Ultrasound Monitoring of Embryonic, Follicular, and Uterine Dynamics of Early Pregnancy in the Alpaca Sara Brunsden Introduction: The alpaca, Vicuna pacos, is a member of the Camelidae family, along with llamas, guanacos, vicunas, and Bactrian and Dromedary camels. Traditionally found in the altiplano of South America, the popularity of the alpaca has caused it to spread all over the world, including here in the United States. In South America, they are predominantly used for their fleece, while the industry here revolves mainly around breeding. However, relatively little is known about the reproduction of the alpaca. It is the overall goal of this study to discover more about the gestation of the female, specifically the embryonic stage from conception to forty days of pregnancy. Like the rabbit and cat, the alpaca is an induced ovulator, meaning that the act of copulation triggers the female to ovulate. Differing information has been presented on whether alpacas have waves of follicular development similar to other mammalian species. According to studies by Bravo (1991) and Sumar (2000), the follicles grow, mature, and regress in a distinct pattern. However, a study by Donovan (2011) at the University of Massachusetts Amherst did not find a pattern of definitive follicular waves. Alpacas are considered to have a low fertility rate compared to other domesticated mammals, with the highest rate of early embryonic death (EED) occurring within the first month of pregnancy, possibly due to weak maternal fetal tissue associations (Olivera 2003). The rate of EED has been suggested to be as high as 58% (Fernandez-Baca 1970), with 44% occurring before Day 27 (Ratto 2011). Ratto (2011) also suggests that lactating 1 females have a decreased occurrence of early embryonic death as compared to females that did not have a cria the year before. The rate of early embryonic death is investigated in the study animals to compare to these suggested rates. One aspect of alpaca reproduction that is widely agreed on is that nearly all pregnancies implant in the left uterine horn. Ratto (2011) found that the rate for left uterine horn implantation is 98%, while Brown (1999) states that “few embryos that are produced and implant in the right side survive beyond 30 days gestation and none survive after 87 days.” Olivera (2003) believes that this phenomenon is due to “specific surface molecule eXpression on the uterine epithelium allowing embryo apposition and adhesion only in restricted areas.” This study also investigates which uterine horn the embryonic vesicle first appears in the study animals. Another feature of the alpaca gestation that has consistently been found to be true is that the corpus luteum (CL) is of utmost importance in maintaining the pregnancy (Olivera 2003). It was found by both Fernandez-Baca (1970) and Brown (1999) that the CL reaches its maXimum size by Day 8-10 of pregnancy. Olivera (2003) also found that the corpus luteum is more prevalent on the left ovary, suggesting that more ovulations, and therefore more large follicles, occur on the left ovary. The development of the corpus luteum is followed throughout the pregnancies in this study. The gestation of the horse has been well studied and a great deal of information is known concerning early events in pregnancy. Because of the similar gestation length of 345 days and the similar epitheliochorial placenta of the alpaca and the horse, it would stand to reason that many of the events that occur during pregnancy would also be similar. According to Allen (2001), the equine embryo should be seen in the uterus between Day 12 2 and 14 and remains spherical in shape until implantation. The uterine horns will be closely monitored during this study to see when the alpaca embryo appears and what shape it takes. Methods: Five females were studied over the course of a year, from January 2011to December 2011. The females ranged in age from 13 years to 4 years and were all proven breeders with at least one previous cria (Figure 1). Animal Age Dates Observed Number of Crias Time Since Last Cria A 13 September 12-December 16 8 2 months B 5 June 20-September 9 1 12 months C 4 January 28-November 16 1 4 months D 6 August 1-September 9 2 11 months E 8 January 31-September 30 2 18 months Figure 1: Study Females. The females were studied three days per week with at least one day between observations. At the start of each session, an intact male was brought to the group of females to determine their receptivity in a process known as behavior testing. If the females were receptive, they would drop to the ground in sternal recumbency, or “kush”. If they were not receptive, they would exhibit nonreceptive behavior, such as kicking, spitting, and/or running away. Different males were used each day in order to keep females from becoming used to a certain male. Females will also react differently to a more aggressive male as compared to a more subordinate male. Receptivity was graded on a scale of 1 to 3, as described in Figure 2 and shown in Figure 3. 3 Grade Behavior 1 Not receptive: Spat, kicked, ran way, did not allow male to mount 2 Not receptive: Allowed male to mount but did not drop into kush position 3 Receptive: Dropped into kush position after mounted by male Figure 2: Receptivity Grading Scale. Figure 3: Receptivity Grading Scale. A: Grade 1, Female (right) spitting at Male (left). B: Grade 2, Female standing to be mounted by Male. C: Grade 3, Female dropped in kush position. After behavior testing, each female was then haltered and brought into the lab for eXamination. The female was restrained in a chute and any manure found in the caudal rectum was removed. In order to gain good contact for the ultrasound, 60 mL of water- soluble lubricant was inserted into the rectum. The ultrasound eXamination was then performed transrectally using a 7.5 MHz ultrasound probe. If the female was receptive and had a significantly sized follicle, she was bred. Significant follicles were determined to be those ≥5 mm in diameter, as stated by Brown (2000). Seven males were used for the Male Age Proven Left Testis Length X Width Right Testis Length x Width (cm) (cm) A Unknown Yes 3.9 X 2.4 3.95 X 2.5 B 2 No 4.1 X 3.3 4.8 X 3.2 C 3 Yes 3.8 X 2.3 3.6 X 2.2 D 8 Yes 4.7 X 2.6 4.6 X 2.6 E 6 Yes 4.0 X 3.1 4.1 X 3.1 F 4 Yes 3.9 X 2.2 3.4 X 2.7 G Unknown Yes 4.6 X 3.1 4.6 X 3.0 Figure 4: Study Males. Teste dimensions were measured with calipers. 4 breedings (Figure 4). Six were proven and one was not. After the female was bred, the observations continued every other day for forty days, which is when the embryonic stage of gestation ends and the fetal stage begins using the convention associated with the horse. Pregnancy was determined to be established when the embryonic vesicle was seen. Photos and video were taken of the ovaries, uterine horns, and embryonic vesicle when present. The diameter of each ovary was recorded, as well as the diameter of any significant follicles. If any follicles under 5 mm in diameter were present, they were recorded as multiple small follicles (msf). The diameter of the corpus luteum was measured and the ultrasound appearance was noted. The contractility, or movement, of each uterine horn was graded on a scale from 1 to 3, with 1 being low and 3 being high. The diameter of each uterine horn was also recorded and the amount of curvature was noted. When the embryonic vesicle and embryo proper became apparent, its location and size was noted, as well as when the heartbeat was first seen. When the forty days was reached, the females were used for another study looking at fetal development, after which the pregnancies were terminated with a subcutaneous injection of prostaglandin F2α. When the females came back into receptivity, they were rebred and used again for this study. Results: In total, ten pregnancies were achieved during this study. Early embryonic death occurred in four of them, at a rate of 40%. This is lower than the rate of 58% suggested by Fernandez-Baca (1970). When the rate of EED in the two lactating females (Female A and C) was calculated, it was found to occur in two of the five pregnancies at a rate of 40% (Figure 5). The rate of early embryonic death in the three females (Females B, D, and E) 5 that did not have a nursing cria was also 40%, as two out of five pregnancies had early embryonic death. This is in contrast to findings by Ratto (2011) who found that lactating females had a lower rate of EED at 30% and the females without a cria had a higher rate of 46% EED. However, it must be noted that the sample size in the Ratto study is much larger than in the present study. Figure 5: Lactational Status of Female Versus Outcome of Pregnancy. Ratto (2011) also found that 44% of early embryonic death occurred before Day 27 of pregnancy. Our data concludes otherwise (Figure 6). One of the pregnancies did stop by Day 12, but the other three occurred at Days 30, 31, and 41. Animal Pregnancy Day Early Embryonic Death Noted A 1 30 B 1 41 B 2 12 C 1 31 Figure 6: Day Early Embryonic Death Noted.