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Biology of Reproduction 66, 291-296 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Reproductive Characteristics of Cloned Heifers Derived from Adult Somatic Cells1

B.P. Enrighta, M. Tanejaa, D. Schreibera, J. Riesena, X.C. Tiana, J.E. Fortuneb, and X. Yang2,,a

a Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269 b Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study examined the onset of puberty, follicular dynamics, reproductive hormone profiles, and ability to maintain pregnancy in cloned heifers produced by somatic cell nuclear transfer. Four adult somatic cell-cloned heifers, derived from a 13-yr-old Holstein cow, were compared to 4 individual age- and weight-matched heifers produced by artificial insemination (AI). From 7 to 9 mo of age, jugular venous blood samples were collected twice weekly, and from 10 to 11 or 12 mo of age, blood sampling was carried out every other day. After the heifers reached puberty (defined as the first of 3 consecutive blood samples with peripheral plasma progesterone concentrations of >1 ng/ml), ultrasound examination of ovaries and jugular plasma sample collection were carried out daily for 1 estrous cycle. Cloned heifers reached puberty later than controls (mean ± SEM, 314.7 ± 9.6 vs. 272 ± 4.4 days and 336.7 ± 13 vs. 302.8 ± 4.5 kg for clones and controls, respectively; P < 0.05). However, cloned and control heifers were not different in estrous cycle length, ovulatory follicle diameter, number of follicular waves, or profiles of hormonal changes (LH, FSH, estradiol, and progesterone). Three of the 4 clones and all 4 control heifers became pregnant after AI. These results demonstrate that clones from an aged adult have normal reproductive development.

developmental biology, embryo, follicular development, ovary, pregnancy, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal cloning with cultured somatic cells has potential applications in agriculture and biomedicine. It allows targeted genetic manipulations, genome preservation, and may also be used as a tool for basic research [1–5]. Presently, clones have been successfully produced from somatic cells of sheep [4], mice [6], pigs [7], goats [8–10], cows [11–16], and gaur [3]. However, successful somatic cell cloning has resulted in small numbers of live animals due to the low efficiency of the overall cloning process [17]. Pregnancy loss and fetal death from respiratory distress, pulmonary hypertension, and morphological abnormalities of the kidney or cacomelia as well as immunological deficiency are common problems associated with somatic cloning [18–24]. Live-born clones generally require intensive assistance at parturition [18, 19, 22]. Furthermore, controversies have arisen regarding telomere lengths of animals cloned from adult cells. Telomeres of cloned animals have been reported as being shorter [23], longer [24, 25], or indifferent [26, 27] from those of animals obtained using natural reproduction. These observations raise the questions of whether the genome of somatic clones is completely reprogrammed and of whether healthy clones from aged animals can grow and reproduce normally. Also, the long-term effects of somatic cell cloning are unknown, because it is a relatively new technology. To date, and to our knowledge, no systematic study on the reproductive potential of cloned animals has been reported.

Previous studies in our laboratory produced 10 cloned heifers from an aged dairy cow [21, 26]. Four of those heifers remain alive and provided an opportunity to study reproduction in clones produced from adult somatic cells. An early checkpoint in reproduction is puberty and the associated changes in follicular and hormone profiles. In Holstein heifers, the average age at the first estrus falls between 10 and 12 mo, depending on growth rate, body condition, and season [28]. Follicular development in cattle occurs in waves, in which a cohort of 5–7 follicles develops simultaneously, with one of them becoming the dominant follicle. During each estrous cycle, 2 or 3 waves of follicular activity occur, with the last wave containing the ovulatory follicle [29–31]. Heifers of identical genotypes, such as somatic clones, are an ideal model to study the effects of genotype on the onset of puberty and follicular wave patterns. This study was designed to compare reproductive characteristics, including puberty, follicular dynamics, and hormone profiles during an estrous cycle, of cloned heifers with controls produced by artificial insemination (AI).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Design

This project was approved by the Institutional Animal Care and Use Committee of the University of Connecticut. Four 7- to 9-mo-old cloned heifers derived from somatic cells of a 13-yr-old Holstein dairy cow of high production merit were age and weight matched with contemporary heifers produced by AI (n = 4) [21, 26]. At the beginning of the study, cloned and control heifers weighed 210 ± 10 (mean ± SEM) and 222 ± 12 kg, respectively. Heifers were stall fed under standard conditions at the Kellogg Dairy Center of the University of Connecticut. The heifers were not treated with any drugs except regular vaccinations before this study. All observations and experiments were conducted during the months of February to September 2000. Twice-daily observations were carried out, both before and after puberty, to detect external signs of estrus, such as mucous discharge, mounting, and standing. Puberty was defined as the first ovulation, and plasma progesterone concentrations were used as a marker [32]. To examine follicular development in cloned and control heifers, ultrasound monitoring of their ovaries was performed. The plasma LH, FSH, estradiol, and progesterone concentrations were measured throughout one estrous cycle. Postovulatory FSH concentrations were also determined. Finally, the breeding performances of clones and control heifers were compared.

Ultrasound Examinations

Ovarian follicular dynamics were determined using a real-time, B-mode, linear-array ultrasound scanner equipped with a 5.0-MHz transrectal transducer (Aloka, Wallingford, CT) [33]. Ultrasound examinations were carried out twice weekly from 9 or 10 mo of age, every other day from 10 to 11 or 12 mo of age, and then daily from 11 or 12 mo of age for one complete estrous cycle starting at estrus (Day 0) after all heifers had reached puberty. The reproductive tract was not palpated before ultrasonography. The same technician carried out all observations. Diagrams depicting the relative location of follicles were made for each ovary, and their growth and regression were monitored individually. The antral cavity of follicles was measured on the ultrasound screen with a ruler calibrated against the built-in scale of the ultrasound unit. This procedure allowed observation of growth and regression of the corpus luteum and individual follicles of >=4 mm during the entire estrous cycle. Although follicles of <4 mm could be detected, their individual development could not be followed accurately; thus, they were not included in this study.

Small, medium, and large follicles were defined as having the following antral diameters, as determined by ultrasound: small (4 to <6 mm), medium (6–10 mm), and large (>10 mm). When a follicle maintained the maximum diameter for more than 1 day, the first day was taken as the day of maximum diameter. The persistence of a dominant follicle within the ovary was defined as the interval of time (days) elapsed between its appearance as a follicle of >=4 mm and its maximum size. The growth rate of each dominant follicle (mm/day) was calculated as the maximum size divided by the number of days between its appearance as a follicle of >=4 mm and its maximum size [33, 34].

Blood Sampling

Blood was collected into heparinized tubes from the jugular vein from 7 to 11 or 12 mo of age to determine when puberty was reached. After reaching puberty, which was defined as the first of 3 consecutive blood samples in which the peripheral plasma progesterone concentrations were >1 ng/ml, heifers were synchronized with 25 mg i.m. of prostaglandin F2{alpha} (PGF2{alpha}; Lutalyse; Pharmacia-Upjohn Co., Kalamazoo, MI) at 11–12 mo of age [32]. Blood sampling for LH, FSH, estradiol, and progesterone was carried out twice daily for one complete estrous cycle after PGF2{alpha} administration. At the end of this full cycle, heifers were bled every 4 h from 12 to 60 h after standing estrus to characterize the secondary FSH surge.

Radioimmunoassay

After blood collection, samples were centrifuged at 2100 rpm for 15 min, and the supernatant plasma was stored at -20° C until assayed. Radioimmunoassay was used to measure plasma concentrations of LH, FSH, estradiol, and progesterone. Procedures and assay sensitivities were as described previously [35, 36].

Breeding

At 14–15 mo of age, cloned and control heifers were bred by AI to determine if pregnancy could be established and maintained. Heifers were synchronized with 25 mg i.m. of PGF2{alpha} before breeding. Semen from the same Holstein sire was used for AI.

Statistical Analysis

Differences between duration of estrous cycle, growth rate and maximum size of dominant follicles, and persistence and mean number of follicles were tested using one-way analysis of variance. The tadpole III statistical system (Elsevier-Biosoft, Cambridge, U.K.) was used for analysis of the follicle data. For hormone data, all analyses were carried out using the SAS proc mixed model with repeated measurements (SAS version 6; SAS Institute, Cary, NC). The main effects were treatment (clone vs. control) and days of the cycle. For testing the heterogeneity of variance of hormones within treatment groups, a two-tailed F test was used [37]. The values presented are mean ± SEM unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
As shown in Figure 1, clones reached puberty later than controls (314.7 ± 9.6 days vs. 272 ± 4.4 days, P < 0.01). The average weight of clones (336.7 ± 13 kg) and controls (302.8 ± 4.5 kg) at puberty was also different (P < 0.01).



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  		FIG. 1. Progesterone concentrations in plasma and age at puberty for a representative clone (•) and paired control ({diamondsuit}) heifer. Puberty was defined as the three consecutive sampling periods where plasma progesterone levels were >1 ng/ml. Arrows indicate puberty

Ultrasound examination of ovarian activity before puberty showed that follicle turnover occurred in all heifers. This was demonstrated by the appearance and disappearance of medium (6–10 mm) follicles. However, in all animals, follicle growth did not exceed 10 mm in diameter during the prepubertal period (data not shown).

All heifers displayed patterns of either 2 or 3 follicular waves (Fig. 2). Two of the 4 clones had 3 waves of follicular development per cycle, whereas the other 2 clones had 2 waves per cycle. In controls, 3 heifers had 3 waves of follicular development per cycle, whereas the fourth heifer had 2 waves per cycle. No differences were found between clones and controls in the duration of estrous cycle (19.5 ± 0.9 days vs. 19.3 ± 0.9 days), maximum size of the dominant follicle, appearance of the follicle on day of the cycle, follicle growth rate, or follicle persistence in the first or ovulatory waves (Table 1).



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  		FIG. 2. Follicular wave pattern of the dominant follicles for a representative (A) control and (B) cloned heifer. An asterisk indicates an ovulated follicle


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  		TABLE 1. Characteristics of dominant follicles during an estrous cycle in cloned (n = 4) and control (n = 4) heifers

More small (4 to <6 mm) follicles were found in clones compared to controls (9.7 ± 1.1 vs. 7.3 ± 0.8, P < 0.0001) (Fig. 3) when averaged over the entire cycle. Also, an interaction existed between treatment and day (P < 0.05). In contrast, clones had fewer medium (6–10 mm) follicles than controls (0.4 ± 0.05 vs. 0.8 ± 0.1, P < 0.05) (Fig. 3) when averaged over the entire cycle. Furthermore, a treatment x day interaction was observed (P < 0.01). Clones had fewer large (>10 mm) follicles compared to controls (0.7 ± 0.08 vs. 0.8 ± 0.09, P < 0.01) (Fig. 3). No differences were found between clones and controls for total number of follicles combined over an entire estrous cycle (data not shown).



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  		FIG. 3. Numbers (mean ± SEM) of small (4 to <6 mm), medium (6–10 mm), and large (>10 mm) antral follicles per day over 1 complete estrous cycle in cloned (•, n = 4) and control ({diamondsuit}, n = 4) heifers

Daily hormone profiles of LH, FSH, estradiol, and progesterone during the estrous cycle in clone and control groups are shown in Figure 4. No differences were found in LH, FSH, estradiol, and progesterone between clones and controls. However, effects of day on estradiol, progesterone, and LH were found in both groups, indicating a rise and fall in these hormone levels over the estrous cycle (P < 0.01). The mean LH surge occurred on Day 18.8 ± 1.1 of the estrous cycle, with a mean peak concentration of 12.4 ± 2.8 ng/ml in the cloned heifers. The LH surge in controls occurred on Day 17.8 ± 1.5 of the estrous cycle, with a mean peak concentration of 15.9 ± 5.5 ng/ml (Fig. 4).



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  		FIG. 4. Daily LH, estradiol, FSH, and progesterone concentrations in plasma (mean ± SEM) of cloned (•, n = 4) and control ({diamondsuit}, n = 4) heifers during 1 complete estrous cycle

The FSH levels after estrus were analyzed in both treatment groups in samples collected every 4 h. No differences were found between treatment groups in these levels (Fig. 5). However, a time effect was found on FSH after estrus (P < 0.05). The secondary FSH surge occurred between 20 and 28 h after estrus in cloned and control animals, with a mean peak concentration of 18.8 ± 3.6 and 15.5 ± 3.0 ng/ml, respectively (Fig. 5).



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  		FIG. 5. FSH profiles after estrus (mean ± SEM) for cloned (•, n = 4) and control ({diamondsuit}, n = 4) heifers

The heterogeneity of variance for daily progesterone, LH, FSH, and estradiol within treatment groups was also examined. Treatment groups had similar variation in terms of LH, FSH, and estradiol, but clones had less variation than controls for progesterone values (P < 0.05).

Three of the four clones and all 4 controls became pregnant within 3 AI services. One clone has experienced problems at breeding. This heifer has also shown poor signs of estrus, although her estradiol, progesterone, LH, and FSH profiles have been normal. A veterinary examination showed no apparent abnormalities in the reproductive tract.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The objectives of this study were to investigate developmental differences in reproduction between heifers from AI and from adult somatic cell cloning and to test if cloned heifers with the same genotype can be used as a model to study genetic influence on reproductive parameters. For reproductive parameters, we examined onset of puberty, follicle growth and hormonal parameters, and the ability of heifers to establish and maintain pregnancy. The number of clones used in this study, although small, is, to our knowledge, among the largest number of live clones reported at the time of their birth from a single donor [11, 13, 16]. All animals were born within 1 mo of each other, which made studies conducted here more feasible.

Dairy heifers reach puberty when their body weight is between 30% and 40% of adult weight, with the average age of puberty falling between 10 and 12 mo of age [38]. The onset of puberty can also be influenced by physical environment, photoperiod, age and breed, heterosis, environmental temperature, and body weight. In this study, clones were observed to reach puberty later compared to the age- and weight-matched controls. Both groups were exposed to a similar environment, and heifers were, as mentioned, age and weight matched and also of the same breed. These results indicate that clones may need to reach a higher critical weight and age for onset of puberty [39]. If the same also occurred in the donor is unclear, because no puberty record is available for her. The late onset of puberty in clones may suggest genetic influence on the age of puberty, which has been shown among different breeds [40]. However, all 4 cloned heifers in this study were derived from the same donor cow. Therefore, further studies are required to investigate the genetic linkage to the onset of puberty.

Follicle emergence, growth, and persistence are indicators of follicle health [41]. In general, a follicle that grows slowly and persists for an extended period of time during the estrous cycle is of poor quality [35, 42]. In this study, the values obtained for each follicle health parameter were within the normal range and were not different between the 2 treatment groups [34]. In general, cattle have either 2 or 3 waves of follicle development per estrous cycle [29–31]. That both 2- and 3-wave heifers were observed in the cloned group indicates that clones have normal follicular recruitment, selection, and dominance.

Follicle populations change with age, with small and medium follicles being observed before puberty, in the absence of large follicles [43]. This is in agreement with the results presented here, which showed no follicle of >10 mm in diameter before puberty. In this study, clones had more small follicles over the estrous cycle than controls. However, controls had more intermediate and large follicles over the entire estrous cycle. The follicle classifications were based on those previously described [33]. However, follicle growth occurs across a continuum rather than being discrete data [34], so differences can be associated with where the scale for follicle size is set. Also, it is logical to expect more large follicles in the control animals, because the majority of these animals had 3 waves per cycle whereas the clone group had the same number of 2- and 3-wave animals. Additionally, total follicle numbers were not different between clones and controls, indicating that clones have normal follicle dynamics.

In this study, no differences were observed between groups in the profiles of all hormones measured during a complete estrous cycle. The hormones analyzed in this study were found to be within the normal ranges for heifers before puberty in both clone and control groups [44–47]. These results indicate that clones have a normal development of the pituitary-gonadal axis and of the feedback control mechanisms.

Whether reproductive hormones are governed by genotype is unknown. Evidence in poultry suggests that concentration of growth hormone is heritable, albeit at a low level [48]. Because clones are genetically identical, it is reasonable to expect that their hormonal patterns are less variable among themselves. The test of homogeneity of variance showed that clones had less variation in progesterone values than controls. This finding supports the expectation that clones, by having a single genotype, should have values that are more similar than those of controls. Absence of homogeneity of variance in the other hormones tested may have been due to the pulsatile nature of their secretory patterns or the small number of animals used. However, these preliminary data suggest that clones may be used as a model to study genetic influence on hormone secretion.

With a number of breeding attempts, all control heifers in this study became pregnant; however, only 3 of the 4 cloned heifers became pregnant. Breeding of the fourth clone is currently being conducted. Overall, these results suggest that clones from an aged adult are capable of conceiving and of maintaining pregnancy.

In this study, the reproductive development in somatic clones derived from adult cells was examined in relation to the attainment of puberty, estrous cyclicity, and pregnancy. To our knowledge, this is the first systematic description of ovarian follicular dynamics and hormone profiles during the estrous cycle in cloned cattle. The main findings of this study were that clones from an aged adult cow have normal development of reproduction and can potentially be used as a valuable model to test genetic influence on reproductive patterns, such as follicular waves and reproductive hormone profiles. However, it is important to note that this study was carried out on a limited number of available animals of a single genotype, and further studies, both with a larger sample size and with clones derived from different cell types and genetic status, are required to answer these questions conclusively.


   ACKNOWLEDGMENTS
 
The authors thank B. Hansen, C. Keator, and Dr. R. Milvae for assistance with hormone assays as well as Dr. T. Hoagland for assistance with statistical analysis, Dr. J. McCracken for reading of the manuscript, Dr. W. Hansel for progesterone antibodies, Dr. G. Niswender for LH antiserum, Dr. L.E. Reichert for purified LH, Dr. D.J. Bolt for FSH antibody and purified FSH, M. Julian for ordering chemicals and preparing animal protocols, and A. Nieminen for care of the animals.


   FOOTNOTES
 
First decision: 18 July 2001.

1 This manuscript is a scientific contribution (no. 2055) of the Storrs Agricultural Experiment Station at the University of Connecticut and was supported by University of Connecticut Research Foundation grants to X.C.T. and X.Y. Back

2 Correspondence: X. Yang, Agricultural Biotechnology Laboratory, 1390 Storrs Road, U 4163, University of Connecticut, Storrs, CT 06269. FAX: 860 486 0534;jyang{at}canr.cag.uconn.edu Back

Accepted: September 11, 2001.

Received: June 27, 2001.


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 DISCUSSION
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