1 OBSERVATION OF FETAL DEVELOPMENT IN ALPACAS AT SPECIFIC GESTATIONAL AGES USING ULTRASOUND, FETAL DISSECTION, AND COMPUTER AIDED TOMOGRAPHY TECHNIQUES A Capstone Experience Manuscript Presented by Lauren Melle Completion Date: May 2011 Approved By: Dr. Stephen Purdy, Department of Veterinary and Animal Sciences Dr. Alan Richmond, Department of Biology 2 ABSTRACT Title: Observation of Fetal Development in Alpacas at Specific Gestational Ages using Ultrasound, Fetal Dissection, and Computer Aided Tomography Techniques Author: Lauren Melle, Animal Science CE Type: Independent Capstone Thesis Approved By: Dr. Stephen Purdy, Department of Veterinary and Animal Science Approved By: Dr. Alan Richmond, Department of Biology The alpaca, Vicugna pacos is a valued species in South America and of growing popularity in the United States. Relatively little is understood about the reproduction of this species. This study attempts to observe the specific developments of the fetal alpaca throughout early gestation. The identification of these developmental events using three methods: ultrasonography, actual fetal dissections, and computer-aided tomography scans, will aid in identifying trends in development. Six fetal specimens were followed using ultrasound, which is the common field method, up until different gestational ages (154 days, 126, 103, 102, 74, and 42 day twins). Fetuses were then terminated at specific gestational ages in order to observe the development that had occurred up until time of termination and compare findings with that observed during gestation using ultrasound. CAT scan images of the specimens were then used to compare to ultrasound and dissection data. Comparisons among the three methods resulted in the determination of normal ranges of development at specific gestational ages and the accuracy of the ultrasound field method as a accurate method in following the developments of alpaca fetal development. Lastly, this study presents previously unknown findings on the developments of the fetal alpaca. 3 1. Introduction The alpaca, Vicugna pacos, is a domesticated species of South American camelid that is extensively used as a source of livelihood. While the importance of the alpaca for meat, hides, and fiber in South America dates back thousands of years, the popularity of the alpaca has only recently grown in the United States. In the United States alpacas are primarily raised as investment animals and bred for superior genetics relating to fiber and show conformation. While the popularity of the alpaca has significantly increased and continues to grow, relatively little is understood about reproduction in this species. In order to continue the growth and improvement of the alpaca industry it is necessary to explore the reproductive cycle and fetal development of this species. Alpacas are induced ovulators, which means that copulation and penetration of the cervix must occur for ovulation and subsequent fertilization in order to obtain a pregnancy. Due to the fact that alpacas are induced ovulators, they do not show a defined estrous cycle, which can result in varying lengths of receptivity and non-receptivity. The uterine horn is similar to that of Bovine with long horns and a short uterine body. The uterine horns and ovaries are unfixed due to lack of ligamentous structures. The placentation is epithelochorial and fills both horns (Aiello, 1998). The detection of ovulation can be confirmed through transrectal ultrasound around 3-4 days1 after breeding when a corpus luteum is visible and the female shows signs of sexual non- receptivity. Pregnancy can be determined at approximately 15 days using transrectal ultrasound through the identification of a pre-implantation conceptus. According to a study in 2003, at day 15 after copulation, the blastocyst is completely free in the uterine lumen and can transverse the 4 two uterine horns until implantation occurs at approximately day 20 and placentation is observed on ultrasound at day 45 (Olivera, 2003). While the early part of gestation has been studied to an extent, little is know about the development of the fetus as it relates to growth and structural development after 40 days. As mentioned in the previous paragraph, there is a definitive field method for confirming pregnancy; however, there is no field method for determining gestational age or to determine if proper growth of the fetus is occurring. Determining gestational age is a tool that would be useful during abortions, when the breeding date is unknown, or, as in the case with many South American alpaca producers, males and females are kept in the same pasture, and occasionally without a breeding management program (Herrera, 2002). In addition, knowledge of gestational age and proper fetal development is useful for studies using alpacas as a animal model. These studies often require normal developing fetuses at a particular age in gestation (Gazitua, 2001). In previous research gestational age has been determined in the Equine and in the llama using the measurements of the limbs, skull, and abdomen on aborted fetal specimens (Herrera, 2002). Other studies used ultrasound examinations in order to obtain measurements (Grazitua, 2001). However, no study has attempted to correlate both ultrasound data and aborted fetal measurements in order to show progression of fetal development and growth at specific gestational ages. This study will attempt to show normal alpaca fetal development as defined by the studied specimens. It will also attempt to determine trends of development and growth of the fetal alpaca at several different time points throughout gestation from day 42 through day 154 using trans-rectal ultrasound during gestation, and then fetal measurements post-termination. In addition, this study seeks to show the accuracy of ultrasound examinations, the current field 5 method, as it relates to viewing specific fetal structures that represent the progression of fetal development. This will be accomplished through the comparison of ultrasound images to CAT scan images, and actual fetal dissections and measurements. 1Unpublished findings from the University of Massachusetts Amherst 2. Methods and Materials This study followed five female alpacas from the time of breeding through the time of termination of the pregnancy. Animals were all healthy and had at least one normal pregnancy prior to the study. Females were followed for two semesters where some had multiple pregnancies. The animals were kept in a group pen and turned out on pasture during daylight. Sexual receptivity was tested every other day in order to determine if the females were receptive. All females were tested even if a pregnancy was already established. This was to reaffirm the continuing presence of the corpus luteum (CL). Males were haltered and brought to the female alpaca pen where they were allowed to approach the females for breeding. If the female alpaca assumed the sternal recumbent position, also known as cushing, she was considered to be receptive. The degree of receptivity was also noted. In certain cases females would cush immediately after being mounted by the male, but in other instances, the male would mount the female for an extended time before the female cushed. The male was ultimately removed from the female if cushing did not occur after one minute. Females that did not cush immediately were considered to be “less” receptive. Every female that cushed during the sexual 6 receptivity testing was examined by transrectal ultrasound using a 5 MHz linear probe. The left and right ovaries were examined for the presence of follicles. Follicles that exceeded 5mm in diameter were considered to be of ovulatory size. Dominance follicles that suppressed the growth of neighboring follicles were not seen. Several observations revealed ovaries with multiple large follicles. Females that displayed receptive behavior and had a follicle that exceeded 5 mm were bred. Breeding lasted 15-20 minutes and a semen sample was collected post-copulation and evaluated for a separate study. Post-breeding females were examined using transrectal ultrasound every other day throughout the study. Ovulation for bred females was confirmed when the presence of the CL was documented and the female no longer displayed sexual receptivity. Contractility of the uterine horns was also documented and graded on a scale of 1-3 where 3 is highest amount of contractility. The presence of the embryonic vesicle was recorded, as well as, the embryonic vesicle as it moved between the uterine horns prior to implantation. The embryonic vesicle was compressed by the uterine horn contracting. This phenomenon was documented on film. The embryo proper was measured in length as seen in Figure 14, in order to show the progression of fetal growth. When cephalization was visible via ultrasound, the fetus was measured from the crown of the head to the rump as seen in Figure 15. The skull diameter was measured in two ways during the ultrasound examination. The biparietal skull diameter (BPD) measured the distance between the parietal bones of the skull as illustrated in Figure 13. The incisor to occipital diameter (IOD) measured from the distal part of the incisor to the occipital as illustrated in Figure 14. The diameter of the orbit (OD) was measured as shown in Figure 22 and ossification was observed (Figure 21). This structure has 7 been studied in Equine in order to determine gestational age using transrectal ultrasonographic measurements and was found to be a method of estimating gestational age, (Turner, 2006). The neck length (NL) was determined as Figure 23 indicates and ossification of the cervical vertebrae (Figure 27). Abdominal length (Abl), height (Abh), and width (Adw) were not measured during the ultrasound examinations due to the positioning of the fetus in utero, however, these structures were measured on the actual fetuses (Figure 37). Total limb length (TLL) was measured as is illustrated in Figure 28; however, individual bones were not observable. The stomach diameter (SD) was measured as shown in Figure 31. Each alpaca was examined every other day during gestation and the above structural measurements recorded when they were visible.