Ultrasound Monitoring of Embryonic, Follicular, and

Uterine Dynamics of Early 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 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 , 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 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 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 (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 , 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 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 , 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. When the degree of uterine contractility was compared between the pregnant and nonpregnant females, inconclusive data was found, as shown in Figure 7. In two of the ten pregnancies, the contractility was increased during gestation, but in two, the contractility

6 decreased. There was no change in three of the pregnancies, and in three of them, the contractility varied from observation to observation and no trend could be established. Different pregnancies in the same female could also vary in the amount of contractility as compared to that observed in the female when she was not pregnant. It does not appear that uterine contractility correlates with Figure 7: Trends in Uterine outcome of pregnancy. As shown in Contractility from Nonpregnancy to Pregnancy. Figure 8, both the six pregnancies that were successfully carried past the forty-day mark and the four pregnancies that resulted in early embryonic death were associated with all four of the categories of contractility.

Figure 8: Uterine Contractility Versus Outcome of Pregnancy.

7 It was also found that the embryonic vesicle tended to appear in the left uterine horn first before spreading into the right horn. In seven out of the nine pregnancies that data was available for, the vesicle was found in the left horn at least three days before it was seen to expand into the right horn as well. According to Brown (2000) and Ratto et al

(2011), the right horn has a luteolytic effect on embryos, which must migrate to the left horn in order to implant. The data shown in Figure 9 supports these findings in 78% of the pregnancies in this study. The time period during which the embryonic vesicle appears in the uterus does not appear to follow that of the horse. Allen (2001) stated that the horse embryo should appear between Day 12 and 14, while only one of our alpaca pregnancies fell into that time range, with one embryonic vesicle being seen before and the others after.

Animal Day First Seen in Left Day First Seen in Right Uterine Horn Uterine Horn A Pregnancy 1 7 18 Pregnancy 2 34 25 B Pregnancy 1 18 29 Pregnancy 2 12 Pregnancy Lost1 C Pregnancy 1 19 Pregnancy Lost2 Pregnancy 2 Data not available3 Data not available3 Pregnancy 3 16 19 D Pregnancy 1 20 15 E Pregnancy 1 18 21 Pregnancy 2 29 36 Total 10 Pregnancies Left Horn First: 7/9 (78%) Right Horn First: 2/9 (22%)

Figure 9: Day of Pregnancy when the embryonic vesicle first appears. 1: Last observation Day 19. 2: Last observation Day 28. 3: Embryonic vesicle location not recorded.

8 Throughout the observations, follicles of significant size (≥ 5 mm diameter) were

found on the ovaries of all five of the females during all pregnancies. The largest recorded

was 12 mm in diameter, although the average diameter was 7 mm. On several occasions, a

single ovary even had more than one significant follicle present (Figure 10A). Multiple

small follicles (< 5 mm diameter) were also noted but not measured (Figure 10B). Although

a greater number of follicles developed on the ovary opposite the corpus luteum, follicles

were also seen on the same ovary as the corpus luteum, as shown in Figure 10C. In contrast

to nonpregnant follicular growth as reported by Donovan (2011), there are waves of

follicular development and regression during pregnancy. It is a possibility that a large

amount of follicular development may be indicative of early embryonic death (EED). In

three of the four pregnancies that resulted in EED, at least one significantly sized follicle

was found during almost every observation. In contrast, in five of the six pregnancies that

were successfully carried past the forty-day mark, large follicles were found only a few

times. The initial hypothesis was that the follicles seen at the beginning of pregnancy were

remnants of follicular development from before the female was bred. However, follicles

were also found throughout the forty days being observed, not just at the beginning,

making the hypothesis unlikely.

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Figure 10: Follicular development during pregnancy. A: 6 mm (purple) and 5 The mm (white) follicles on the left ovary on Day 20 of pregnancy. B: 7 mm follicle (orange) and multiple small follicles on the left ovary (green) on Day 6 of appearance of the pregnancy. C: 12 mm corpus luteum (blue) and 7 mm follicle (red) on the left ovary on Day 25 of pregnancy. corpus luteum

(CL) was tracked in consistently in Female A in this study. In the second pregnancy, the CL

showed a bright white center and a dense white outline on the periphery for the first two

weeks after ovulation. By the third week, the CL interior became darker with a white line

through the middle, which continued throughout pregnancy (Figure 11A). In the first

pregnancy that had early embryonic death, the CL became uniformly white in appearance

(Figure 11B) instead of developing the echodense line in the center, which could perhaps

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!"#$%$$ !"#$&$$ !"#$%'$ !"#$%($ !"#$)*$ be used as a sign of early embryonic death. The appearance of the corpus luteum was not

regularly followed in the other pregnancies, though the CL seems to follow the same trend

in the other four pregnancies for which data is available.

10 Figure 11: Development of the corpus luteum during pregnancy. A: Pregnancy survived past forty days. B: Early embryonic death occurred approximately Day 30.

The corpus luteum was found on the right ovary on five of the pregnancies and on the left ovary in four pregnancies, shown in

Figure 12. The tenth pregnancy (not shown) resulted in twins and had a corpus luteum present on each ovary. This contrasts with data found by Olivera (2003) that the CL is more prevalent on the left ovary. This data does support the findings from Brown Figure 12: Location of the Corpus Luteum.

(1999) that pregnancies originating from the right ovary migrate to the left uterine horn.

Out of the eight pregnancies for which both embryonic vesicle location and corpus luteum location is available, four pregnancies originated from the right ovary and moved to the left uterine horn (50%) (Figure 13). Of the three pregnancies from the left ovary, two remained in the left horn (67%), while one traveled to the right uterine horn (33%). One pregnancy from the right ovary stayed in the right uterine horn.

Figure 13: Location of Corpus Luteum and Uterine Horn in Which the Embryonic Vesicle is First Seen.

11

It has also been suggested by Ratto (2011) that the location of the corpus luteum could have an effect on embryo survivability. While the data from that study did not support this hypothesis, our data does. Of the four pregnancies resulting in early embryonic death, three originated from the right ovary and one from the left. In contrast, three of the successful pregnancies came from the left ovary and two from the right. It

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!"#$%&'#(#)** +(,*-** would appear that early embryonic death occurs more frequently when the ovulatory follicle is located on the right ovary, while successful pregnancies have a relatively equal chance of originating from the left or right ovary.

An observation of note is that the curvature of the uterine horns increased during pregnancy, most likely due to the influence of progesterone. The amount by which the horn

12 curved varied with each observation (Figure 14), and it did not appear that the curvature increased as pregnancy progressed.

Figure 14: Curvature of the Uterine Horn. A: Nonpregnancy right uterine horn (25 mm diameter). B: Pregnant right uterine horn at Day 7 (22 mm diameter). C: Pregnant right uterine horn at Day 12 (19 mm diameter). According to Allen (2001), the embryo of the horse remains spherical in shape until implantation occurs. This was not found in the alpaca. The contractility of the uterine horns caused the embryonic vesicle to constantly mutate and change shape, demonstrated in

Figure 15.

Figure 15: Embryonic vesicle at Day 23 in the left uterine horn. Picture A was taken two minutes before Picture B.

13

Using the data collected during our observations, we were able to create a timeline of the events that occur during early pregnancy (Figure 16). The is present at Day 2 before becoming the corpus luteum by Day 4. The embryonic vesicle can be seen as early as Day 7 of pregnancy, while the embryo proper is not evident until Day

25. The heartbeat appears at Day 27.

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Figure 16: Timeline of Early Pregnancy Events.

Individual Animals:

Female A:

Pregnancy 1: Bred to Male A: September 12-October 17: Early Embryonic Death

Pregnancy 2: Bred to Male B: November 3-December 16: Successful Pregnancy

14

Figure 17: Uterine Contractility During Pregnancy, Female A.

15 Figure 18: Follicular Development During Pregnancy, Female A.

Female A was bred successfully twice during the course the study. The first pregnancy resulted in early embryonic death, while the second was successfully carried past forty days. Uterine contractility was consistent throughout both of her pregnancies

(Figure 17). In Pregnancy 1, contractility stayed at Grade 1. In Pregnancy 2, uterine contractility was Grade 1, except for Days 18 and 20, when it was Grade 2. Significant follicular development was observed during both of these pregnancies. During the first pregnancy, there were significant follicles consistently found on both ovaries, while in the second pregnancy, significant follicles were found mostly on the left ovary, as shown in

Figure 18. It should be noted that the corpus luteum was also present on the left ovary. The corpus luteum was also located on the left ovary during the first pregnancy. It was found that the embryonic vesicle appeared in the left uterine horn first in Female A’s first pregnancy at Day 7, but appeared in the right uterine horn first at Day 25 in the second pregnancy. In the first pregnancy, the embryonic vesicle spread into the right horn by Day

18, and in the second pregnancy, it was seen in the left horn on Day 34.

16 Female B:

Pregnancy 1: Bred to Male C: June 20- August 3: Early Embryonic Death

Pregnancy 2: Bred to Male D: August 12- September 9: Early Embryonic Death

Figure 19: Uterine Contractility During Pregnancy, Female B.

Female B was successfully bred twice during the study. Both of the pregnancies resulted in early embryonic death. As seen in Figure 19, uterine contractility in the first pregnancy increased dramatically from Grade 0 to Grade 3, while contractility during the second pregnancy had an overall decrease from Grade 3 to Grade 1. Follicular development was not followed closely in this female, though several follicles of significant size were noted throughout both pregnancies. The corpora lutea for both pregnancies were located on the right ovaries. The embryonic vesicle was seen in the left uterine horn first in both

Pregnancy 1 and 2, at Days 18 and 12, respectively. The embryonic vesicle appeared in the right uterine horn at Day 29 in the first pregnancy, but the second pregnancy was lost before the vesicle could be seen in the right uterine horn.

Female C:

Pregnancy 1: Bred to Male E: January 28- March 2: Early Embryonic Death

17 Pregnancy 2: Bred to Male E: April 13- May 24: Successful Pregnancy

Pregnancy 3: Bred to Male D: October 4- November 16: Successful Pregnancy

Figure 20: Uterine Contractility During Pregnancy, Female C.

Figure 21: Follicular Development During Pregnancy from Pregnancy 1, Female C. Female C had three successfully pregnancies during the study period. The first resulted in early embryonic death, but the second and third were both carried past the forty-day mark. As shown in Figure 20, the uterine contractility for Pregnancies 1 and 3 varied between Grade 1 and Grade 2, with one day recorded as Grade 3, on Days 24 and

18 30, respectively. The contractility for Pregnancy 2 was not consistently recorded. Female C consistently had follicles present on both the left and right ovaries during her first pregnancy (Figure 21). The corpus luteum was located on the right ovary, which may account for the greater follicle size on the left ovary. The follicular development was not closely monitored during the second and third pregnancies, though significant follicles were seen on several occasions. The corpora lutea for these pregnancies were located on the left ovary. The embryonic vesicle was observed in the left uterine horn first in

Pregnancy 1 at Day 19 and Pregnancy 3 at Day 16. It spread into the right horn at Day 19 in

Pregnancy 3, but Pregnancy 1 was lost before this occurred. The location of the embryonic vesicle was not recorded during the second pregnancy.

Female D:

Pregnancy 1: Bred to Male D: August 1- September 9: Successful Pregnancy

Figure 22: Uterine Contractility During Pregnancy, Female D.

19 Figure 23: Follicular Development During Pregnancy, Female D.

Female D was bred once during the study period, which resulted in a successful pregnancy carried past forty days. The uterine contractility for this pregnancy, as seen in

Figure 22, increased greatly from Grade 0 to Grade 3. There was some follicular development during the pregnancy, particularly on the left ovary (Figure 23). This might have been due to the location of the corpus luteum on the right ovary. The embryonic vesicle appeared in the right uterine horn first at Day 15 and spread into the left horn by

Day 20.

Female E:

Pregnancy 1: Bred to Male F: January 31- March 14: Successful Pregnancy

Pregnancy 2: Bred to Male G: August 22- September 30: Successful Pregnancy

Figure 24: Uterine Contractility During Pregnancy, Female E. 20 Figure 25: Follicular Development During Pregnancy, Female E.

Female E carried both pregnancies past forty days. Uterine contractility for the first pregnancy varied between Grade 1 and Grade 3, as seen in Figure 24. There was a slight decrease in contractility in the second pregnancy, from Grade 3 to Grades 1 and 2. Female E did not have as many significant follicles as the other females. She did however have multiple small follicles in almost every observation, represented by the data points at zero on the y-axis in Figure 25. The first pregnancy resulted in twins, with a corpus luteum located on each ovary. In the second pregnancy, the corpus luteum was on the right ovary.

In both of the pregnancies, the embryonic vesicle was first seen in the left uterine horn at

Days 18 and 29 and in the right uterine horn at Days 21 and 36.

21 Conclusion:

The observations made in this study tend to disagree with some of the published literature. The rate of early embryonic death has been reported as being 58% by

Fernandez-Baca (1970), while our rate was lower at 40%. It was stated by Ratto (2011) that lactating females had less occurrence of early embryonic death than females who were not currently nursing a cria (30% compared to 46%). The rates for these two groups found here were both also 40%. Ratto (2011) also stated that 44% of embryo loss occurs before

Day 27 of pregnancy. Our observations disagree here as well, as only 25% of embryo loss occurred before Day 27. Established literature also states that 98% of pregnancies implant in the left uterine horn (Brown 2000). In contrast, out study found the embryonic vesicle to first appear in the left horn only 78% of the time.

Discrepancies in data were additionally seen when observing the corpus luteum.

Olivera (2003) stated that the corpus luteum occurs on the left ovary more often than the right. Our observations found that it appears to occur on the either ovary approximately half of the time and actually occurred slightly more on the right ovary than on the left (56% to 44%). This does however support the idea that embryos originating from the right ovary must migrate to the left uterine horn, as four out of the eight pregnancies for which this data was recorded originated from the right ovary and were first seen in the left horn.

However, three of these four pregnancies resulted in early embryonic death, contrasting with findings by Ratto (2011) that the location of the corpus luteum does not have an impact of the rate of embryo mortality. During our observations of the corpus luteum, it was detected that the CL changes in appearance throughout the pregnancy and develops a blackish, fluid-filled outer ring with a white echodense line of tissue in the middle. When

22 early embryonic death occurred, however, the black ring did not develop and the CL instead remained homogeneously white in color.

Due to the similarities in gestation and type of placenta between the alpaca and the horse, we compared some observations between the two species. While Allen (2001) found that the equine embryonic vesicle appears in the uterine horns between Days 12 and 14, the range in which we detected the alpaca embryonic vesicle was wider, from 7 to 29 days.

We also found that the shapes of the two embryonic vesicles do not match. The horse vesicle was reported by Allen (2001) to remain spherical in shape, while the alpaca vesicle is constantly deformed by the contractions of the uterus.

The contractions of the uterine horns were followed during this study. We originally hypothesized that the influence of progesterone from the corpus luteum would cause the contractility of the uterus to increase. No discernable trends were found. Two of the pregnancies did increase in their amount of contractility; however two also decreased in their amount, while three had no change and three pregnancies were too varied to detect a trend. The contractility also varied between different pregnancies in the same female.

There does not appear to be an association between the amount of contractility and the outcome of the pregnancy. While observing the contractility of the uterine horns, it was also noticed that the horns increase their curvature during pregnancy.

Interestingly, follicles were seen to develop throughout all of the pregnancies in this study. Significantly sized follicles greater than or equal to 5 mm in size were observed on several occasions, as well as multiple small follicles. More than one significant follicle was found on the same ovary, in addition to the same ovary as the corpus luteum. Unlike the nonpregnant female as reported by Donovan (2011), there do appear to be waves of

23 follicular growth, maturation, and regression in the pregnant female. It is a possibility that having a larger amount of significant sized follicles is a sign of early embryonic death.

This research has a great deal of potential for future uses. If signs of early embryonic death can be recognized, the breeder could prepare appropriately in case the pregnancy is indeed lost. Knowing the parameters for a successful breeding versus an unsuccessful one will also help alpaca owners determine the optimal time to breed their females. Having this information available will not only improve the alpaca industry here in the United States but also in South America, where people’s livelihoods rely on the alpaca. Future studies are currently in progress at the University of Massachusetts Amherst to answer these questions and more.

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25