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Trop Anim Health Prod (2010) 42:11351141 DOI 10.

1007/s11250-010-9536-z

ORIGINAL RESEARCH

Embryo transfer, a useful technique to be applied in small community farms?


Marco A. Alarcn & Carlos S. Galina & Manuel D. Corro & Marco A. Asprn

Accepted: 12 February 2010 / Published online: 9 April 2010 # Springer Science+Business Media B.V. 2010

Abstract The objective of this study was to determine if the technique of embryo transfer in cattle can be commercially feasible in a region situated in the humid tropics of Mexico. Twenty-six Bos taurus and twenty-six Bos indicus cows were estrous synchronized and superovulated to obtain a total of 80 embryos of both sub-species. Embryos were classified using stereoscopic microscopy based on established criteria. Nine dual-purpose farms situated in the tropics of Mexico were chosen to provide ten recipient cows each to transfer one embryo per cow. The females were transferred using a fixed-time protocol after verifying the presence of a corpus luteum on the seventh day after the end of hormonal treatment. Pregnancy diagnosis was carried out 28 days after embryo transfer by ultrasonography. Estimation of the cost was determined by calculating the expenses for preparation of the donor and embryo recovery, which were US $633 and US $589 for B. taurus and B. indicus,
M. A. Alarcn : C. S. Galina (*) Departamento de Reproduccin, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autnoma de Mxico, Mexico, D.F., Mexico e-mail: cgalina@servidor.unam.mx M. D. Corro Centro de Enseanza, Investigacin y Extensin en Ganadera Tropical, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autnoma de Mxico, Tlapacoyan, Veracruz, Mexico M. A. Asprn Posgrado en Ciencias de la Produccin y de la Salud Animal. FES Cuautitln, Universidad Nacional Autnoma de Mxico, Ajuchitln, Quertaro, Mexico

respectively. The cost of each embryo was determined considering the number of transferable embryos recovered, which was 3.8 on mean. The cost of each conception was calculated taking into account the percentage of pregnant animals (27% on mean), and the cost for preparing donor and recipient cows, for transferring embryo. The overall cost per gestation was US $1,447. Considering a 50:50 ratio of male to female born, the cost for a replacement heifer calf was US $2,894, which surpassed by far the commercial cost of a crossbred ready-to-bred heifer normally used as replacement (approximately US $900). Keywords Embryo Transfer . Cost . Superovulation . Bos taurus . Bos indicus . Donor cows . Recipient cows

Introduction Milk production in tropics has been hampered by inadequate nutrition, poor health management procedures, and insufficient selection of animals for breeding. The heterosis in the herds is so variable that the expression of the production characters such as milk is quite unpredictable. Cunningham (1989) was one of the first to suggest that a steady production of F1 animals among small community farmers would be an efficient tool to improve milk production under the prevailing conditions. Previously, McDowell (1985) demonstrated the benefits of heterosis in dairy cattle in relation to health, milk production, and general fitness of the animals raised in the harsh environmental conditions of the tropics. Madalena (1993) carried out a study in Brazil in 65 cooperative farms involving 500 heifers, varying from 1/4 Holstein to nearly purebreds (31/32). The farms were grouped in two classes: "high" and "low" management.

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The author found that the F1 crossbred surpassed their 3/4 or 5/8 contemporaries whether the program favored the line Bos taurus or Bos indicus or the management was of good or poor quality. In this comprehensive study, the author not simply compared milk production, also parameters such as resistance to tick infestation and reproductive efficiency. Following this and other evidences and with the advent of embryo transfer, several government organizations in the developing world have launched programs to promote the use of embryo transfer (ET) among small community farmers to encourage the diffusion of F1 females. The success of the initiative seems related to the extent on the subsidy and the infrastructure to operate the program, rather than quantifiable results. Obviously, when the program ensues, the disappointment among farmers is evident (Molina, 2003). One possibility to avoid government programs which are not sustainable is to encourage dairy farmers (with a mean herd size of around 40 cows) to either have their embryos produced elsewhere or produce their own. However, the cost of programs such as these, jointly with the selection of recipient cows needs to be assessed in order to estimate their feasibility under tropical conditions, being this the objective of the present study.

Material and methods Embryo production This was undertaken using 52 adult females, half of them of B. taurus (BT) origin (Holstein Friesian) located in a large dairy cooperative situated at Tizayuca, Hidalgo, between parallels 19 50 and 19 55 north latitude and between 90 00 and 99 00 longitude west, at 2,260 m above sea level. The climate is temperate with an annual mean temperature of 14.9C and an annual pluvial precipitation of 60 mm1. The other half were B. indicus (BI) females origin (Brahman) and were located at El Clarn farm, it belongs to the Faculty of Veterinary Medicine, National Autonomous University of Mexico, located in Tlapacoyan, Veracruz. This unit is situated in the Central part of Veracruz State at 19 58 latitude north and 97 13 longitude west at an altitude of 151 m above sea level. The climate in this area is hot and humid with a mean annual temperature of 23.4C and a mean of 1, 840 mm of rain distributed in about 10 months of the

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www.hidalguia.com.mx/tizayuca/ubicacion.htm www.e-local.gob.mx/work/templates/enciclo/veracruz/municipios/ 30183a.htm

year2. These locations were chosen as a model for farmers to purchase embryos produced in other locations, to ascertain the feasibility of this system of production, rather than embarking the farmers themselves in a long-term project to produce embryos in their own small units. Animals selected were at least 90 days postpartum and had a mean of five calvings without gross pathological conditions. The animals were cycling at the onset of the superovulatory treatment and had a body condition score of 2.53.5 on a scale of 15 (Pullan, 1978). Estrus of all cows was synchronized with a double intramuscular injection, 7 days apart, of 100 g of GnRH (Ovalyse, Pfizer Mexico), followed 7 days later by 25 mg of prostaglandin F2 (PGF2; Lutalyse, Pfizer Mexico) given intramuscularly. Twenty four hours after PGF2, the animals were observed continuously for estrous signs for 72 h. Ten days after the onset of estrus, a rectal examination was done to ascertain the presence of a corpus luteum (CL). The following day, animals were subjected to a regime of superovulation using decreasing intramuscular doses of follicle-stimulating hormone (FSH; Folltropin-V Bioniche Animal Health, Canada) every 12 h (AM-PM) during 4 days. At the third day, two injections (AM-PM) of 25 mg of PGF2 were given to assure the lysis of the CL. Animals were inseminated at 0, 12, and 24 h after the onset of estrus. The total dose of FSH-P for B. indicus cows was 280 mg and 400 mg for B. taurus. The reason for this disparity is that B. indicus cows are more susceptible to the gonadotrophin treatment as there is a pronounced effect on the follicular milieu and more immature follicles are recruited following treatment (Baruselli et al. 2006). Cows were inseminated with semen from a proven bull of the other species, i.e., cows BT were artificially inseminated (AI) with BI and vice versa, in order to procure F1 embryos. Seven days after AI, a rectal examination was done to assess the number of corpora lutea formed and thus establish the superovulatory response. Cows with more than two CLs were flushed with ViGro complete flush solution (Bioniche Animal Health, Canada) and the embryos were collected in a filter (Em-con, Bioniche Agtech, USA). The filter was taken to the laboratory and embryos were searched for in a Petri dish using stereoscopic microscope (Stereo Zoom6 Karl Zeiss, Switzerland), in order to determine the stage of development and quality according to the criteria proposed by the International Embryo Transfer Society (Robertson and Nelson, 1998). After evaluation, transferable freezable embryos were placed individually in 0.25-ml straws IMV, France using ViGro ethylene glycol 1.5 M (Animal Health, Canada) as cryoprotectant. Embryos were frozen using an automatic freezer CL5500 (Control Freeze, Australia) and stored at 196C in a tank with liquid nitrogen until they were transferred to recipient cows.

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Criteria for selecting the producers Nine commercial dual-purpose farms were selected for the study. All the enterprises were part of a cooperative established in the region of Macuspana, Tabasco, located at 17 45 latitude north and 9232 longitude west and at an altitude of 32 m above sea level. The climate is hot and humid with an annual temperature mean of 23.6C. Pluvial precipitation is about 3 186 mm3. Selection of farms was based on location, facility of access from the main road, and interviews with the farmers to ascertain their commitment to use the ET technique. This selection procedure was as careful as possible in order to minimize the desertion of the participants and was based on previous experiences (Montiel et al. 2006). Selection and preparation of the recipient cows The main criteria for selecting recipient cows were animals not pregnant and with more than 90 days postpartum, fewer than five calvings, and without any gross pathological features in genital tract. Only animals with a CL detected by rectal palpation and a body condition score of 2.53.5 in the scale 15 proposed by Pullan (1978) were selected. Once elected, the animals were separated from the rest of the herd to guarantee they were away from the bulls. The animals were estrous synchronized 20 days later and an ultrasound examination was performed to reassure that the animals were not pregnant. Embryos were transferred between July and August, months considered optimal for fertility. A fixed-time bovine embryo transfer protocol was used and consisted of an injection of 100 g of a GnRH analog (Ovalyse, Pfizer, Mexico) and the insertion of a progesterone-releasing device (CIDR, Pfizer, Mexico), which remained for 7 days. After implant withdrawal, an intramuscular injection of 25 mg of prostaglandin (Lutalyse, Pfizer, Mexico) was given. Forty eight hours after this event, a second injection of 100 g of GnRH analog followed. ET was performed 7 days after the last GnRH analog application.

Embryo production From the 26 B. taurus donor cows, 81 transferable and 25 non-transferable embryos were obtained; four cows were not flushed because they have a superovulatory response. As for the 22 cows remaining, another four (18%) did not produce any embryos, while three of the 18 cows (17%) producing embryos gave exclusively non transferable, whereas four of 18 (22%) produced exclusively transferable embryos. Embryos of both types were produced by 11 of 18 donor cows (61%). The mean production of embryos per donor cow was 3.1, whereas the mean calculated only from the cows where embryos were collected was 4.5 (Fig. 1). In the case of B. indicus, out of 26 cows, a total of 80 transferable and 48 non-transferable embryos were obtained. From this group, eight cows did not respond to the superovulatory treatment. As for the 18 responding to treatment, three (17%) did not produced any embryos, the remaining four of 15 (27%) only gave transferable embryos, and one of 15 (7%) solely non-transferable. Embryos of both types were produced by ten of 15 (67%) donor cows. The mean production of embryos per donor cow was 3.1 and the mean calculated only from the cows where embryos were collected was 5.3 (Fig. 2). Cost related to donor cows Table 1 shows the costs related to the production of embryos. As can be observed, the investment related to drugs utilized for estrous synchronization, the material used to recover the embryos and the labor accounts for most of the costs. Other investments are extra feed and preventive medicine measures. Production costs for F1 embryos For the donors of B. taurus origin, a total of 81 transferable F1 B. taurus x B. indicus were obtained from the 26 donor cows. The cost for embryo production was calculated at US $203. Similar results were obtained in the case of B. indicus donor cows with 80 transferable embryos at a cost of US $191. Fixed-time embryo transfer in recipient cows It was necessary to synchronize 165 recipient cows in the nine farms. This program was divided in two stages. In the first stage, the ovulatory response was 57%, i.e., from the 90 cows treated 51 had a palpable CL. (Fig. 3). Assuming a similar response, another 75 potential recipients were synchronized and 39 responded (Fig. 4).

Results Superovulatory response The B. taurus donors had a total of 195 CLs (mean=7.5) and 24 follicles, whereas the B. indicus donors had 157 CLs (mean=6) and 27 follicles.
www.e-local.gob.mx/work/templates/enciclo/tabasco/mpios/27012a. htm
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1138 Fig. 1 Embryo production of Bos taurus cows

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Costs related to the preparation of the recipient cows and embryo transfer Most of the cost was attributed to the feeding of the animals (US $64), hormones and materials utilized in the estrous synchronization procedure (US $19/animal). Hence, the cost for preparing each recipient cow was US $91. The cost related to the actual transfer of the embryo was calculated in US $22 based on materials and labor for the transfer. Total cost of the operation After the initial response following estrous synchronization, the number of animals had to be increased; hence, the cost for animals not responding was US $6,825. Therefore, the cost for preparation and embryo transfer to the recipient cows increased to US $10,170. If the two costs are added, the total investment needed to transfer 90 embryos was US $16,995.
Fig. 2 Embryo production of Bos indicus cows

The mean cost to produce each embryo either from B. taurus or B. indicus was US $197; hence, the price for producing 90 embryos was US $17,730. If to this cost, is added the investment for the preparation of the recipients, which was US $16,995; the total investment for the program was US $34,725. Cost for each pregnancy Considering the number of animals pregnant with a fertility index of 27% (24 gestations out of 90 cows) thus the total cost per gestation using ET was US $1,447.

Discussion According to the superovulatory response, the variability was quite marked in relation to the FSH treatment. However, in the case of B. indicus, 27% of the cows

Trop Anim Health Prod (2010) 42:11351141 Table 1 Cost related to donor cows Synchronization and superovulation material GnRH PgF2 Needles 181 1/2 Syringes 5ml Syringes 10ml FSH-p Estrous alert Semen Sub total Flushing material Tygon hose Em con filter Foley catheter Complete flush Syringes 10ml HSW Syringes 20ml HSW Embryo maintenance medium Ethylene glycol Straw Albumin Petri dish Pipette ivf Xylocaine 2% Sub total Labor Preventive medicine Feed TOTAL Unitarian cost per cow

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$6,00 $6,80 $2,10 $1,20 $1,60 $105,00 $1,92 $30,00 $154,62 $9,21 $25,39 $5,39 $25,25 $1,92 $1,74 $35,00 $23,48 $1,65 $13,57 $16,52 $7,13 $1,49 $167,74 $200,00 $7,00 Bos taurus $105,00* Bos indicus $60,50* Bos taurus $634,36 Bos indicus $589,86

produced basically 50% of the freezable embryos. Similarly, 67% of the cows produced embryos either viable or not viable for freezing. It is interesting that in spite of difficulties in cows of the B. indicus type not responding to treatment, the number of embryos selected for freezing were similar. Results on embryo production agree with a previous report of Mapletoft et al. 2002. These authors have shown a high variability in the response as 24% of their superovulated cows did not produced viable embryos. Moreover, 64% of the cows produced an inadequate number of embryos and merely 30% produced 70% of the total number of good quality embryos. Our results agree with a previous report (Lerner et al. 1986) as in the B. taurus group, we were able to secure 22% of the donor cows producing 23% of the viable embryos for freezing and 61% of the cows delivered embryos of good freezing quality whereas the remaining were of poor quality and discharged. Thus, based in the results for B. taurus and B. indicus donor cows, one can safely assume that between 20% and 30% of them will not produce embryos at all, similar to what has been reported earlier (Baruselli et al. 2006). Production of embryos in B. taurus females seems to be an established technique and a mean of 78 transferable embryos are expected as response (Chebel et al. 2008). This norm has been established for cattle raised under temperate conditions and with a better level of nutrition. The response in B. indicus is more unpredictable (Barros and Nogueira 2001). In the present study, results were rather similar when B. taurus or B. indicus donors were used. It is difficult to explain the incongruence of our results with data available for B. taurus in the literature. One possible explanation of this is the quality of the donor cows selected. Most of these cows were chosen from the production line in the enterprise without a strict criterion for selection, just animals in good health and cycling (the

60 days of treatment

Fig. 3 First program of synchronized recipients and embryo transfer

1140 Fig. 4 Second program of synchronized recipients and embryo transfer

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type of animal a small community farmer might have access to). There are results in B. taurus cattle, especially in animals raised in the tropical highlands (Chebel et al. 2008) where the mean number of embryos was not more than four per flushing. In the case of B. indicus donors, there is abundant body of evidence of differences in the follicular dynamics when is compared to B. taurus (B et al. 2003; Barati et al. 2006). These authors have demonstrated that B. indicus cows are more susceptible to the gonadotrophin treatment (Baruselli et al. 2006). This effect is more evident when the dose of exogenous FSH is increased and more poor quality embryos are observed following flushing (Greve et al. 1995). This susceptibility of B. indicus cattle unable to respond to the superovulatory treatment at all was evident in the present study as 30% of the cows selected did not respond to FSH stimulation. With reference to the response obtained in the recipient cows following estrous synchronization, Montiel et al. (2006) found that of 215 recipient females prepared for transfer only 69% were detected in estrus. However, after rectal examination 7 days later, only 45% of the 215 females were suitable for transfer. This result is similar to our findings as only 51% of the females had a CL when selected for transfer. The percentage of animals pregnant was 27% concurring with previous data showing a range of 2540% pregnancy rate when using frozen embryos (Montiel et al. 2006). This percentage can be increased about 10% if non-frozen embryos are used (Cutini et al. 2000; Lpez et al. 1995; Hasler et al. 1987). These modest results are not in accordance with data in B. taurus animals raised under temperate conditions where a 60% fertility index can be expected (Spell et al. 2001). Obviously, the environment

plays a major role in the success of the technique and more information is in demand to remedy this shortcoming as there are reports from cows raised under tropical conditions where fertility can be comparable to B. taurus (Baruselli et al. 2006) The production of embryos was less expensive in B. indicus cattle; the cost of feeding reduces the investment needed. An important shortcoming is the selection of the donors. One alternative that might reduce the costs is to purchase the embryos from commercial companies; however, the actual production of F1 females is rather limited. Another constraint in a program such as this is the great variability obtained in the superovulatory response which increases the costs considerably. Producing embryos using in vitro fertilization can be an option. Another major factor to be considered is the failure to obtain a more homogeneous response of cattle selected for recipients. This variability is even more dramatic when cattle in small community farms are used. Daz et al. (2002) found that as much as 30% of the animals failed to ovulate following synchronization. Bolivar and Maldonado (2008) works in Colombia estimates costs for transferable embryo about US $170, comparing three superovulatory protocols and 100% fertility. When the fertility was estimated at 50%, costs soared to US $340. However, the results from this contribution only considered costs assuming two hypothetical fertility indices but not considering the losses of embryos due to freezing, nor the finding that as much as 30% of the cows did not produce embryos. Improvements in fertility, number of embryos suitable for freezing, and an adequate response in the recipient animals are needed before recommending a program such as this to farmers with a low livestock inventory.

Trop Anim Health Prod (2010) 42:11351141 Acknowledgments The authors are grateful to the program PAPIIT IN 200107 (Mexico) for financial support.

1141 Hasler, J.F., McCauley, A.D. and Lathrop, P.N.F., 1987. Effect of donor-embryo-recipient interactions on pregnancy rate in a large-scale bovine embryo transfer program, Theriogenology, 37,139168. Lerner, S.P., Thayne, W.V., Baker, R.D., Henschen, T., Meredith, S., Inskeep, E.K., Dailey, R.A., Lewis, P.E. and Butcher, R.L., 1986. Age, dose of FSH and other factors affecting superovulation in Holstein cows, Journal of Animal Science, 63,176183. Lpez, J.R., Alfaro, M.E. and Holy, L., 1995. Respuesta superovulatoria en ganado Bos indicus y Bos taurus bajo condiciones tropicales, y efecto del desarrollo y calidad del embrin sobre el porcentaje de gestacin, Revista Veterinaria Mxico, 26,189193. Madalena, F.E., 1993. A simple scheme to utilize heterosis in tropical dairy cattle, World Animal Review, 1,1725. Mapletoft, R.J., Steward, K.B. and Adams, G.P., 2002. Recent advances in the superovulation in cattle, Reproduction, Nutrition, Development, 42,601611. McDowell, R.E., 1985. Crossbreeding in tropical areas with emphasis on milk, health and fitness, Journal of Dairy Science, 68,24182435. Molina, J.I., 2003. Aceptacin de la tcnica de transferencia de embriones bovinos en productores adscritos al programa para el mejoramiento gentico de la ganadera del estado de Chiapas, (Tesis de Maestra, Facultad de Medicina Veterinaria y Zootecnia. Universidad Nacional Autnoma de Mxico), Mxico, D. F. Montiel, F., Galina, C.S., Rubio, I. and Corro, M., 2006. Factors affecting pregnancy rate of embryo transfer in Bos indicus and Bos taurus/Bos indicus cows, Journal of Applied Animal Research, 29,149152. Pullan, N.B., 1978. Condition scoring of white fulani cattle, Tropical Animal Health and Production, 10,110120. Robertson, I. and Nelson, R.E., 1998. Chapter 9, Certification and identification of the embryo In Manual of the International Embryo Transfer Society. Stringfellow DA, Seidel SM, Third edition, by International Embryo Transfer Society, USA, 103116. Spell, A.R., Beal, W.E., Corah, L.R. and Lamb, G.C., 2001. Evaluating recipient and embryo factors that affect pregnancy rates of embryo transfer in beef cattle, Theriogenology, 56,287297.

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