BIOLOGY OF REPRODUCTION 67, 334–339 (2002)
1
Infigen, Inc., DeForest, Wisconsin 53532
ABSTRACT
Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly
25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.
parturition, pregnancy
INTRODUCTION
Cloning as a form of replicative reproductive technology has received a great deal of attention because of its many potential applications and the impact that it is having on the implementation of other technologies (i.e., transgenic animal bioreactors, xenotransplantation, etc.). For a literature review, refer to the companion paper [1]. Most literature has focused on data culminating with the birth of a few cloned animals [2–5]. An exception was a report comparing birth weight and growth data of bovine clones to their full or half siblings produced through the use of other reproductive technologies [6]. However, in that published article, no information was provided regarding efficiencies of producing animal clones or the physiological condition of the cloned calves born.
Transferring a nucleus from an embryonic blastomere into an enucleated oocyte to produce a calf was first demonstrated in 1986 [7]. For over a decade, use of this technology has generated hundreds of bulls and cows for both beef [6] and dairy (as recorded in the registry database of
Holstein Association USA, Inc., Battleboro, VT) production. It has also been used by cattle artificial insemination organizations to produce high genetic merit bulls for the
1 Correspondence: Michael D. Bishop, Infigen, Inc., 1825 Infinity Dr.,
DeForest, WI 53532. FAX: 608 846 0520; e-mail: mbishop@infigen.com
Received: 14 November 2001.
First decision: 3 December 2001.
Accepted: 4 January 2002.
Q 2002 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
production and sale of semen. However, a thorough evaluation of these clones has not been reported.
The first bovine produced from nonembryonic cells was born in 1997 [8]. Subsequently, several groups have reported production of bovine clones using various nonembryonic cell types [3–5]. These publications report relatively small numbers of births from a limited number of cell lines from which to draw conclusions.
In this article, we summarize and report on the survival, growth, and development of 117 calves produced over a
26-mo period (from 1 January 1998 to 29 February 2000) using our proprietary nuclear transfer technology and nonembryonic cells derived from various tissues. Furthermore, we compare the results obtained from use of our nuclear transfer technology to that obtained and reported for more established reproductive technologies (e.g., artificial insemination [AI] embryo transfer [ET], or transfer of in vitro fertilized [IVF] and matured embryos into recipients).
334
MATERIALS AND METHODS
Recipient Management
Biosecurity.
Holstein heifers weighing 600–800 lbs. were purchased and used as recipients subsequent to negative test results for bovine leukosis virus, brucellosis, bovine viral diarrhea, paratuberculosis (Johne’s), neospora, and tuberculosis prior to arriving at Infigen-operated quarantine facilities. The heifers remained in quarantine for 45–60 days; during this time, they were retested for the above diseases and vaccinated before entering the recipient embryo transfer herd. After a positive pregnancy determination, animals were retested prior to transfer to gestation and calving facilities. In addition, all recipients were tested annually in an attempt to maintain a high level of biosecurity.
Feeding.
Rations were formulated using the Cornell Net Carbohydrate and Protein System (CNCPS) [9]. All rations contained Fermentin (Applied Biotech, Evansville, WI).
Estrus detection.
Visual observations for estrus were made at approximately 12-h intervals and recorded as first being observed in the morning or afternoon. In addition, an observed estrus of a recipient during other times of the day was recorded. Prostaglandin was used only as a reproductive management tool to reset cycles where the current estrus did not fit the ET protocol. Recipients receiving prostaglandin were observed in estrus before any ET event occurred.
Embryo transfer.
Embryos resulting from nuclear transfer (NT) were cultured for 5–8 days. Prior to transferring them into recipients, they were visually graded using International Embryo Transfer Society [10] standards. Grade 1 and 2 blastocyst embryos with stages that correspond to days in culture were loaded into mini-French straws (IMV Corporation,
Minneapolis, MN) for transfer purposes. Each straw had 2 or, on a few occasions, 1 or 3 embryos. The recipient’s days postestrus was synchronized with that of the NT embryo’s age. Specifically, fusion of nuclear donor cells into enucleated oocytes and the predicted ovulation of the recipient were matched to within
6
48 h. Embryos were transferred to the uterine horn ipsilateral to the corpus luteum using standard ET techniques.
Occasionally, an embryo was placed in each uterine horn of the recipient.
Ultrasonographic pregnancy evaluation.
Seventeen to 20 days after
ET, recipient heifers were examined for pregnancy using transrectal ultrasonography with a 5-MHz linear array probe and an Aloka 500 ultrasound machine (Aloka Co., Ltd., Wallingford, CT) connected to a video printer.
Recipients were either classified as pregnant or not pregnant based on

ONTOGENY OF CLONED CATTLE TO LACTATION
TABLE 1. Comparison of pregnancy initiation rate, fate of pregnancies, birth, and fate of cloned calves.
Number % Parameter
Transfers and initiations
Embryo transfers
Pregnancy initiations
Fate of pregnancies
Abortions
Hydrops
Calved
Fate of calves
Born
Born alive
Remain alive
2170
535
402
30
103
117
106
82
24.7
75.1
5.6
19.3
90.6
77.4
335
FIG. 1. Number and cumulative percentage of aborted pregnancies by fetal age.
whether or not a fetal heartbeat was observed. Open (not pregnant) recipients were recycled for future ET. Fetuses were measured for crown-rump length every 7 days until Day 70 of gestation. Later stage pregnancies were evaluated transabdominally using a 3.5-MHz probe.
Prepartum management of recipients.
Pregnant recipients were moved from the gestation facilities to appropriate calving facilities between Day
210 and Day 265 of gestation. The decision to induce parturition and deliver the calves by cesarean section was based on days in gestation, breed of the calf, calf viability, observed changes in appearance of placenta during ultrasound, and urine ketone level of the recipient. Parturition was induced approximately 24 h prior to scheduled delivery using 25 mg dexamethazone and 25 mg of prostaglandin (Lutalyse, Pharmacia Co., Kalamazoo, MI).
Clone Management
Birth.
Prior to delivery, the size of the umbilicus was assessed. Calves with a larger than normal umbilicus tended to have umbilical separation very close to the abdominal wall if allowed to break naturally. Over the course of this study, several different approaches for handling the umbilicus were used that included stretching until a natural break occurred, holding the umbilical stock and stretching several centimeters away from the body wall, cutting the umbilicus several centimeters away from the body wall, and clamping off the umbilicus with emasculators. When breaks occurred close to the body wall, attempts were made to tie off the blood vessels before they retracted into the body wall. An umbilicus of normal size was allowed to stretch and break naturally. After removal from the recipient, the calf’s vital functions were determined. The calf was rubbed dry with toweling and manually tested for a nursing reflex. A blood sample was taken prior to feeding colostrum to determine total serum protein levels. The calves were generally given 2 h to establish a nursing reflex. If this did not occur, they were fed using an esophageal feeder. The goal was to get the calf to drink at least 4 L of colostrum and to elevate total serum protein by 1 g/dl by 12 h. If serum total protein was not 5.5
g/dl and the IgG level was less than 1200 mg/dl at 24 h, 1 L of bovine plasma was administered intravenously (i.v.). As soon as the calf’s physiological functions were stable (1–14 days), it was transferred to appropriate calf-rearing facilities.
Calf rearing.
Calves were reared for 60 days in either individual polydome calf huts or an environmentally controlled biosecure facility with individual stalls. Calves were fed milk replacer (instant nursing formula
DC medicated 22% crude protein, 20% crude fat; Land O’Lakes, Inc.,
Fort Dodge, IA) equal to 10% of their daily body weight. The volume of milk replacer for each day was fed in two or three feedings, depending on environmental conditions or other management considerations. The calves had constant access to fresh water and an 18% protein calf starter pellet (Calf Vantage Texturized Starter [B28.5] [MT] Medicated; Cargill,
Minneapolis, MN).
At approximately 60 days of age, calves were weaned from milk replacer if they were consuming at least 2 kg of concentrate/day. After weaning, 2–3 calves were grouped and housed together. As they developed, calves were gradually regrouped into 8–10 head per pen. The rations fed were formulated using CNCPS [9]. All rations contained Fermentin. The goal of the feeding program for the cloned animals was for them to achieve maximum skeletal growth without putting on excess body condition.
Weights and hip heights were recorded at regular intervals, starting at approximately 90–120 days of age. Weight per day of age (WDA) was calculated by dividing total weight gain by days of age. Average daily gain (ADG) was calculated by subtracting birth weight from the recorded weight of the calf and dividing by days of age. Individual period weight gains were also calculated by determining the amount of weight gained in that period and dividing by the number of days since the last recorded weight.
Breeding, parturition, and lactation.
Sexual maturity was determined by observing estrus activity of the cloned dairy heifers. Starting at approximately 14 mo of age, cloned heifers were artificially inseminated.
Periparturient heifers were transitioned to a milking ration (Catapult Closeup Complete; Cargill) approximately 30 days prior to calving. Prior to calving, heifers were fitted with a Calf Alert monitor (Foalert, Inc.,
Acworth, GA) that telephonically signaled animal caretakers that parturition had begun. Cloned heifers were allowed to give birth unassisted if possible; however, human assistance was given when needed.
RESULTS
Thirty-four genetically unique cell lines were used to produce nuclear transfer embryos for nonsurgical transfer into 2170 recipients (Table 1). Both fetal and adult cells, some of which had been transfected, were used to produce the cloned embryos [1]. Five hundred thirty-five recipients
(24.7%) initiated pregnancy. Early fetal abortion rate was
67% prior to Day 60 of gestation (Fig. 1). Interestingly, prior to abortion, aborted fetuses grew at the same rate as those that were not aborted (Fig. 2).
Overall, 75.1% of the recipients that initiated a pregnancy aborted the fetus, 5.6% developed a condition called hydrops, and 19.3% completed pregnancy (Table 1). There were 14 recipients that gave birth to 2 calves of the 103 recipients that calved. One hundred six (90.6%) of the 117
FIG. 2. Comparison of crown-rump growth. Comparison of crown-rump growth of fetuses that produced a calf (calved), those that aborted after pregnancy initiation (aborted), and previously report crown-rump data of noncloned fetuses [15] (noncloned). There were 1179 measurements taken on fetuses from 401 pregnancies that did abort and 816 measurements taken from 133 pregnancies that did not abort.

336 PACE ET AL.
TABLE 2. Growth of 17 cloned calves from the same genetic line.
a
Days of age
1
120
180
365
540
Weight
(kg 6 SD)
51 6 14
137 6 25
208 6 28
399 6 51
497 6 51 a NA, Not applicable.
Growth parameter
Weight/day of age
(kg 6 SD)
NA
1.14
6 0.2
1.16
6 0.16
1.09
6 0.14
0.92
6 0.10
Hip height
(cm 6
NA
SD)
95 6 4
105 6 4
132 6 4
141 6 7 calves were alive at birth. Eighty-two (77.4%) of the 106 live healthy calves were from 20 unique genetic cell lines.
Birth weight of live calves ranged from 11 to 72 kg (Fig.
3), with an average birth weight of 51
6
11 kg. Fifty-two cloned calves raised at the same facility had similar weight gain during the first 120 days after birth. Thirty-eight calves that had birth weights within 1 SD of the mean birth weight averaged 0.63
6
0.18 kg/day weight gain. Nine calves born with birth weights at least 1 SD below the mean weight gain had gains of 0.64
6
0.13 kg/day, and five calves born
1 SD above the mean gained 0.71
6 0.17 kg/day.
Weights and hip heights of 17 clones from the same genetic line were measured at 120, 180, 365, and 540 days of age (Table 2). Differences in weight and hip height were minimal among calves except for one that was approximately 2 SD below the average weight and 3 SD below the average hip height. Average WDA was similar at 120 and
180 days (1.14
6
0.20 vs. 1.16
6
0.16 kg, respectively), declining to 1.09
6 0.14 and 0.92
6 0.10 kg at 365 and
540 days, respectively.
Twenty-four calves born alive either died or were killed during this study. They were grouped as preventable deaths or nonpreventable deaths. The physiological system failures that likely caused the deaths are provided in Table 3. The
11 nonpreventable deaths were caused by dysfunctions of the placenta, respiratory failure, digestive abnormality, circulatory failure, or nervous or musculoskeletal system dysfunctions. Three calves had multiple systemic dysfunctions and died. The 13 potentially preventable deaths involved
FIG. 3. Birth weights of the 106 live cloned calves (74 Holstein, 26
Brown Swiss, 3 Angus, and 3 Holstein 3 Jersey cross).
complications associated with digestive, placental, respiratory, musculoskeletal, and urinary systems.
Cloned heifers started to display signs of reaching puberty at approximately 10–11 mo. All of the 22 cloned heifers that were inseminated became pregnant. Age at calving was 23–25 mo, similar to noncloned cattle. The milk from cloned cattle appears to be normal; however, complete lactation data and milk component analyses are being accumulated for subsequent publication.
DISCUSSION
We demonstrate that the majority of cloned cattle are born healthy and grow, reproduce, and lactate similarly to noncloned cattle. This study is the largest and most in-depth study in which cultured cells of several different types were used to produce clones from 34 genetically unique lines.
The cultured cells came from both adult and fetal tissues, and many of them were transfected [1]. There were 2170 transfers of one to three embryos per recipient. The data were accumulated from 26 mo of nuclear transfer research and an evaluation of resulting cattle clones that range in age from 11 to 35 mo.
Proper statistical analysis of the data was not practical
TABLE 3. Nonpreventable and preventable deaths of cloned calves and the major physiological system(s) involved in death.
Physiological system involved
A. Nonpreventable calf deaths
Multiple dysfunctions
Placenta
Respiratory
Digestive
Circulatory
Nervous
Musculoskeletal
B. Preventable calf deaths
Placental
No. of calves
3
2
1
2
1
1
1
3
Age at death
(days)
1–2
1
3
78–122
42
154
298
1
Weight at birth
(kg)
11–63
50–59
62
52–60
52
51
44
53–69
Observations
Failure of most systemic functions
Apparent premature separation of placenta
Lung immaturity, meconium aspiration at birth
Chronic diarrhea (1), small intestinal intussusception with obstruction (1)
Congenital heart defect
Hydrocephalus
Developmental orthopedic disease
Respiratory
Digestive
Musculoskeletal
Urinary
3
5
1
1
1–5
5–90
328
112
48–66
59–72
42
59
Extensive internal bleeding from enlarged umbilicus
Developed pneumonia (2), premature induction of labor 16 days early, immature lungs (1)
Clostridial infection (1), developed abomosal ulcers from eating wood chips
(2), bloated (2)
Injury, patellar luxation
Pyelonephritis probably secondary to umbilical infection

because of a myriad of variables that were introduced during the study. The focus of our research was to develop cloning technology using cultured cells. Comparisons of our cloning efficiency data to that of other reproductive technologies could be beneficial in elucidating the advances that have been made in the development of nuclear transfer technologies and have been included in this discussion.
The overall pregnancy initiation rate was 24.7% in this study and included results from all experimental research regimens conducted over the 26-mo period. This initiation rate is a little lower than what has been reported by others in selected small studies [3–5, 11]. However, in our study, approximately 75% of the embryo transfers were embryos derived from transfected donor cells, which appear to give lower pregnancy initiation rates [1]. A comparison of pregnancy initiation rate across studies is difficult because different methods and times are used to determine pregnancy.
In the present study, pregnancy initiation was defined as the presence of a fetal heartbeat at 17–20 days postembryo transfer. Until the development of ultrasound, most studies evaluating pregnancy initiation rates were made using rectal palpation at 50–60 days after fertilization or were estimated using 90-days nonreturn to estrus. For example, in a study of 7652 transfers of in vivo derived embryos, 71% of the recipients were pregnant upon rectal palpation at 50–60 days [12]. By comparison, transfer of 2268 IVF embryos resulted in a 54% pregnancy rate by palpation at 50–60 days [13]. In a large artificial insemination study involving
15 884 first services, the estimated pregnancy rate using 90days nonreturn to estrus data was 67% [14]. Therefore, while methods used to evaluate pregnancy initiation were different, it would appear that recipients receiving cloned embryos are 2–3 times less likely to become pregnant than when using other reproductive methods.
Early fetal loss was high in this study, with 66.5% of initiated pregnancies being lost by the 60th day of gestation
(Fig. 1). Our data are consistent with that from others performing nonembryonic cell cloning [3–5, 11]. An evaluation of fetal growth during the first 70 days of gestation indicated that, as long as a fetus had a heartbeat, growth as measured by crown-rump length was the same for cloned and noncloned fetuses [15] (Fig. 2). It is striking that aborted fetuses had sudden growth cessation rather than a gradual slowing of growth before death. These data might appear to be in conflict with that reported earlier [11], when it was found that fetuses that eventually aborted were growing slower than those that did not. However, the estimated day of pregnancy failure was midway between the day of no detectable heartbeat and the previous estimation of crown-rump length. Averaging the day of death could make it look like aborted fetuses were growing slower.
There is also fetal wastage during early pregnancy of noncloned fetuses produced by other reproductive technologies. In two studies where cattle were examined using ultrasound at 28 days after AI and then reexamined 14–28 days later, the abortion rates were 17% and 28%, respectively [16, 17]. The use of blood progesterone taken on the
22nd day of gestation to determine pregnancy estimated that 8.5% of the pregnant recipients were no longer pregnant on the 60th day of gestation [18]. Also, 5.3% of 862
Holstein heifers determined to be pregnant by measuring bovine pregnancy-specific protein B in their blood at 30–
45 days after AI had aborted by the 60th day of gestation
[19]. Therefore, while abortions are substantial in noncloned cattle during the first 60 days of gestation, fetal wastage
ONTOGENY OF CLONED CATTLE TO LACTATION 337 of cloned embryos appeared to be from 3 to 12 times greater than for noncloned fetuses.
An additional 25% of the pregnancies aborted after the
60th day of gestation in the present study (Fig. 1). This rate of abortion is higher than reported for pregnancies established using other reproductive technologies. Abortions after the 60th day of gestation were 3%–11% for AI [16, 18,
20], 5% for in vivo matured embryos transferred into recipients [12, 21], and 8%–12% when IVF embryos were transferred [13].
There is a rare physiological condition in pregnant cattle where the placenta cannot regulate fluids. This condition has been termed hydrops, and the frequency has been estimated to occur in 1 in 7500 pregnancies [22]. Reproductive technologies that involve manipulation of embryos have noted an increased frequency of this condition [13,
23]. Several groups using cultured cells during cloning have observed this condition but have not had enough observations to estimate the frequency of the condition [3–5, 11].
Hydrallantois and hydramnios were not differentiated in our study; however, the occurrence was 5.6% of all initiated pregnancies (Table 1) or 17% of those pregnancies reaching
60 days gestation. It is not known whether the increase in the frequency of this condition is the result of placentation anomalies or interruption of early gene expression, but it is a condition that must be further elucidated prior to a major use of this technology for agricultural purposes. It is interesting to note that, in our cloning research with porcine, which has different placentation than the bovine, neither this condition nor large birth weight piglets have been observed (unpublished observation).
Pregnancy initiations, abortions, and frequency of development of the hydrops condition play a major role in the efficiency of this reproductive technology; however, they do not play a role in the physiological well-being of the calves born. Recent popular press releases have led to confusion of the public by mixing efficiency of production and the physiological well-being of the clones from data regarding as few as 13 animals [3]. In this study, there were
117 calves born from 103 pregnant recipients. Eleven were dead at birth, 10 calves died or were killed within 48 h of birth, an additional 6 calves died prior to weaning (60 days), and 8 calves died postweaning. Therefore, 77% of the clones that were born alive are still alive and healthy
(or 85% of the clones that were alive 48 h after birth). By comparison, an analysis of U.S. Holstein data indicates that
11%–13% of primiparous births were born dead or died within 48 h [24, 25]. A U.S. government survey of dairy producers indicated that 87% of the dairy heifers born alive remain alive and, of those that died, 10.8% died prior to weaning and 2.15% died between weaning and giving birth to their own first calf [26]. Preweaning death losses were
9% when in vivo matured embryos were transferred [21] and 14%–16% using IVF embryos [13, 23]. Therefore, the percentage of cloned calves surviving birth, preweaning, and postweaning is very similar to other reproductive technologies.
There are several reports that manipulation and/or in vitro culture of embryos [5, 27–31] increase birth weights.
An excellent study comparing 418 cloned cattle produced from embryonic cells to full or half siblings that were produced using either embryo transfer or AI and natural mating (AI/NM) reported that the clones were 36% heavier than those from AI/NM [6]. The variation in birth weight for clones was also 3–5 times greater than calves born as a result of AI/NM. It is also interesting to note that calves

338 PACE ET AL.
resulting from ET were 8% heavier than AI/NM, supporting the idea that even short time exposure to in vitro culture and manipulation has an effect on birth weight. The average birth weight in the present study was 51
6
11 kg with a range from 11 kg to 72 kg. While there were no noncloned calves born in the present study, data from the nearby US
Dairy Forage Research Center at Prairie du Sac, WI, had an average birth weight of 42
6
5.9 kg for 521 female
Holstein calves born alive during 1992 to 1994 with a range from 26 to 76 kg (personal communication with Len I.
Strozinski, dairy herd manager). Since most of our cloned animals are Holstein, it would appear that the cloned animals born in the present study are approximately 20% heavier and have twice the amount of variation in birth weight as calves born at the Forage Research Center.
While the cloned animals undoubtedly received better management attention than the average calf might receive in the U.S. population of calves, management error did play a role in some of the deaths. Therefore, we chose to categorize calf deaths as either preventable or nonpreventable.
There were 11 (10.4%) calves born with physiological dysfunctions that would not allow these calves to function and grow into normal adult animals. These calves were killed and necropsies were performed to determine the cause of death (Table 3). In the cattle population, it has been reported that 2%–3% of the calves born exhibit some type of congenital defect [32]. In a study involving embryo transfer, 5% of the calves died because of congenital abnormalities [21]. In another study where the physiological condition of hydrops was included with congenital anomalies,
5.2%, 2.2%, and 0.6% of the calves born died as a result of these anomalies using IVF embryos, in vivo cultured and then transferred embryos (ET), or AI to produce the calves, respectively [23]. Therefore, it appears that techniques involving handling or culturing as well as manipulation of the embryo increases the chance for congenital anomalies of the neonatal calf that results in death.
There were 13 (12.3%) calves that died from problems classified as preventable (Table 3). Three of the deaths were related to complications resulting from an enlarged umbilicus. This condition is seen more frequently in cloned calves but can be managed by tying or clamping off the umbilical arteries. The remaining calves died from complications involving pneumonia, clostridial infection, ulcers from consuming wood shavings, bloat, a patellar luxation, pyelonephritis secondary to an umbilical infection, and early induction of labor.
While no direct comparisons were made between clones and other calves in this study, growth rates of approximately 1 kg/day (Table 2) are normal for well-managed herds [33]. We also found that the rate of gain was similar for light and heavy birth weight calves, indicating that the prenatal conditions that allowed for increased size does not continue after the calf is born. Our results tend to agree with a study involving beef clones that reported only small differences in weight between clones and their full and half siblings at 205 and 365 days of age [6].
Conclusion
The production of normal cloned cattle using cultured cells is a tremendous scientific advancement. We have demonstrated that 85% of the clones that are alive 48 h after birth are still alive and healthy, ranging in age from 5 to
29 mo. These cattle clones grow and reproduce and appear to lactate similarly to noncloned cattle (lactation study is ongoing). Cloning cattle using cultured cells is an emerging reproductive technology where significant research efforts are still needed to develop a better understanding of the molecular mechanisms involved in order to increase efficiency of production. However, even with present technical inefficiencies of production, the cattle produced using this technology offer immediate unique opportunities to meet critical needs in both human health care and agriculture.
ACKNOWLEDGMENTS
The authors wish to acknowledge the excellent technical support of
Dr. Steven Malin in transferring embryos, to Dr. Sandra Curren for ultrasound evaluation, and to Dr. Sheila McGuirk and staff at the University of Wisconsin for performing cesarean sections and early postnatal care of the clones. We also thank Carrie Sparks and Rita Wells for help in preparation of this manuscript and Thom Patterson, Heidi Van Beek, and Karen
Van Beek for data management.
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