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Early pregnancy diagnosis in alpaca (Lama pacos) and llama (Lama glama) by ultrasound
REPeoN SCIENCE
ELSEVIER
Animal Reproduction Science 47 (1997) 113-121
Early pregnancy diagnosis in alpaca (Lama paces) and llama hwza gZama) by ultrasound Victor H. Parraguez *, Sandra Cortkz, Francisca J. GazitGa, Germh Ferrando, Verbnica MacNiven, Luis A. Raggi Facultad de Ciencias Veterinarias y Pecuarias, Unioersidad de Chile, Casilla 2, Correo 15, La Granja,
Santiago, Chile
Accepted 14 October 1996
Abstract An ultrasonography study of early pregnancy diagnosis was carried out in 19 alpacas and 12 llamas, after controlled matings. The aim was to determine the earliest gestational age at which pregnancy diagnosis by transrectal ultrasonography could be achieved, and to generate an empirical formula for gestational sac diameter (GSD) growth as a function of gestational age (GA), allowing an estimate of GA during the first month of pregnancy. We found that pregnancy diagnosis may be carried out as early as 9 days after mating in alpacas and 7 days in llamas. This diagnosis was found to be accurate at 23 days in alpacas and 34 days in llamas. The empirical relations that best describe the relationship between GSD and GA were GA = 1ogGSD + 1.2339/0.0585
r = 0.85,
P < 0.001
in alpacas, and GA = log GSD + 1.2649/0.0546
r = 0.77, P < 0.001
in llamas, where GA is measured in days and GSD in centimeters. Our results also indicate that ultrasonography is a reliable technique for early pregnancy diagnosis. Furthermore, the empirical formulae reliably make it possible to estimate GA from GSD during the first month of pregnancy and their use might improve the efficiency of camelid breeders. 0 1997 Elsevier Science B.V. Keywords: Pregnancy; Alpaca; Llama; Ultrasound
* Corresponding author. Tel.: (562) 6785548: Fax: (562) 5416840; e-mail: [email protected]. 0378-4320/97/$17.00
0 1997 Elsevier Science B.V. All rights reserved.
PII SO378-4320(96)01630-2
114
V.H. Parraguez et al/Animal
Reproduction Science 47 (1997) 113-121
1. Introduction The alpaca and llama are two species of domestic South American camelids which, until 20 years ago, lived exclusively at high altitudes, over 3800 m above sea level (a.s.1) in the Andean high plateau. Owing to the fine qualities of their wool, beneficial characteristics of their meat and recent interest in them as pets, these species have spread around the world. In indigenous communities of Andean farmers, the domestic camelids are of transcendental importance, from both an economic and a social point of view, because these animals play a central role in farming activities. This situation has existed since the Inca empire. The limited development of the camelid livestock is a result of ancestral customs in animal management and production systems. The major limiting factors affecting the development of camelid livestock reflect disadvantages in their reproductive physiology, such as a long gestational period (11.5 months) (Novoa, 19911, the fact that females are uniparous (Bustinza et al., 1988) and only 35% of embryos survive the first month after mating (Femandez-Baca, 1993). There is agreement in the literature that this low fertility rate mainly results from the interaction of unsuitable reproductive management, nutritional deficiencies and high inbreeding (Huanca, 1990; Novoa, 1991; Raggi and MacNiven, 1993). Today, there is a strong foreign demand for South American camelids and the exportation of live animals is increasing. Thus, population size and the genetic quality of animals are in a critical situation. Success in animal production hinges on reproductive efficiency. Thus, it is important to develop methods to detect pregnancy at an early stage. The importance of ultrasound for improving reproductive efficiency in different domestic animals has been clearly established in studies on structural and functional gonads and reproductive tract characteristics (Bourke et al., 1992; Bravo et al., 19931, control of ovulation moment (Bravo et al., 19911, early pregnancy diagnosis (Buckrell et al., 1986; Garcia et al., 1993), evaluation of fetal growth and its relationship with gestational age (White et al., 1985; Kahn, 1989; Haibel and Fung, 1991) and detection of embryonic loss and fetal death or mummification (Bourke et al., 1992; Bretzlaff et al., 1993). The main goal of this study was to determine the earliest gestational age at which pregnancy can be determined by ultrasound in alpacas and llamas. Another important aim was to obtain an empirical relationship between gestational sac diameter (GSD) and gestational age (GA) during the first month of gestation. 2. Materials and methods Nineteen alpacas and 12 llamas, maintained under normal breeding conditions in the central zone of Chile (33”12’ and 33”28’S, 70”43’ and 70”38’W; altitude 730 m a.s.l.), were used. All animals included in the study had had at least one normal pregnancy and parturition, and were clinically healthy. Before beginning the protocol, normal uterine ultrasound characteristics were established. The protocol consisted of controlled matings with proved males. Males were introduced into the female pen and the presence of females in estrus was apparent by their
V.H. Parraguez et al./Animal Reproduction Science 47 (1997) 113-121
115
assumption of the typical prone position. Effective matings (day 0) were those in which penis introduction in the female genitalia occurred and the duration of intercourse was over 12 min. After the effective mating, ultrasound examination once a day was performed until appearance of the gestational sac. The examinations were done using a linear 5 MHz transrectal probe connected to an ultrasound apparatus (Aloka, model Echo Chamera SSO-210DXII). Photographs of the observed structures were obtained on a video printer (Sony, model UP8700MD) coupled to the ultrasound apparatus. Subsequently, the examination was repeated once a week until 34 days of gestation. Pregnancy was determined by observation of an anechogenic gestational sac on the uterine lumen. Dorsoventral gestational sac diameter was measured and its evolution followed during the first month of pregnancy. In addition, the appearance of the embryo and its heartbeat were monitored. Simultaneously with the ultrasound examination, jugular blood samples (3 ml) were obtained to measure plasma progesterone concentrations, to confirm pregnancy in the animals. The blood samples were centrifuged at 1200 X g for 10 min at 4°C. Plasma was separated and stored at -20°C until progesterone determination by heterologous radioimmunoassay (Coat-a-count, Diagnostic Products Corporation, Los Angeles, CA), previously validated for alpacas and llamas (Sumar et al., 1988; Leon et al., 1990). Gestational sac diameter measurements were analyzed by descriptive statistics. Curves of GSD vs. GA were also obtained by calculating a linear regression from the data. Results are shown as X k SEM.
3. Results 3.1. Alpacas Ultrasound examination of alpacas before pregnancy shows that the uterus may be observed immediately anterior to the bladder. This latter structure is easily recognized as an anechogenic sphere, appearing immediately after probe introduction into the rectum. The uterus appears as a spherical structure with a medium echographic density, viewed as a gray sphere containing several dark dots, with an external diameter of 3.10 + 0.30 cm. No lumen was observed in the uterus before pregnancy (Fig. 1). After mating, if pregnancy occurs, the uterus will be characterized by the appearance of a gestational sac with a center of very low echographic density and clear external limits, the size of which increases with gestational age (Fig. 2). The results show that pregnancy diagnosis may be done in some animals at 9 days of gestation, when the gestational sac is 0.6 cm in diameter, or 100% of animals at 23 days of gestation, when the gestational sac is 1.00 + 0.14 cm in diameter. Pregnancy diagnosis date and respective measurements are shown in Table 1. The results obtained in all animals from the day of gestational sac appearance until 30 days of gestation, were analyzed to obtain a representative linear regression. This regression allows the calculation of the gestational age from gestational sac diameter as follows GA = log GSD + 1.2339/0.05&j r = 0.85; P < 0.001 where GA is expressed
in days and GSD in centimeters.
116
V.H. Parraguez et al./Animal Reproduction Science 47 (1997) 113-121
Fig. 1. Echographic image of non-pregnant uterus (U); B, bladder. Scale 1:1.03.
uterus from an adult alpaca:
+, dorsal and \ rentral limits of the
Fig. 2. Echographic image of pregnant uterus (U) in an alpaca at 9 days gestation: gestational sac; B, bladder. Scale 1: 1.
X , dorsoventral diameter of
V.H. Parraguez et al./Animal Reproduction Science 47 (1997) 113-121
117
Table 1 Gestational
age (GA) and gestational
sac diameter (GSD) in ultrasound
pregnancy
diagnosis
in alpacas
GA (days)
GSD km) n
9
10
11
12
13
15
16
17
23
0.60 1
OSOf0.04 5
0.60 1
0.40 1
0.60 1
0.50 1
0.70+0.05 3
0.58 +0.03 4
1.00+0.14 2
At 30 days of gestation, all animals showed an embryo, typically observed as a small spot of high echographic density located on the basal zone of the gestational sac. After day 25 of gestation, some embryos showed a clear visible heartbeat; 100% of embryos showed a heartbeat from day 30. 3.2.
Llamas
The observed echographic characteristics of the empty uterus in the llama are similar to those of the alpaca, both in position relative to the bladder and in echographic density. However, the uterine diameter is slightly larger than in the alpaca (3.43 + 0.78 vs. 3.10 + 0.30 cm for llama and alpacas, respectively) with a larger variation range as demonstrated by the SEM. A gestational sac was observed as early as 7 days after mating in one llama, with a diameter of 0.30 cm (Fig. 3). A gestational sac was observed in 100% of animals at day
Fig. 3. Echographic image of pregnant uterus (U) in a llama at 7 days gestation: gestational sac; B, bladder. Scale 1:O.g.
x , dorsoventral
diameter of
V.H. Parraguez et al./Animal Reproduction Science 47 (1997) 113-121
118 Table 2 Gestational
age (GA) and gestational
sac diameter (GSD) in ultrasound
pregnancy
diagnosis
in llamas
GA (days)
GSD (cm) n
I
10
13
14
21
34
0.3 1
0.54 * 0.03 5
0.4 1
0.4 1
0.87*0.03 3
2.2 1
34, when it was 2.2 cm in diameter. The subsequent date of pregnancy diagnosis and respective measurements are shown in Table 2. As in the case of alpacas, a linear regression was calculated from the measurements of gestational sac diameter in llamas. The following equation allows the calculation of gestational age from gestational sac diameter GA = 1ogGSD + 1.2649/0.0546
I = 0.77, P < 0.001
An embryo with visible heartbeat was seen at 31 days of gestation, and was located in a similar position to that in alpacas (Fig. 4). Plasma progesterone concentration, in alpacas and llamas, was always higher than 5 nmol 1-l) a concentration considered indicative of pregnancy in those animals (Leon et al., 1990; Sumar, 1991).
Fig. 4. Echographic image of pregnant ute~s (U) in a llama at 31 days gestation: + , dorsoventral embryo. A dark spot in the left part of the embryo shows the heart (arrow). Scale 1:1.2.
limits of the
V.H. Parraguez et al./Animal Reproduction Science 47 (1997) 113-121
119
4. Discussion Transrectal ultrasonography is an invaluable tool for early pregnancy diagnosis in alpacas and llamas. We found that this technique is easily applicable without significant risk for domestic South American camelids. In fact, no distress, no rectal bleeding or interruption of pregnancies were seen after ultrasonography examinations, in contrast with transrectal ultrasonography in ewes (Schrick and Inskeep, 1993). In addition, all the animals became accustomed to this procedure after two or three sessions, facilitating management by the operator. Results show that pregnancy diagnosis in alpacas could be carried out from 9 days after mating, with 100% accuracy at day 23. In the case of llamas, the test could be carried out from 7 days after mating, with 100% accuracy at 34 days. It is noteworthy that these results are the most precocious in South American camelids or in other domestic ruminants. In llamas, Bourke et al. (1992) detected pregnancy from day 19 after mating (range 19-28 days). Other authors (Mialot and Villemain, 1994), using alpacas and llamas, have detected early pregnancy between 20 and 100 days after mating. In the case of ewes, pregnancy was detected from day 15 after mating, with 100% accuracy at day 40 (Schtick and Inskeep, 1993) or from day 17 after mating, with 95% accuracy on day 32 (Garcia et al., 1993). In cows, White et al. (1985), detected pregnancy from day 30, with 98% accuracy. Other pregnancy diagnosis techniques have been used in domestic South American camelids. Using the Doppler technique, pregnancy can be detected from day 80 after mating, with 92% accuracy in alpacas, and at day 75 with 92% accuracy in llamas (Alarcon et al., 1990). Using the estrous behavior method, the same researchers diagnosed pregnancy from day 125 in both species, with 88% accuracy in alpacas and 95% in llamas. Using rectal palpation, they detected pregnancy from day 165 in both species with 100% accuracy. However, Bravo and Varela (1993) using this method in alpacas, detected pregnancy from day 60, but they did not report the percentage accuracy. In llamas, using plasma progesterone measurements, pregnancy can be detected 5 days after mating (Leon et al., 1990) and confirmed by a second plasma progesterone measurement at 1 l- 12 days after mating. The second measurement is necessary because an important proportion of females can develop a corpus luteum which is functional until days 9-10 after mating in the absence of pregnancy (Sumar, 1988). Preliminary results from our laboratory in llamas and alpacas are consistent with those of Leon et al. (1990). However, the technique requires at least one blood sample (which is invasive), a plasma progesterone measurement, which is relatively time consuming (at least 24 h), and the method is expensive compared with ultrasound. During evaluation of the initial stages of pregnancy, the observation of the embryo and its heartbeat in the gestational sac is important. In this study the presence of an embryo heartbeat was detected as early as 25 and 31 days of gestation in alpacas and llamas, respectively. This is the first report of this anatomical and functional event in domestic South American camelids. Only the detection of an embryo image, between days 21 and 41 of gestation, has been reported in llamas (Bourke et al., 1992). In cows, the embryo image was visible between 26 and 29 days of gestation (White et al., 1985). Studies in ewes have reported the detection of an embryo heartbeat from 18 (Schrick
120
V.H. Parraguez et al. /Animal Reproduction Science 47 (1997) 113-121
and Inskeep, 1993), 21 (Garcia et al., 1993) and 30 days of gestation (Buckrell et al., 1986). Therefore, it appears that the early developmental processes are temporally similar in these ruminant species, independent of the length of gestation. Our data allow the calculation of linear regressions and their corresponding equations, making it possible to calculate the gestational age from ultrasonographic measurements of the gestational sac during the first month of pregnancy. Although the correlation coefficients obtained (0.85 and 0.77 for alpacas and llamas, respectively) may not allow a precise estimation of gestational age, an error of about 1520% in the estimated GA during the first month of pregnancy is probably not significant considering the total length of gestation (11.5 months). Early detection of pregnancy in South American camelids is important, especially because of the high percentage of embryo death during the first month of gestation. Early pregnancy diagnosis would be an aid to develop special care for pregnant females, including nutritional aspects and minimizing stress. The results presented here indicate that ultrasonography is the most adequate technique for early pregnancy diagnosis in domestic camelids. In spite of generally unsuitable breeding conditions, typical of the Andean high plateau, ultrasonographic diagnosis can be done with satisfactory results, using portable equipment. In addition, ultrasonography can be applied to other successful reproductive management practices such as artificial insemination, embryo transfer and controlled matings. The use of ultrasonography techniques will help to improve the reproductive efficiency of camelid livestock.
Acknowledgements We thank Andri5s Salinas for his skillful technical assistance and Dr. Illani Atwater (NIH, Bethesda, MD) for revision of the manuscript. This study was supported by grant FONDECYT 1940292 and Universidad de Chile-Cia. Minera Disputada de Las Condes-Criadero El Litral.
References Alarc6n, V., Sumar, J., Riera, G.S. and Foote, W.C., 1990. Comparison of three methods of pregnancy diagnosis in alpacas and llamas. Theriogenology, 34: 1119-l 127. Bourke, D.A., Adam, CL. and Kyle, C.E., 1992. Ultrasonography as an aid to controlled breeding in the llama. Vet. Rec., 130: 424-428. Bravo, P.W. and Varela, M.H., 1993. Prenatal development of the alpaca (Lama paces). Anim. Reprod. Sci., 32: 245-252. Bravo, P.W., Stabenfeldt, G.H., Lasley, B.L. and Fowler, M.E., 1991. The effect of ovarian follicle size on pituitary and ovarian responses to copulation in domesticate south american camelids. Biol. Reprod., 45: 553-559. Bravo, P.W., Stabenfeld, G.H., Flower, M.E. and Lasley, B.L., 1993. Ovarian and endocrine patterns associated with reproductive abnormalities in llamas and alpacas. J. Am. Vet. Med. Assoc., 202: 268-272. Bretzlaff, K., Edwards, J., Forrest, D. and Nuti, L., 1993. Ultrasonographic determination of pregnancy in small ruminants. Vet. Rec., 88: 12-24. Buckrell, B.C., Bonnett, B.N. and Johnson, W.H., 1986. The use of real-time ultrasound rectally for early pregnancy diagnosis in sheep. Theriogenology, 25: 665-673.
V.H. Parraguez et al/Animal Bustinza, A.V., Burfening, P.J. and Blackwell, paces). J. Anim. Sci., 66: 1139-l 143.
Reproduction Science 47 (1997) 113-121 R.L., 1988. Factors affecting
121
survival in young alpacas (Lama
Femandez-Baca, S., 1993. Manipulation of reproductive functions in male and female New World camelids. Anim. Reprod. Sci., 33: 307-323. Garcia, A., Neary, M.K., Kelly, G.R. and Pierson, R.A., 1993. Accuracy of ultrasonography in early pregnancy diagnosis in ewe. Theriogenology, 39: 847-861, Ha&cl, G.K. and Fung, E.D., 1991. Real time ultrasonic biparietal diameter measurement for the prediction of gestational age in llamas. Theriogenology, 3 1: 847-86 1. Huanca, T., 1990. Manual de1 Alpaquero. Segunda Edici6n. Proyecto ALPACAS-INNIA-CORPUNOCORTESU/IC. Puno, Peru, 233 pp. Kahn, W., 1989. Sonographic fetometry in the bovine. Theriogenology, 31: 1105-1121. Lebn, J.B., Smith, B.B., Timm, K.I. and le Cren, G., 1990. Endocrine changes during pregnancy, parturition and the early post-partum period in the llama (Lama glama!. J. Reprod. Fertil., 88: 503-511. Mialot, J.P. and Villemain, L., 1994. Diagnostic de gestation par ecographie chez le lama et l’alpaca. Rec. Med. Vet., 170: 23-27. Novoa, C., 1991. Fisiologia de la reproduccicin de la hembra. In: S. Femandez-Baca (Editor), Avarices y Perspectivas de1 Conocimiento de 10s C.S.D.A. FAO, Olicina Regional para America latina y el Caribe. Santiago, Chile, pp. 91-109. Raggi, L.A. and MacNiven, V., 1993. Antecedentes fisiologicos y productivos de 10s camelidos sudamericanos domesticos. Document0 de Extensibn. Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, 45 pp. Schrick, F.N. and Inskeep, E.K., 1993. Determination of early pregnancy in ewes utilizing transrectal ultrasonography. Theriogenology. 40: 295-306. Sumar, J., 1988. Removal of the ovaries or ablation of the corpus luteum and its effect on the maintenance of gestation in the alpaca and llama. Acta Vet. Stand., 83: 133-141. Sumar, J., 1991. Contribution of the radioimmunoassay technique to knowledge of the reproductive physiology of South American camelids. In: Proc. of a Symp. on Isotope and Related Techniques in Animal Production and Health. International Atomic Energy Agency, Vienna, Austria, pp. 353-370. Sumar, J., Fredriksson, G., Alarcon, V., Kindahl, H. and Edqvist, L.E., 1988. Levels of 15-keto-13,14-dihydro-PGF2o, progesterone, and oestradiol-17B, after induced ovulations in llamas and alpacas. Acta Vet. Stand., 29: 339-346. White, I.R., Russel, A.J.F., Wright, LA. and Whyte, T.K., 1985. Real-time ultrasonic scanning in the diagnosis of pregnancy and estimation of gestational age in cattle. Vet. Rec., 117: 5-8.
ELSEVIER
Animal Reproduction Science 47 (1997) 113-121
Early pregnancy diagnosis in alpaca (Lama paces) and llama hwza gZama) by ultrasound Victor H. Parraguez *, Sandra Cortkz, Francisca J. GazitGa, Germh Ferrando, Verbnica MacNiven, Luis A. Raggi Facultad de Ciencias Veterinarias y Pecuarias, Unioersidad de Chile, Casilla 2, Correo 15, La Granja,
Santiago, Chile
Accepted 14 October 1996
Abstract An ultrasonography study of early pregnancy diagnosis was carried out in 19 alpacas and 12 llamas, after controlled matings. The aim was to determine the earliest gestational age at which pregnancy diagnosis by transrectal ultrasonography could be achieved, and to generate an empirical formula for gestational sac diameter (GSD) growth as a function of gestational age (GA), allowing an estimate of GA during the first month of pregnancy. We found that pregnancy diagnosis may be carried out as early as 9 days after mating in alpacas and 7 days in llamas. This diagnosis was found to be accurate at 23 days in alpacas and 34 days in llamas. The empirical relations that best describe the relationship between GSD and GA were GA = 1ogGSD + 1.2339/0.0585
r = 0.85,
P < 0.001
in alpacas, and GA = log GSD + 1.2649/0.0546
r = 0.77, P < 0.001
in llamas, where GA is measured in days and GSD in centimeters. Our results also indicate that ultrasonography is a reliable technique for early pregnancy diagnosis. Furthermore, the empirical formulae reliably make it possible to estimate GA from GSD during the first month of pregnancy and their use might improve the efficiency of camelid breeders. 0 1997 Elsevier Science B.V. Keywords: Pregnancy; Alpaca; Llama; Ultrasound
* Corresponding author. Tel.: (562) 6785548: Fax: (562) 5416840; e-mail: [email protected]. 0378-4320/97/$17.00
0 1997 Elsevier Science B.V. All rights reserved.
PII SO378-4320(96)01630-2
114
V.H. Parraguez et al/Animal
Reproduction Science 47 (1997) 113-121
1. Introduction The alpaca and llama are two species of domestic South American camelids which, until 20 years ago, lived exclusively at high altitudes, over 3800 m above sea level (a.s.1) in the Andean high plateau. Owing to the fine qualities of their wool, beneficial characteristics of their meat and recent interest in them as pets, these species have spread around the world. In indigenous communities of Andean farmers, the domestic camelids are of transcendental importance, from both an economic and a social point of view, because these animals play a central role in farming activities. This situation has existed since the Inca empire. The limited development of the camelid livestock is a result of ancestral customs in animal management and production systems. The major limiting factors affecting the development of camelid livestock reflect disadvantages in their reproductive physiology, such as a long gestational period (11.5 months) (Novoa, 19911, the fact that females are uniparous (Bustinza et al., 1988) and only 35% of embryos survive the first month after mating (Femandez-Baca, 1993). There is agreement in the literature that this low fertility rate mainly results from the interaction of unsuitable reproductive management, nutritional deficiencies and high inbreeding (Huanca, 1990; Novoa, 1991; Raggi and MacNiven, 1993). Today, there is a strong foreign demand for South American camelids and the exportation of live animals is increasing. Thus, population size and the genetic quality of animals are in a critical situation. Success in animal production hinges on reproductive efficiency. Thus, it is important to develop methods to detect pregnancy at an early stage. The importance of ultrasound for improving reproductive efficiency in different domestic animals has been clearly established in studies on structural and functional gonads and reproductive tract characteristics (Bourke et al., 1992; Bravo et al., 19931, control of ovulation moment (Bravo et al., 19911, early pregnancy diagnosis (Buckrell et al., 1986; Garcia et al., 1993), evaluation of fetal growth and its relationship with gestational age (White et al., 1985; Kahn, 1989; Haibel and Fung, 1991) and detection of embryonic loss and fetal death or mummification (Bourke et al., 1992; Bretzlaff et al., 1993). The main goal of this study was to determine the earliest gestational age at which pregnancy can be determined by ultrasound in alpacas and llamas. Another important aim was to obtain an empirical relationship between gestational sac diameter (GSD) and gestational age (GA) during the first month of gestation. 2. Materials and methods Nineteen alpacas and 12 llamas, maintained under normal breeding conditions in the central zone of Chile (33”12’ and 33”28’S, 70”43’ and 70”38’W; altitude 730 m a.s.l.), were used. All animals included in the study had had at least one normal pregnancy and parturition, and were clinically healthy. Before beginning the protocol, normal uterine ultrasound characteristics were established. The protocol consisted of controlled matings with proved males. Males were introduced into the female pen and the presence of females in estrus was apparent by their
V.H. Parraguez et al./Animal Reproduction Science 47 (1997) 113-121
115
assumption of the typical prone position. Effective matings (day 0) were those in which penis introduction in the female genitalia occurred and the duration of intercourse was over 12 min. After the effective mating, ultrasound examination once a day was performed until appearance of the gestational sac. The examinations were done using a linear 5 MHz transrectal probe connected to an ultrasound apparatus (Aloka, model Echo Chamera SSO-210DXII). Photographs of the observed structures were obtained on a video printer (Sony, model UP8700MD) coupled to the ultrasound apparatus. Subsequently, the examination was repeated once a week until 34 days of gestation. Pregnancy was determined by observation of an anechogenic gestational sac on the uterine lumen. Dorsoventral gestational sac diameter was measured and its evolution followed during the first month of pregnancy. In addition, the appearance of the embryo and its heartbeat were monitored. Simultaneously with the ultrasound examination, jugular blood samples (3 ml) were obtained to measure plasma progesterone concentrations, to confirm pregnancy in the animals. The blood samples were centrifuged at 1200 X g for 10 min at 4°C. Plasma was separated and stored at -20°C until progesterone determination by heterologous radioimmunoassay (Coat-a-count, Diagnostic Products Corporation, Los Angeles, CA), previously validated for alpacas and llamas (Sumar et al., 1988; Leon et al., 1990). Gestational sac diameter measurements were analyzed by descriptive statistics. Curves of GSD vs. GA were also obtained by calculating a linear regression from the data. Results are shown as X k SEM.
3. Results 3.1. Alpacas Ultrasound examination of alpacas before pregnancy shows that the uterus may be observed immediately anterior to the bladder. This latter structure is easily recognized as an anechogenic sphere, appearing immediately after probe introduction into the rectum. The uterus appears as a spherical structure with a medium echographic density, viewed as a gray sphere containing several dark dots, with an external diameter of 3.10 + 0.30 cm. No lumen was observed in the uterus before pregnancy (Fig. 1). After mating, if pregnancy occurs, the uterus will be characterized by the appearance of a gestational sac with a center of very low echographic density and clear external limits, the size of which increases with gestational age (Fig. 2). The results show that pregnancy diagnosis may be done in some animals at 9 days of gestation, when the gestational sac is 0.6 cm in diameter, or 100% of animals at 23 days of gestation, when the gestational sac is 1.00 + 0.14 cm in diameter. Pregnancy diagnosis date and respective measurements are shown in Table 1. The results obtained in all animals from the day of gestational sac appearance until 30 days of gestation, were analyzed to obtain a representative linear regression. This regression allows the calculation of the gestational age from gestational sac diameter as follows GA = log GSD + 1.2339/0.05&j r = 0.85; P < 0.001 where GA is expressed
in days and GSD in centimeters.
116
V.H. Parraguez et al./Animal Reproduction Science 47 (1997) 113-121
Fig. 1. Echographic image of non-pregnant uterus (U); B, bladder. Scale 1:1.03.
uterus from an adult alpaca:
+, dorsal and \ rentral limits of the
Fig. 2. Echographic image of pregnant uterus (U) in an alpaca at 9 days gestation: gestational sac; B, bladder. Scale 1: 1.
X , dorsoventral diameter of
V.H. Parraguez et al./Animal Reproduction Science 47 (1997) 113-121
117
Table 1 Gestational
age (GA) and gestational
sac diameter (GSD) in ultrasound
pregnancy
diagnosis
in alpacas
GA (days)
GSD km) n
9
10
11
12
13
15
16
17
23
0.60 1
OSOf0.04 5
0.60 1
0.40 1
0.60 1
0.50 1
0.70+0.05 3
0.58 +0.03 4
1.00+0.14 2
At 30 days of gestation, all animals showed an embryo, typically observed as a small spot of high echographic density located on the basal zone of the gestational sac. After day 25 of gestation, some embryos showed a clear visible heartbeat; 100% of embryos showed a heartbeat from day 30. 3.2.
Llamas
The observed echographic characteristics of the empty uterus in the llama are similar to those of the alpaca, both in position relative to the bladder and in echographic density. However, the uterine diameter is slightly larger than in the alpaca (3.43 + 0.78 vs. 3.10 + 0.30 cm for llama and alpacas, respectively) with a larger variation range as demonstrated by the SEM. A gestational sac was observed as early as 7 days after mating in one llama, with a diameter of 0.30 cm (Fig. 3). A gestational sac was observed in 100% of animals at day
Fig. 3. Echographic image of pregnant uterus (U) in a llama at 7 days gestation: gestational sac; B, bladder. Scale 1:O.g.
x , dorsoventral
diameter of
V.H. Parraguez et al./Animal Reproduction Science 47 (1997) 113-121
118 Table 2 Gestational
age (GA) and gestational
sac diameter (GSD) in ultrasound
pregnancy
diagnosis
in llamas
GA (days)
GSD (cm) n
I
10
13
14
21
34
0.3 1
0.54 * 0.03 5
0.4 1
0.4 1
0.87*0.03 3
2.2 1
34, when it was 2.2 cm in diameter. The subsequent date of pregnancy diagnosis and respective measurements are shown in Table 2. As in the case of alpacas, a linear regression was calculated from the measurements of gestational sac diameter in llamas. The following equation allows the calculation of gestational age from gestational sac diameter GA = 1ogGSD + 1.2649/0.0546
I = 0.77, P < 0.001
An embryo with visible heartbeat was seen at 31 days of gestation, and was located in a similar position to that in alpacas (Fig. 4). Plasma progesterone concentration, in alpacas and llamas, was always higher than 5 nmol 1-l) a concentration considered indicative of pregnancy in those animals (Leon et al., 1990; Sumar, 1991).
Fig. 4. Echographic image of pregnant ute~s (U) in a llama at 31 days gestation: + , dorsoventral embryo. A dark spot in the left part of the embryo shows the heart (arrow). Scale 1:1.2.
limits of the
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4. Discussion Transrectal ultrasonography is an invaluable tool for early pregnancy diagnosis in alpacas and llamas. We found that this technique is easily applicable without significant risk for domestic South American camelids. In fact, no distress, no rectal bleeding or interruption of pregnancies were seen after ultrasonography examinations, in contrast with transrectal ultrasonography in ewes (Schrick and Inskeep, 1993). In addition, all the animals became accustomed to this procedure after two or three sessions, facilitating management by the operator. Results show that pregnancy diagnosis in alpacas could be carried out from 9 days after mating, with 100% accuracy at day 23. In the case of llamas, the test could be carried out from 7 days after mating, with 100% accuracy at 34 days. It is noteworthy that these results are the most precocious in South American camelids or in other domestic ruminants. In llamas, Bourke et al. (1992) detected pregnancy from day 19 after mating (range 19-28 days). Other authors (Mialot and Villemain, 1994), using alpacas and llamas, have detected early pregnancy between 20 and 100 days after mating. In the case of ewes, pregnancy was detected from day 15 after mating, with 100% accuracy at day 40 (Schtick and Inskeep, 1993) or from day 17 after mating, with 95% accuracy on day 32 (Garcia et al., 1993). In cows, White et al. (1985), detected pregnancy from day 30, with 98% accuracy. Other pregnancy diagnosis techniques have been used in domestic South American camelids. Using the Doppler technique, pregnancy can be detected from day 80 after mating, with 92% accuracy in alpacas, and at day 75 with 92% accuracy in llamas (Alarcon et al., 1990). Using the estrous behavior method, the same researchers diagnosed pregnancy from day 125 in both species, with 88% accuracy in alpacas and 95% in llamas. Using rectal palpation, they detected pregnancy from day 165 in both species with 100% accuracy. However, Bravo and Varela (1993) using this method in alpacas, detected pregnancy from day 60, but they did not report the percentage accuracy. In llamas, using plasma progesterone measurements, pregnancy can be detected 5 days after mating (Leon et al., 1990) and confirmed by a second plasma progesterone measurement at 1 l- 12 days after mating. The second measurement is necessary because an important proportion of females can develop a corpus luteum which is functional until days 9-10 after mating in the absence of pregnancy (Sumar, 1988). Preliminary results from our laboratory in llamas and alpacas are consistent with those of Leon et al. (1990). However, the technique requires at least one blood sample (which is invasive), a plasma progesterone measurement, which is relatively time consuming (at least 24 h), and the method is expensive compared with ultrasound. During evaluation of the initial stages of pregnancy, the observation of the embryo and its heartbeat in the gestational sac is important. In this study the presence of an embryo heartbeat was detected as early as 25 and 31 days of gestation in alpacas and llamas, respectively. This is the first report of this anatomical and functional event in domestic South American camelids. Only the detection of an embryo image, between days 21 and 41 of gestation, has been reported in llamas (Bourke et al., 1992). In cows, the embryo image was visible between 26 and 29 days of gestation (White et al., 1985). Studies in ewes have reported the detection of an embryo heartbeat from 18 (Schrick
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and Inskeep, 1993), 21 (Garcia et al., 1993) and 30 days of gestation (Buckrell et al., 1986). Therefore, it appears that the early developmental processes are temporally similar in these ruminant species, independent of the length of gestation. Our data allow the calculation of linear regressions and their corresponding equations, making it possible to calculate the gestational age from ultrasonographic measurements of the gestational sac during the first month of pregnancy. Although the correlation coefficients obtained (0.85 and 0.77 for alpacas and llamas, respectively) may not allow a precise estimation of gestational age, an error of about 1520% in the estimated GA during the first month of pregnancy is probably not significant considering the total length of gestation (11.5 months). Early detection of pregnancy in South American camelids is important, especially because of the high percentage of embryo death during the first month of gestation. Early pregnancy diagnosis would be an aid to develop special care for pregnant females, including nutritional aspects and minimizing stress. The results presented here indicate that ultrasonography is the most adequate technique for early pregnancy diagnosis in domestic camelids. In spite of generally unsuitable breeding conditions, typical of the Andean high plateau, ultrasonographic diagnosis can be done with satisfactory results, using portable equipment. In addition, ultrasonography can be applied to other successful reproductive management practices such as artificial insemination, embryo transfer and controlled matings. The use of ultrasonography techniques will help to improve the reproductive efficiency of camelid livestock.
Acknowledgements We thank Andri5s Salinas for his skillful technical assistance and Dr. Illani Atwater (NIH, Bethesda, MD) for revision of the manuscript. This study was supported by grant FONDECYT 1940292 and Universidad de Chile-Cia. Minera Disputada de Las Condes-Criadero El Litral.
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