a result of mares entering seasonal anestrus, 2 treated mares were not inseminated and were omitted from the analyses. The number of mares pregnant after 1 cycle and after 2 cycles of insemination was similar (P>.05) for mares in the 2 groups. Nineteen of 21 treated mares and 15 Table 5. Effect of administering altrenogest during pregnancy on weight, height, heart girth, cannon length and width of foals Parameters
Age (wk)
Weight (kg)
Height (cm)
Heart girth (cm)
1"
1
1
2b
0 38.4 38.3 2 60.6 62.5 4 77.3 77.7 6 88.5 90.5 8 98.7 99.9 12 124.1 124.8 16 147.9152.6
91.4 98.9 104.6 108.1 111.3 114.6 118.4
2
Cannon length (cm) 2
9 4 . 3 76.0 76.8 99.7 88.1 90.1 105.7 95.6 95.9 109.9 100.9 100.0 112.7 104.3 105.2 116.0 111.9 111.6 118.9 118.1 118.6
1 20.2 21.3 22.3 22.8 23.1 23.4 23.5
2
Cannon width (cm) 1
20.6 2.9 21.3 3.0 22.1 3.0 22.8 3.1 23.1 3.1 23.5 4.3 23.6 3.2
2 2.9 3.1 3.2 3.1 3.2 3.2 3.3
Altrenogesttreatedmares-N=I 5 to 19. Controlmares- N=I 2.
of 16 control mares were confirmed pregnant on day 15 post-ovulation after 2 cycles. By 35 days, 16 of 21 treated mares and 13 of 16 control mares remained pregnant. There were 3 embryonic losses in treated mares and 2 in control mares (P>.05) Previous studies in our laboratory have reported an embryonic loss of 13% between days 15 and 50 of gestation. 17 Therefore, it would appear that the embryonic loss in both treated and control mares was comparable to previous reports. Number of cycles per pregnancy was similar (P>.05) for mares in treated (1.37) and control (1.13) groups as was duration of estrus (6.30 vs 6.13 days). Number of inseminations per cycle was notdifferent (P>.05) between treated and control mares (2.92 vs 3.00). Based on these data, reproductive efficiency was not affected in 72
mares previously treated with altrenogest. Fifty-five to 65% pregnancy rate on first cycle is considered normal reproductive efficiency.1° In this study, the pregnancy rate on first cycle was 76.2% for treated mares and 93.8% for control mares. In summary, there was no effect of treatment with altrenogest from day 20 to 320 of gestation on periparturient events, viability and growth of offspring and subsequent reproductive performance of mares.
1. Douglas RH, Bums PJ, Hershman L: Physiological and commercial parameters for producing progeny from subfertile mares by embryo transfer. Eq Vet J, Suppl 3, 111-114, 1985. 2. Ganjam VK, Kenney RM: Peripheral blood plasma levels and some unique metabolite aspects of progesterone in pregnant and non-pregnant mares. Proc 21st Ann Con AAEP, pp 263-276, 1975. 3. Hinrich K, Sertich PL, Cummings MR, Kenney RM: Pregnancy in ovarlectomized mares achieved by embryo transfer: A preliminary study. Eq Vet J, Suppl 3, 74-75, 1985. 4. Hinrich K, Sertich PL, Kenney RM: Use of altrenogest to prepare ovariectomized mares as embryo transfer recipients. Theriog 26, 455-460, 1986. 5. Holtan DW, Squires EL, Lapin DR, Ginther OJ: Effect of ovariectomy on pregnancy in mares. JReprodFert, Supp127, 457463, 1979. 6. Hughes JP, Stabenfeldt GH: The use of hormones in reproductive management of the mare. Aust Vet J 53, 258-261, 1977. 7. Huntsberger DV, Billingsley P: Elements of Statistical Inference. Allyn and Bacon, Inc., Boston, 1981. 8. McKinnon AO, Squires EL, Carnevale EM and Hermenet MJ: Pregnancy rates after surgical transfer into steroid-treated ovariectomized mares. Therio 19, 1055-1064, 1988. 9. McKinnon AO, Squires EL, Voss JL: Ultrasonic evaluation of the mare's reproductive tract - Part I1. Comp Cont Ed Pract Vet 9, 472-482, 1987. 10. Pickett BW, Squires EL, McKinnon AO: Procedures for Collection, Evaluation and Utilization of Stallion Semen for Artificial Insemination. Colorado State Univ Anita Reprod Lab Bull No 03, 1987. 11. Shideler RK, Squires EL, Voss JL, Eikenberry DJ, Pickett BW: Progestagen therapy of ovariectomized pregnant mares. J Reprod Fert, Suppl 32, 459-464, 1982. 12. Steel RGD, Torrie JH: Principles and Procedures of Statistics. A Biomedical Approach. McGraw-Hill Book Co., New York, 1980. 13. Squires E L, Ginther OJ: Collection technique and progesterone concentration of ovarian and uterine venous blood in mares. J Anita Sci 40, 275-281, 1975. 14. Squires EL, Hessemann CP, Webel SK, Shideler RK, Voss JL: Relationship of altrenogest to ovarian activity, hormone ooncontrations and fertility of mares. JAnim Sci4, 901-909, 1983. 15. Squires EL, Stevens WB, McGIothlin DE, Pickett BW: Effect of an oral progestin on the estrous cycle and fertility of mares. J Anita Sci 49, 729-735, 1979. 16. Squires EL, Voss JL, Cook VM: Collection and Transfer of Equine Embryos. Colorado State Univ Anita Reprod Lab Bull No 01, 1985. 17. Villahoz MD, Squires EL, Voss JL, Shideler RK: Some observations on early embryonic death in mares. Theriog 23, 915924, 1985. 18. Webel, Squires EL: Control of the oestreus cycle in mares with altrenogest. J Reprod Fert, Suppl 32, 193-198, 1982. EQUINE VETERINARY SCIENCE
SUMMARY Fifty-one light-horse mares were utilized to evaluate the safety of an oral progestin, altrenogest, administered throughout gestation on: gestation length, embryonic and fetal loss, periparturient events, health and development of offspring, and future reproductive capabilities of the mares. Pregnancies were established by inseminating mares with 250 x 106 progressively motile spermatozoa from the same stallion every other day throughout estrus or by non-surgical transfer of embryos. Mare s were randomly assigned to 1 of 2 treatments upon confirmation of pregnancy on day 20: 1) controls, 2 ml of neobee oil orally per 44.5 kg of body weight; and 2) treated, 2 ml of altrenogest dissolved in neobee oil at a concentration of 2.2 mg/ml orally per 44.5 kg of body weight. Treamaents were administered dally from day 20 to 320 of gestation. There were no significant differences between treatment groups for duration of gestation, placental weight, time to placental expulsion and incidence of retained placental membranes. Number of female foals born from altrenogest treated mares (14 of 23) was greater (P<.05) than the number from untreated control mares (4 of 16). Female foals born from altrenogest treated mares had larger clitori (P<.05) than those from control mares. Times to sternal recumbency, standing and nursing were similar for the 2 groups (P>.05). Body weight and height at withers, heart girth circumference and length and width of cannon were measured at time of birth and at 2, 4, 6, 8, 12 and 16 weeks of age. Measurements did not differ (P>05) between treated and control foals for any development parameters. Authors' address: Animal Reproduction Laboratory, Colorado State University, Fort Collins, CO 80523. V o l u m e 9, N u m b e r 2, 1989
Beginning on day 20 postpartum, mares were teased dally. During estrus, mares were inseminated every other day with 250 x 10 6 motile spermatozoa. Teasing and/or insemination was continued for 2 cycles or until mares were 35 days pregnant. The number of mares pregnant after 1 cycle and after 2 cycles of insemination was similar (P>.05) for treated and control mares. Nineteen of 21 treated mares and 15 of 16 control mares were pregnant after 2 cycles of insemination. Number of cycles per pregnancy was similar (P>.05) for treated and control mares (1.37 vs 1.13) as was number of days mares exhibited estrus (6.30 vs 6.13). Number of inseminations per cycle did not differ (P>.05) between treated and control mares (2.92 vs 3.00). In summary, there was no effect of treatment with altrenogest from day 20 to 320 of gestation on periparturient events, viability and growth of offspring and subsequent reproductive performance of mares.
INTRODUCTION Pregnancy maintenance in mares is largely dependent on adequate production and secretion of progesterone. Initially, the primary corpus luteum and subsequently secondary corpora lutea are solely responsible for production of progesterone. Based on measurements of progesterone in the uterine vein 13 and successful maintenance of pregnancy after ovariectomy,s it would appear that the placenta becomes a source of progesterone at approximately 60 to 80 days of gestation. Inadequacies in production or secretion of progesterone due to failure of luteal function has been suggested as a cause of early embryonic death (EED) and termination of pregnancy. I Pregnant mares are frequently given injections of progesterone in an attempt to prevent abortion.2.6 Mares ovariectomized on day 25 of gestation maintained preg69
nancies when injected intramuscularly with 100 mg of progesterone in oil daily.5Pregnancy was terminated 2-6 days after treatment ended. Both injectable progesterone and a syntheticprogestin, altrenogest, effectivelymaintainedpregnancy in mares ovariectomized on day 34 or 35 of gestation.u Intramuscular injections of 1000 mg of progesterone in propylene glycol every 4 days were required to maintain endogenous levels sufficient to maintain pregnancy. Unfortunately this is an impractical and inconvenient way of administering progesterone to mares, primarily due to local inflammatory reactions at the injection site. Oral administration of a progestin would seem to be more desirable. Altrenogest, an orally active progestin, has been used effectively for suppression of eslrous behavior 14,~5,asand pregnancy maintenance. ~ Use of ovariectomized progesterone treated mares as recipients for embryo transfer has recently been reported. 3,4Thirty-five day pregnancy rates for ovariectomized progestin-treated recipients (28 of 40, 70%) were similar to those of synchronized intact mares (16 of 20, 80%). s Despite the apparent widespread use of altrenogest for pregnancy maintenance, adequate studies have not been performed to determine safety with long term supplementation. This experiment was designed to determine effect of altrenogest therapy throughout pregnancy on gestation length, pregnancy loss, periparturient events, further reproductive capabilities of mares, and overall health and development of their offspring.
MATERIALS AND METHODS General Fifty-one light-horse mares were utilized. Mares selected were 3 to 15 years of age, and weighed between 386 and 500 kg. Pregnancies were established by artificially inseminating mares with 250 x 106 progressively motile spermatozoa every other day throughout estrus (N=24) or by non-surgical transfer of embryos (N=15)? 6 The same stallion sired all foals and donor mares were from the same herd. Mares were cont'umed pregnant on day 15 and 20 post-ovulation via ultrasonography9and randomly assigned to 1 of 2 groups: 1) controls, 2 ml of neobee oil • (carder for altrenogest) orally per 44.5 kg body weight; 2) treated, 2 ml of altrenogest b dissolved in neobee oil at a concentration of 2.2 mg/ml orally per 44.5 body weight: this was twice the recommended dose for estrous suppression. Treatments were administered daily from day 20 to 320 of gestation. Originally, 27 treated and 24 control mares were assigned to this study using a table of random numbers. 7 However, the number of animals available for evaluation changed over the course of the study. Early embryonic death in 5 control and 2 altrenogest treated mares occurred between 30 and 60 days. Two control mares were excluded from the trial, due to maintenance of twin pregnancies and 1 control aNeobee Oil, Hoechst-Roussel Agri-Vet., Somerville, NJ. bReguMate, Hoechst-Roussel Agri-Vet., Somerville, N,J.
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mare died of an undetermined cause. Abortions occurred in 2 altrenogest treated mares on days 165 and 250 of gestation. One abortion was apparently due to a traumatic kick, while the cause of the second was unknown. After these losses, total number of mares per treatment group was 23 treated and 16 control.. All mares were maintained in a single pasture and fed 12 kg of alfalfa and 2 kg of grain mixture dally. Water and trace mineralized salt were available ad libitum. Mares were weighed monthly and dosages were adjusted. Ultrasonography was used to assess viability of the conceptus on day 25, 30, 35, 40, 50, 60, 80, 100 and 120 of gestation. Palpation per rectum was performed monthly thereafter to assess pregnancy status.
Periparturient events Beginning approximately 1 month prior to scheduled foaling, each mare was observed for signs of impending parturition. Those mares estimated to be within one week of foaling were placed in 4.9 x 3.7m foaling stall and closely observed. After parturition, mares and foals were examined and initial assessments made regarding general health. Assessment of foal health was determined by: 1) hair coat - normal or short; 2) attitude - aggressive, slow, dull/sleepy or comatose; 3) maturity - mature (gestation length > 320 days and normal foal), premature (gestational length <320 days) or dismature (gestational length >320 days and abnormal foal). Data collected included sex of foal, time from birth to: 1) sternal recumbency, 2) standing, 3) nursing, and 4) placental expulsion. Weight of placenta and incidence of membrane retainment were also recorded. Placental membranes were classified as retained if not expelled within 3 hours after foaling. Body weight and height at withers, bean girth circumference and length and width of cannon bone were also measured at time of birth and at 2,4,6,8, 12 and 16 weeks of age. Progestin-treated and control mares were compared to determine if treatment had an effect on duration of pregnancy. Clitori of fdlies were measured at birth and subsequent measurements were taken at 1, 4, 6, 10, 12, 15, 18 and 21 months of age. The width and length (mm) of each clitoris was measured twice by 2 technicians. The average clitoral area (mm2) was used for statistical analysis. Comparisons were made between recipient mares and natural mothers to determine effects of embryo transfer on gestational length, sex ratio and placental weight. Time from birth to: 1) sternal recumbency, 2) standing, and 3) nursing; and birth weight and height, bean girth circumference and length and width of cannon bone were also compared between foals from embryo recipients and natural dams. An outbreak of foal septicemia occurred at the Equine Reproduction Laboratory in some foals in both groups and 6 foals died as a result of identified microorganisms and were not available for progressive measurements. In addition, 1 control mare delivered a stillborn foal. Foal losses EQUINE VETERINARY SCIENCE
were similar (P>.05) between treated (N=4) and control groups (N=3). Number of live foals per group after these los ses was 19 treated foals and 13 control foals. Because of the time involved with foal observation and neonatal care, some data points were not collected. Reproductive S o u n d n e s s
Mares and foals were maintained in 3 pastures and grouped according to age of foal. Beginning 20 days postpartum, mares were teased daily until they had been inseminated 2 cycles or were 35 days pregnant. Mares were artificiallyinseminated with 250 x 106progressively motile spermatozoa from 1 stallion beginning on day 2 or 3 of estrus and continuing every other day until cessation of estrus. Data collected were: 1) number of cycles per pregnancy; 2) number of inseminations per cycle; 3) duration of estrus for cycle I and 2; 4) pregnancy rate per cycle; and 5) overall pregnancy rate for treated and control mares. Statistical Analysis
Chi-square analyses 12were used to compare: 1) number of foals from treated and control mares; 2) number of male and female foals from treated and control mares and embryo transferdams versus natural mothers; and 3) number of treated and control mares pregnant per cycle and after 2 cycles. Quantitative data were analyzed by two-sample ttests to compare differences between treatment groups. RESULTS AND DISCUSSION
in utero. Differences were statistically significant(P<.05) at all ages except I and 4 months. Presented in Table 2 are times to sternal recumbency, standing and nursing for foals in the two treatment groups. Table 2. Effects of administering altrenogest during pregnancy on per±parturient events (mean ¢S.E.) Parameters Stermal recumbancy (rain)
Groupa Altrenogest Control
6.4+3.5 5,0±1.1
Birth to stand (rain)
Birth to nurse (min)
44.1:1~..8 38.3±.2.8
77.1¢4.9 75.6±4.3
• Number of observations per mean ranged from 11 to 12 for the treated group and N=9 for controls.
Treatment of mares with altrenogest had no effect on any of these parameters. Health of foals at birth did not differ (P>.05) due to treatment. Since some pregnancies were established by embryo transfer (7 treated, 8 controls) and others by artificial insemination (16 treated, 8 controls), comparisons were made between foals from natural mothers and those from embryo Cansfer recipients. No differences were found between groups for gestational length and sex ratio. Placental weight, time to sternal recumbency, time to standing and time to nursing (Table 3) were also not affected (P>.05) by type of dam.Measurements taken at time of birth are presented in Table 4.
Duration of gestation was similar (P>.05) between treated and control mares (324.7 vs 326.9 days). Treatment of mares with altrenogest during gestation had no effect on placental weight, expulsion of placenta or incidence of retained placental membranes (Table 1). The number of female foals born from mares treated with altrenogest (14 of 23) was greater (P<.05) than the number from untreated control mares (4 out of 16). The reason for the apparent alteration of sex ratio was most likely due to small numbers and chance alone, since treat-
3.7+.19
7.6+3.8
44.0-J:1.9 79.0±4.0
Embryo transfe# 325.2.d:2.4 3.7+22
3.9i.77
39.0±3.6
Table 1. Effect of administering altrenogest during pregnancy on per±parturient events (mean ±S.E.)
aNumberof observationsper mean ranged from 10 to 24. bNumberof observationsper mean ranged from 10 to 15.
Parameters Group a
Placental weight (kg)
Placental explusion
(min)
Inddence retained placentab
Altrenogest
3.6±.22
28.4±5.9
0
Control
3.8+.15
27.1+4.9
1
aNumber observationsper mean ranged from 17 to 23 for the treated group and 13 to 16 for controls. bRetained if not expelledwithin 3 hours
ment started after pregnancy had been established. Females from treated animals had larger clitori at each measurement period indicating that altrenogest had some virilizing effect Volume 9, Number 2, 1989
Table 3. Effect of type of dam on periparturient events (mean ±S.E.) Parameters Type of dam
Gestation length (days)
Natural" 325.4+1.4
Placental Sternal weight recumbency (kg) (min)
Birth to stand (min)
Birth to nurse (min)
738+5.2
There was no effect of type of dam on birth weight or height, heart girth and cannon length, however, cannon width was smaller (P<.05) in embryo transfer foals versus natural mothers. The significance of this finding was unknown. Presented in table 5 are measurements taken on foals in treated and control groups at birth and at weeks 2, 4, 6, 8, 12, and 16. Weight changes over the time period were similar (P>.05) between treated and control animals. Similarly, changes in height at withers and heart girth measurements were not different (P>.05) between treated and controls at each of the measurement periods. In addition, width and length of cannon increased similarly (P>.05) in both groups 71
Table 4. Effect of type of dam on birth weight and height, heart girth and length and width of cannon (mean:t:S.E.) Parameters Birth weight (kg)
Type o f dam
Birth height (cm)
Heart girth (cm)
Cannon length (cm)
Cannon width (cm)
Natural a 38.2+1,5 92.8+_1.4 75.2+1.1 19.9+.28 Embryo transfed' 38.2+1.4 92.1+1.2 74.7+.92 19.95:3.0
2.9%,04 2.7d+.03
"Numberof observationsrangedfrom 23 to 24. bNumberof observationswas N=13, ='~P<.05
REFERENCES
throughout the measurement period. Thus it appeared, based on data coUected at time of birth, and measurements collected over a 16-week period, that there was no effect of treatment of pregnant mares with altrenogest on periparturient events or viability and growth of offspring. As a result of mares entering seasonal anestrus, 2 treated mares were not inseminated and were omitted from the analyses. The number of mares pregnant after 1 cycle and after 2 cycles of insemination was similar (P>.05) for mares in the 2 groups. Nineteen of 21 treated mares and 15 Table 5. Effect of administering altrenogest during pregnancy on weight, height, heart girth, cannon length and width of foals Parameters
Age (wk)
Weight (kg)
Height (cm)
Heart girth (cm)
1"
1
1
2b
0 38.4 38.3 2 60.6 62.5 4 77.3 77.7 6 88.5 90.5 8 98.7 99.9 12 124.1 124.8 16 147.9152.6
91.4 98.9 104.6 108.1 111.3 114.6 118.4
2
Cannon length (cm) 2
9 4 . 3 76.0 76.8 99.7 88.1 90.1 105.7 95.6 95.9 109.9 100.9 100.0 112.7 104.3 105.2 116.0 111.9 111.6 118.9 118.1 118.6
1 20.2 21.3 22.3 22.8 23.1 23.4 23.5
2
Cannon width (cm) 1
20.6 2.9 21.3 3.0 22.1 3.0 22.8 3.1 23.1 3.1 23.5 4.3 23.6 3.2
2 2.9 3.1 3.2 3.1 3.2 3.2 3.3
Altrenogesttreatedmares-N=I 5 to 19. Controlmares- N=I 2.
of 16 control mares were confirmed pregnant on day 15 post-ovulation after 2 cycles. By 35 days, 16 of 21 treated mares and 13 of 16 control mares remained pregnant. There were 3 embryonic losses in treated mares and 2 in control mares (P>.05) Previous studies in our laboratory have reported an embryonic loss of 13% between days 15 and 50 of gestation. 17 Therefore, it would appear that the embryonic loss in both treated and control mares was comparable to previous reports. Number of cycles per pregnancy was similar (P>.05) for mares in treated (1.37) and control (1.13) groups as was duration of estrus (6.30 vs 6.13 days). Number of inseminations per cycle was notdifferent (P>.05) between treated and control mares (2.92 vs 3.00). Based on these data, reproductive efficiency was not affected in 72
mares previously treated with altrenogest. Fifty-five to 65% pregnancy rate on first cycle is considered normal reproductive efficiency.1° In this study, the pregnancy rate on first cycle was 76.2% for treated mares and 93.8% for control mares. In summary, there was no effect of treatment with altrenogest from day 20 to 320 of gestation on periparturient events, viability and growth of offspring and subsequent reproductive performance of mares.
1. Douglas RH, Bums PJ, Hershman L: Physiological and commercial parameters for producing progeny from subfertile mares by embryo transfer. Eq Vet J, Suppl 3, 111-114, 1985. 2. Ganjam VK, Kenney RM: Peripheral blood plasma levels and some unique metabolite aspects of progesterone in pregnant and non-pregnant mares. Proc 21st Ann Con AAEP, pp 263-276, 1975. 3. Hinrich K, Sertich PL, Cummings MR, Kenney RM: Pregnancy in ovarlectomized mares achieved by embryo transfer: A preliminary study. Eq Vet J, Suppl 3, 74-75, 1985. 4. Hinrich K, Sertich PL, Kenney RM: Use of altrenogest to prepare ovariectomized mares as embryo transfer recipients. Theriog 26, 455-460, 1986. 5. Holtan DW, Squires EL, Lapin DR, Ginther OJ: Effect of ovariectomy on pregnancy in mares. JReprodFert, Supp127, 457463, 1979. 6. Hughes JP, Stabenfeldt GH: The use of hormones in reproductive management of the mare. Aust Vet J 53, 258-261, 1977. 7. Huntsberger DV, Billingsley P: Elements of Statistical Inference. Allyn and Bacon, Inc., Boston, 1981. 8. McKinnon AO, Squires EL, Carnevale EM and Hermenet MJ: Pregnancy rates after surgical transfer into steroid-treated ovariectomized mares. Therio 19, 1055-1064, 1988. 9. McKinnon AO, Squires EL, Voss JL: Ultrasonic evaluation of the mare's reproductive tract - Part I1. Comp Cont Ed Pract Vet 9, 472-482, 1987. 10. Pickett BW, Squires EL, McKinnon AO: Procedures for Collection, Evaluation and Utilization of Stallion Semen for Artificial Insemination. Colorado State Univ Anita Reprod Lab Bull No 03, 1987. 11. Shideler RK, Squires EL, Voss JL, Eikenberry DJ, Pickett BW: Progestagen therapy of ovariectomized pregnant mares. J Reprod Fert, Suppl 32, 459-464, 1982. 12. Steel RGD, Torrie JH: Principles and Procedures of Statistics. A Biomedical Approach. McGraw-Hill Book Co., New York, 1980. 13. Squires E L, Ginther OJ: Collection technique and progesterone concentration of ovarian and uterine venous blood in mares. J Anita Sci 40, 275-281, 1975. 14. Squires EL, Hessemann CP, Webel SK, Shideler RK, Voss JL: Relationship of altrenogest to ovarian activity, hormone ooncontrations and fertility of mares. JAnim Sci4, 901-909, 1983. 15. Squires EL, Stevens WB, McGIothlin DE, Pickett BW: Effect of an oral progestin on the estrous cycle and fertility of mares. J Anita Sci 49, 729-735, 1979. 16. Squires EL, Voss JL, Cook VM: Collection and Transfer of Equine Embryos. Colorado State Univ Anita Reprod Lab Bull No 01, 1985. 17. Villahoz MD, Squires EL, Voss JL, Shideler RK: Some observations on early embryonic death in mares. Theriog 23, 915924, 1985. 18. Webel, Squires EL: Control of the oestreus cycle in mares with altrenogest. J Reprod Fert, Suppl 32, 193-198, 1982. EQUINE VETERINARY SCIENCE