Successful Embryo Transfers from Heifers

Near Puberty: Opportunities for the Future.(1)

Janice Oyarzo,a Ross Tappan,b David Selner,c Mary Bellin,a Roy Axa

a Department of Animal Sciences, University of Arizona,
P. O. Box 210038, Tucson, AZ 85721-0038, U.S.A.
b Arizona Dairy Co., 19135 E. Elliot Road, Higley, AZ 85236, U.S.A.
c P. O. Box 127, Shawano, WI 54166, U.S.A.

Take Home Message

Young Holstein dairy heifers just a few months prior to puberty (peripuberal) can be used as embryo transfer (ET) donors. The hormonal therapy and ET did not adversely affect milk production, reproductive performance, or udder conformational traits. This enables their first sons and daughters to be reaching puberty when the donor heifers acquire their production phenotypes as 2-year-olds. These animals can serve as an additional resource for attaining genetic gain within a dairy herd.


Development of bulls for an artificial insemination (AI) program typically utilizes contract matings of young elite cows to proven sires. Since the advent of ET, a high percentage of prospective sires for sampling are produced in this manner, resulting in full-sibs which may or may not be sampled by an AI company. Approximately half of these contract matings result in heifers. The potential exists that, prior to normal breeding of these heifers at 15 months of age, they could serve as ET donors. Granted, they have no performance data, but their pedigree indices would be the same as their full-sib brothers.

If a peripuberal heifer could serve as an ET donor, an interesting scenario emerges. Let's assume we flush her for an ET at six months of age. When she is 15 months of age, her ET offspring are being born. When she calves at 24 months of age and starts to compile a performance record, she has sons and/or daughters that are approaching nine months old and soon ready for breeding.

In the dairy industry, many have contended that hormonal treatments and flushing embryos from peripuberal heifers might impair the ability of the heifer to breed normally, affect lactational performance, or be detrimental to type classification scores. This paper summarizes experiments performed to evaluate physiological responses of peripuberal heifers subjected to a superovulation regimen. Corresponding control heifers were full-sibs to the ET donor heifers. The full-sib heifers were originally obtained by ET of Holstein cows in the herd. Both groups of heifers were maintained in the same herd so reproductive, lactational, and type traits could be compared.

Materials and Methods

Preliminary Studies

Initial studies were performed to evaluate whether Syncro-Mate-B (SMB) implants were needed in addition to superovulation regimens. The SMB implants were intended to mimic the progestational dominance of a normal luteal phase of the estrus cycle. Questions existed as to whether or not untreated reproductive tracts of the prepubertal heifers could induce the changes in sperm that are needed prior to fertilization of the egg (capacitation).

Prior to AI, frozen sperm in straws were thawed, and heparin (10 µg/ml) was added to induce capacitation of sperm. These preliminary experiments determined that following the SMB implants in both ears, a dose of 3 mg of FSH given twice a day for 3 days stimulated adequate follicular development. Results also confirmed that sperm were capacitated in the reproductive tracts of heifers that received SMB implants prior to FSH injections. Therefore, the addition of heparin was not required to induce capacitation of sperm.

Experimental Design

Ten pairs of full-sib Holstein heifers at Arizona Dairy Co., Higley, AZ, were used for this study. The animals were 10 to 12 mo. old when the SMB implants were initiated. One heifer was assigned to receive the hormonal treatment and her full-sib was not. Heifers assigned for treatment received a SMB implant at day 0, and a second implant was inserted in the other ear on day 7. Both implants were removed on day 16.

The hormonal treatment regimen is presented in Table 1. Ovaries of heifers were superstimulated with twice daily injections of 3 mg FSH (Schering-Plough Animal Health Corp., Kenilworth, NJ) on days 13, 14, and 15. On the morning of day 16, the SMB implants were removed and 10,000 I.U. of hCG were administered (Steris Laboratories, Inc., Phoenix, AZ). All treated heifers were artificially inseminated following signs of estrus in the evenings of days 16-18. On day 22, each animal was palpated per rectum, and was flushed transcervically. Following uterine flushings, each heifer received 25 mg Lutalyse (UpJohn, Kalamazoo, MI) to induce luteolysis of developing corpora lutea.

Table 1. Treatments to superovulate peripuberal heifers.
Day Treatment
0 Implant Syncro-Mate-B (left ear)
7 Implant Syncro-Mate-B (right ear)
13 3 mg FSH a.m. and p.m. (i.m.)
14 3 mg FSH a.m. and p.m. (i.m.)
15 3 mg FSH a.m. and p.m. (i.m.)
16 Remove both implants.
16 hCG (10,000 I.U., i.m.)
16 AI (p.m.)
17 AI (p.m.)
18 AI (p.m.)
22 Transcervical flush
22 Lutalyse (25 mg i.m.)

Control and treated heifers remained in the herd and were bred at 15 months of age. Embryos were scored (1) and healthy embryos were transferred to recipients at the dairy. Recipients had been visually observed in heat one week prior to the transfer.

Reproductive data for age at first calving, and days to conception (days open) were calculated for each heifer. First lactation milk records were obtained from Dairy Herd Improvement Cooperative records and were adjusted for a 305d mature equivalent (M.E.). Udder composite linear scores and other type traits were evaluated for each heifer between 32 and 38 months of age and were adjusted for age and stage of lactation (2). Differences for each trait among control and treated siblings were analyzed using a student t-test.


The outcome of embryo transfers of heifers that were subjected to the superovulation regimen are summarized in Table 2. Nine of the ten heifers responded to hormonal treatment. Of those 9 heifers, a total of 12 embryos were retrieved during the transcervical flushes. Seven of those embryos (58%) were transferred into recipients, and five of seven (71%) embryos transferred resulted in pregnancies. Those pregnancy results are comparable to outcomes utilizing ET in a commercial herd (3, 7).

Table 2. Number of embryos retrieved and transferred.
Heifer I.D. Embryos Retrieved Embryos Transferred* Recipient Pregnancies
1346 0 -- --
4230 1 0 --
3254 0 -- --
4224 0 -- --
4235 2 2 2
4236 2 1 1
4227 2 2 0
3257 4 2 2
2257 0 -- --
2252 1 0 --
Total 12 7 (58%) 5 (71%)

*Excellent and good quality embryos only were transferred to recipients.

The reproductive and lactational traits of heifers subjected to ET and their non-treated full-siblings used as controls are summarized in Table 3. Milk production adjusted for a 305d M.E., age at first calving, or days open in the postpartum interval following their first calving did not differ among treatment groups. Daily peak milk in the first lactation also did not differ significantly (data not shown).

Udder conformation traits for the ET donor and control siblings were determined when heifers were between 32 and 38 months of age. No statistical differences were detected among the treatment groups for each of the udder traits evaluated (Table 4).

Table 3. Reproductive and first lactational performance of Holstein cows that had undergone hormonal treatments and subsequent embryo transfers as peripuberal heifers (ET Donors) compared to non-treated full-sib sisters (Sibling Controls).

Trait Mean (SEM)
ET Donors Sibling Controls
Milk, 305d M.E. (kg) 14,136 (704) 14,318 (600)
Age at first calving 24.4 (.4) 24.2 (.30)
Days open after first 132 (17) 146 (21)

Table 4. Udder conformational traits of 3-year-old Holstein cows that had undergone hormonal treatments and subsequent embryo transfers as peripuberal heifers (ET donors) compared to non-treated full-sib sisters (Sibling Controls).

Udder Trait Mean (S.E.M.)
Sibling Controls ET Donors
Fore udder 24.2 (1.1) 24.9 (1.5)
Rear udder height 25.9 (1.6) 26.0 (1.1)
Rear udder width 26.6 (1.2) 27.4 (1.3)
Susp. Ligament 30.3 (1.3) 27.4 (1.3)
Udder Depth 28.8 (1.6) 30.2 (1.0)
Teat Placement 26.7 (.7) 28.2 (1.6)
Teat Length 22.2 (.9) 24.8 (.9)


Using peripuberal heifers as ET donors did not affect subsequent reproductive or lactational performance or udder conformational traits. Therefore, utility of peripuberal heifers to capitalize on gaining genetic merit from donors at an early age should not be discouraged.

Other researchers have attempted to retrieve embryos from juvenile donors, and problems were encountered because ovulation rates were low or highly variable and abnormalities in oocyte maturation occurred (1, 4, 5, 6). Furthermore, recovered embryos exhibited low developmental potential both in vitro, as well as in vivo. Though the number of recovered embryos per donor was low in this study, the resulting pregnancy rate of the embryo which were transferred was good. Therefore, we should consider peripuberal heifers as a source of superior genetics that can make an impact early in their lives within the herd.

From the outcome in this study, the prospect of utilizing genetic superior heifers as embryo donors should be considered because they would be commencing a lactational record coincident with puberty of sons or daughters. For an AI sampling program, a son could be sampled immediately. Daughters from these types of donors could subsequently serve as peripuberal ET donors in the same manner that their dams did.

Embryo transfers have made an important impact already in this herd at Arizona Dairy Co. Donor cows are selected on the basis of records exceeding 1,800 kg of milk above herdmates for a 305d M.E. record. The ET daughters have produced an average of 1,600 kg more milk than their contemporary herdmates sired by the same AI sires. Therefore, the genetic superiority was conveyed in the dam's side of the pedigree.

The theoretical genetic and economic gains from being able to split embryos, predetermine gender prior to splitting, and also incorporate DNA markers as a package for genetic selection of embryos in the lab has been calculated (8). Those techniques are commercially available today. Therefore, peripuberal heifers become candidates for dairy herds to capture an additional source of superior genetics so they can make an impact early in their lives within the herd.

Theoretically a replacement animal nearing puberty could be available for ET before the dam calved as a 2-year old. With the increasing shift of AI companies to utilizing multiple ovulation and embryo transfer (MOET) herds, this approach is another tool to assist them in the MOET operation. This becomes a useful methodology to shorten the generation interval in those programs to glean statistically valid genetic information with a short turn around time until the next crop of offspring. Additionally, incorporation of DNA marker-assisted selection, determination of gender and splitting of embryos will accelerate the expected genetic change (8).


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2. Holstein Type-Production Sire Summaries. (1994) Holstein-Friesian Association of America.

Hasler, J.F. (1992) Reproductive technology and genetic improvement. J. Dairy Sci. 72:2857-2879.

4. Lussier, J.G. and T.D. Carruthers. (1989) Endocrine and superovulatory responses in heifers pretreated with FSH or bovine follicular fluid. Theriogenology. 31:779-794.

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Revel, F., P. Mermillod, N. Peynot, J.P. Renard, Y. Heyman. (1995) Low developmental capacity of in vitro matured and fertilized oocytes from calves compared with that of cows. J. Reprod. and Fert. 103:115-120

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Vanderboom, R.J., R. Tappan, T.C. McCauley, and R.L. Ax. (1995) High technology embryo transfer: Economic and genetic gains for a commercial dairy. 50th Distillers Feed Res. Conf., San Antonio, TX.

1. This research was supported by 21st Century Genetics, Shawano, WI, and the University of Arizona Agricultural Experiment Station.