Philippine Journal of Science 137 (2): 99-104, December 2008 ISSN 0031 - 7683
Twin Calf Production in Water Buffaloes Following Non-Surgical Transfer of in vitro-produced-vitrified Embryos
Danilda Hufana-Duran*1,3,4, Prudencio B. Pedro1,2, Apolinario L. Salazar Jr.1 Hernando V. Venturina1, Peregrino G. Duran1, Yoshiyuki Takahashi3, Yukio Kanai4, and Libertado C. Cruz1
1Philippine Carabao Center, Science City of Muñoz, Nueva Ecija, 3120 Philippines 2Benguet State University, La Trinidad, Benguet, Philippines 3Laboratory of Theriogenology, Department of Veterinary and Clinical Sciences, Hokkaido University, Sapporo, 060-0818 Japan 4Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, 305-8572 Japan
Twinning in buffalo cows is only about 0.01%. Effecting twin pregnancies by transferring more than one embryo of different developmental stages was tested. Cumulus-oocyte complexes collected from slaughterhouse-derived ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured, on Day 6-7, developed blastocysts stage embryos were removed, frozen by vitrification, and stored in liquid nitrogen. After 2 to 5 months, 130 embryos were warmed; 72 were cultured in vitro to test for viability while 58 were non-surgically transferred in two's (n= 20 recipients) or three's (n = 6 recipients) to 26 buffalo cows. No significant difference was observed on post-warming survival rate of the embryos at different developmental stages with 85-88% hatched within 72 h of in vitro culture. Embryo transfer resulted to birth of one twin (3.8%) and five single births (19.2%) presenting 23.1% (6/26) calving rate. This demonstrates the possibility of enhancing twinning in buffaloes by embryo transfer techniques.
Key Words: Twinning, embryo transfer, in vitro embryo production, oocytes, in vitro fertilization, vitrification
INTRODUCTION Indonesian Swamp buffaloes is 0.0002%, and in Malaysia is 0.0003%. Chaudhary (1989) reported <0.3% twinning Twinning can be used to improve profitability in the beef in Nili Ravi buffaloes, while Kandasamy et al. (1989) industry (Guerra-Martinez et al. 1990; Herd et al. 1993). reported 0.062% in Murrah buffaloes. Natural twinning occurs in cattle with an up to 4% (Cady & Van Vleck 1978) and increase to 9% (Kinsel et al. Twinning in water buffaloes could be of economic 1998) following intensive management. Rose & Wilton impact especially when high genetic buffaloes are to be (1991) reported a considerable increase in return out of produced. In cattle, some methods have been reported milk production. to increase the proportion of twins; genetic selection (Echternkamp & Gregory 1999), hormonal treatments In water buffalo, 0.01% pregnancies produce twins such as a low dose of pregnant mare serum gonadotropin (Fisher & Adenil 1956). Fischer (1964) contended that (Laster et al. 1973; Yang et al. 1992), follicle stimulating twinning rate in Egyptian water buffaloes is 0.2%, in hormone (Wildt et al. 1975; Davis & Bishop 1992) or *Corresponding author: [email protected] human menopausal gonadotropin (Suzuki et al. 1993)
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injection, immunization against inhibin (Morris et al. In vitro fertilization 1993), and supplemental embryo transfer (Holy et al. The colloidal suspensions of silica particles (45%, 65%, 1981). However, in each of these treatments, neither and 95%) developed to form a discontinuous density the twinning rate was consistent, nor was it greater than gradients on a sterile 15 mL centrifuge tube were used for the rates obtained using embryo transfer (ET) technique the separation of sperm cells (Hufana-Duran et al. 2005b). (Rowson et al. 1971; Holy et al. 1981; Sreenan & Diskin Frozen semen thawed at 37º C for 15 sec was layered gently 1985; 1989; Suzuki et al. 1994). Gordon (1994) reported at the top of the discontinuous layers, and motile sperm cells that twinning by the use of in vitro-produced (IVP) were separated through equilibrium by centrifugation at embryos is beneficial for increasing productivity and 800 x g for 10 min. The supernatant was discarded and the profitability in the production of beef calves because IVP sperm pellet was transferred into a 15 mL centrifuge tube embryos cost less than in vivo-derived embryos. Misra and layered with a pre-incubated Brackett & Oliphant sperm et al. (1999) reported for the first time the production of washing medium (Brackett & Oliphant 1975) containing heterosexual twins out of the transfer of in vivo produced 1.25 mM sodium pyruvate and 13.9 mM glucose (referred embryos collected from live donor. as mBO medium) and centrifuged at 800 x g for 5 min. The supernatant was discarded. Sperm concentration was We have earlier reported the successful vitrification determined by a Neubauer’s counting chamber and the final of in vitro-derived water buffalo embryos that resulted to sperm concentration (1 x 106 sperm cells/mL) was made birth of healthy normal calves after non-surgical embryo by adding (1:1) pre-incubated mBO medium containing transfer (Hufana-Duran et al. 2004; 2005a; 2007). This 10 mg/mL bovine serum albumin (BSA, Fraction V, Wako paper reports the birth of the first set of twins by embryo Pure Chemical Inc., Osaka, Japan), 10 mM caffeine and 4 transfer using in vitro produced-vitrified embryos at IU/mL heparin. In vitro matured oocytes were co-cultured different developmental stages. with the sperm cells for 6-8 h in 5% CO2 incubator at 39º C for in vitro fertilization.
MATERIALS AND METHODS In vitro culture The in vitro culture (IVC) was carried out as described by In vitro production of embryos, vitrification and embryo Tsuzuki et al. (1998). After 6-8 h of co-culture, oocytes transfer were carried out as described earlier (Hufana- were incubated for 0 to ~72 h in 1% PL medium (TCM Duran et al. 2004; 2005a) with few modifications. In vitro 199 with 1% FBS, 0.4 mM sodium pyruvate (P) and 5 embryo production and cryopreservation were performed mM sodium lactate (L)). On the 72 h of in vitro culture, in a satellite embryo biotechnology laboratory of the the culture medium was replaced with 15% PL medium Philippine Carabao Center (PCC) in India using salvaged (TCM 199 containing 15% FBS and same concentration of genetic materials from slaughtered buffaloes. Embryo sodium lactate and sodium pyruvate) and incubated in this transfers were carried out to Bulgarian Murrah buffaloes medium until 130 h. After ~130 h, the culture medium was at the National Gene Pool of the Philippine Carabao Center replaced with 15% PLG medium (15% PL containing 1mg/ in the Philippines. mL glucose (G)). The blastocysts (early, mid & expanded) developed on the 6-7 day of IVC were vitrified. In vitro maturation of oocytes Cumulus-oocyte complexes (COCs) were collected from Cryopreservation ovaries taken from slaughter house. COCs with compact The embryos were cryopreserved by vitrification as cumulus cells and granulated cytoplasm were selected described earlier (Hufana-Duran et al. 2004) at room and cultured for in vitro maturation (IVM) in TCM 199 temperature (25° C). French straws (0.25 mL) were used (Earle’s salts with 25 mM HEPES, Gibco-BRL, Life for loading the embryos. Selected embryos were washed Technologies, Inc., Grand Island, NY, USA) containing with PB1 medium (Dulbecco’s phosphate buffered 10% fetal bovine serum (FBS; Gibco) and antibiotics saline containing 5.56 mM glucose, 0.33 mM pyruvate, (100 units penicillin/mL and 100 µg streptomycin/mL). 100 units penicillin/mL and 3 mg BSA fatty acid free/ IVM medium droplets were prepared in 100 µL droplets mL (Whittingham 1971)). Then, the embryos were pre- in Nunc tissue culture dishes (35mm x 10 mm, Nunclon equilibrated in 10% (vol/vol) ethylene glycol in PB1 153066, Inter-med., Roskilde, Denmark) covered with medium for 5 min, transferred to EFS40 (ethylene glycol, mineral oil (Embryo tested, Sigma) and equilibrated for 40% vol/vol; ficoll, 18% wt/vol; sucrose, 0.3 M) and at least 3 h in a 5% CO incubator (Forma Scientific 3111 2 loaded on the straw within 30 sec and rapidly plunged in Series) at 39° C. Oocytes were matured in vitro in a 5% liquid nitrogen. Straws were kept in liquid nitrogen for 2 CO incubator for 22-24 h at 39° C. 2 to 5 mo before embryo transfer.
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Embryo warming and viability test Pregnancy diagnosis and calving rate One hundred thirty embryos were warmed. Of Recipient animals were subjected to palpation per rectum these, 72 (55.4%) were used as control to check the at 30 and 45 days after ET to check the persistency of viability and survivability in vitro and 58 (44.6%) the corpus luteum present during the transfer of the were transferred to 26 recipients over a period of two embryos. Thereafter, pregnancy was confirmed by months. The 20 recipients were given two embryos another palpation per rectum at least 180 days after the and six were given three embryos. transfer. The number of calves born, birth weight, and withers height were recorded. Embryos were directly warmed in 0.5 M sucrose at 25 °C for 5 min and washed twice with PB1 Results were analyzed using Chi-square and Fisher’s medium followed by once in a pre-warmed culture exact test for any significant difference. T-test was used in medium. Embryos were cultured for 72 h in culture analyzing the significant differences on gestation length
medium on cumulus cell monolayers in 5% CO2 birth weights and heights between twin and single births incubator at 39 °C. Observations of the embryos assuming equal variances. were done daily and the number of embryos with intact zona pellucida during warming, re-expansion of blastocoel within 48 h and hatching rate within 72 h of culture were recorded. RESULTS AND DISCUSSION For embryo transfer, warmed embryos at Results on viability test of the in vitro produced embryos different developmental stages with re-expanded at various developmental stages (Table 1) showed 94- blastocoels were washed in PB1 medium and (2 or 3 100% intact zonae pellucidae after warming, 85-94% had embryos per straw) were loaded in 0.25 mL French blastocoel re-expansion rate after 48 h and 85-88% hatched straw for unilateral transfer. out from their zonae pellucidae within 72 h of in vitro culture. No significant differences were observed on the viability as to their embryonic stage of development. Recipient selection and embryo transfer Recipient animals were riverine buffaloes with Embryos transferred to recipient animals (Table 2) regular estrous cycle and were kept in complete have resulted in 3.8% (1/26) twinning rate and 19.2% confinement system of management for purposes (5/26) single births. The overall calving rate was 23.1% of embryo transfer. Transfer of embryos was done (6/26), which was higher than the previous reports (10.9%, in months of Feb and Mar when temperature varies Hufana-Duran et al. 2004; 13.9%, Hufana-Duran et al. from 30 to 35˚ C. 2005a). Improvement on the success rate was accounted to the improvement of the culture system (use of sequential Embryo transfer was performed on day 7th after media system) and sperm treatment, the use of recipients detection of spontaneous estrus (Day 0=onset of undergoing spontaneous estrus, and the transfer of observed estrus). embryos at different embryonic stages that suits the Before the transfer of the embryo(s), the presence synchrony of the in vivo environment. of functional corpus luteum in the ovaries was checked The twin births occurred when three embryos by palpation per rectum and recipient animals were were transferred while single births occurred when treated with 2% lidocaine hydrochloride (i.m., Ethical two embryos were transferred. The birth of the twins Pharmaceutical Co. Pvt. Ltd., India). could not be directly accounted as a consequence of
Table 1. In vitro survival rate of vitrified in vitro derived water buffalo embryos after warming With re-expanded blastocoel With intact Zona Hatched after 72 h Embryo Stages n after 48 h Pellucida (%) of in vitro culture (%) of in vitro culture (%) Early Blastocyst 20 20 (100%) 17 (85%) 17 (85%)
Mid Blastocyst 18 18 (100%) 17 (94%) 16 (88%)
Exp. Blastocyst 34 32 (94%) 32 (94%) 30 (88%) Total 72 70 (97.2%) 66 (91.7) 63 (87.5) No significant difference was observed among figures in the same column
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Table 2. Twin calf production out of non-surgical transfer of vitrified water buffalo embryos produced in vitro Transfer of 2 Parameter Transfer of 3 embryos Total (%) embryos Number of embryos transferred 40 18 58 Number of recipients 20 6 26 Calving rate Singlet [% per recipient] 5 [25.0] 0 5 [19.2] Twins [% per recipient] 0 1 [16.7] 1[ 3.8] Number of calves born 5 (12.5) [25.0] 2 (11.1) [33.3] 7 (12.1) [26.9] (% per embryo) [% per recipient] Twinning rate [% per recipient] 0 1 [16.7] 1 [3.8] No significant difference was observed transferring three embryos to the recipients because of birth of a single calf of 310±4.3 days (range: 306 the limitation of the number of trials for comparison. to 316 days). Gestation length was significantly However, the transfer of three embryos at three (P<0.05) shorter in twin birth than in single birth. This different developmental stages have higher possibility observation was the same in cattle (Diskin & Sreenan of implantation than only two embryos as it increases 1985; Lambert et al.1996) suggesting an economic the chance of overcoming embryo-recipient asynchrony impact in livestock production. (Misra & Joshi 1991) and effecting higher incidence of twinning (Gordon & Boland 1978). The silent estrus The twin calves were both male. The first calf in water buffaloes made it difficult for perfect timing weighed 26 kg and came out at around 5 A.M. The of ET resulting in low implantation rate. The transfer second calf weighed 28.4 kg born at around 7 A.M. of three embryos at different developmental stages The interval was ~2 h. The birth weight of the twins helped overcome the high incidence of asynchrony was significantly (P<0.01) lighter than the average birth and enhanced twinning. weight of single calf (38.4±3.29 kg). No case of large calf syndrome was observed. Though the present twinning rate (3.8%) in water buffaloes is lower than the cattle, it is still important for The above results indicated that though twinning is higher economic value of buffaloes. very rare in water buffalo (0.01%), it could be enhanced This is the first report of twin derived from vitrified and raised to 3.8% using the present techniques. Other in vitro-produced embryos. In the present study, three factors affecting conception rate and transfer strategies embryos were deposited in the uterine horn epsilateral need to be studied to improve the present efficiency to the ovary bearing a corpus luteum and out of that two rate. Nonetheless, the above results imply that embryo embryos were assumed to survive to produce twin or transfer is a promising tool in enhancing twinning one of the embryos got splitted and produce the twin of incidence in water buffaloes and that vitrified in same sex. vitro-derived embryos can be used for the transfer and enhance twinning with modest overall calving rate of The twin calves were born from their surrogate 23.1%. Thus, farmer’s economic resources and meat mother 301 days from the day of embryo transfer and protein sources may subsequently improve and (Table 3). This was ±9 days earlier than the average supplied, respectively.
Table 3. Average gestation length, birth weight, and birth height of twin and single calves born from vitrified in vitro derived water buffalo embryos Gestation length, Birth weight, Birth height, Births n Sex days kg cm Twin 1 301a 26.7a 67.4±1.41a Both Male
Single 5 310±4.3b 38.4±3.3b 73.8±2.9b 2 F, 3 M Values in the same column with superscripts a and b are significantly different at P<0.05, while column with superscripts c and d are significantly different at P<0.01.
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CONCLUSION ECHTERNKAMP SE, GREGORY KE. 1999. Effects of twinning on gestation length, retained placenta, and Base on the results obtained in this study, it was dystocia. J Animal Sci 77:39–47. concluded that production of twin calves in water buffaloes could be enhanced by embryo transfer FISCHER H. 1964. Water buffalo twins. The MAHA techniques using embryos produced in a complete Magazine 21:63. in vitro production system. The implantation of two FISCHER H, ADENIL C. 1956. A new case buffalo twins or three embryos into the reproductive tract of the on the island of Sumba. Hemera Zoa 63:446-449. recipient animal could enable the survival of these embryos, thereby effecting twinning that is very GORDON I. 1994. Twinning by embryo transfer. In: rare in this species considering its single ovulation Laboratory Production of Cattle Embryos. Oxon: CAB characteristics. The availability of in vitro-derived International. p. 377–386. embryos and the advent of vitrification techniques GORDON I, BOLAND MP. 1978. Towards cattle twins that allows long-term storage and transport of by egg transfer. World Review in Animal Production these embryos ensue high opportunity in enhancing 14: 9-23. twinning in this livestock species which could hasten the propagation of genetically superior animals thus GUERRA-MARTINEZ P, DICKERSON GE, ANDERSON improve livestock production. GB, GREEN RD. 1990. Embryo-transfer twinning and performance efficiency in beef production. J Animal Sci 68: 4039–4050. HERD RM, BOOTLE BW, PARFETT DC. 1993. An ACKNOWLEDGEMENT economic evaluation of traditional, twinning and This work was supported by grants from the Philippine sex-controlled systems of beef production in southern Council for Agriculture and Natural Resources Research Australia. Aus J Agric Res 44:1541–1556. and Development-Department of Science and Technology HOLY L, SIRICEK A, VANATKA F, VRTEL M, (PCARRD-DOST), the Japanese Society for the Promotion FERNANDEZ V. 1981. Artificial induction of twinning of Science-RONPAKU Dissertation Program, and in in cattle by means of supplemental embryo transfer. cooperation with Frigorifico Allana Limited, India. Theriogenology 16:483-488. Appreciation goes to Mr. Rogelio D. Hufana and Mr. HUFANA-DURAN D, PEDRO PB, VENTURINA Roy de Vera for assistance on the transfer of embryos and HV, HUFANA RD, SALAZAR AL, DURAN PG, to Ms. Amy Bansan, Ms. Jennifer B. Fernandez, and Mr. CRUZ LC. 2004. Post-warming hatching and birth Aaron dela Cruz for cooperation and support. of live calves following transfer of in vitro-derived vitrified water buffalo (Bubalus bubalis) embryos. Theriogenology 61:1429-1439. REFERENCES HUFANA-DURAN D, PEDRO PB, VENTURINA HV, DURAN PG, CRUZ LC. 2005a. Full-term BRACKETT BG, OLIPHANT G. 1975. Capacitation of development of in vitro produced-vitrified water rabbit spermatozoa in vitro. Biol Reprod 12:260-274. buffalo (Bubalus bubalis Linn) embryos. Philipp J CADY RA, VAN VLECK LD. 1978. Factors affecting Sci 134:89-94. twinning and effects of twinning in Holstein dairy HUFANA-DURAN D, DURAN PG, KANAI Y, cattle. J Animal Sc 46:950–956. TAKAHASHI T, CRUZ LC. 2005b. Effect of CHAUDHARY MA. 1989. Incidence of twinning in Nili density-gradient sperm separation technique on in Ravi buffaloes. Buffalo Bull 8:87-90. vitro fertilization potential of water buffalo semen with low post-thaw motilities. Philipp Agric Sci DAVIS ME, BISHOP MD. 1992. Induction of multiple 88: 257-267. births in beef cows with FSH: calving rate and subsequent performance. Livestock Production Sci HUFANA-DURAN D, PEDRO PB, VENTURINA HV, 32:41–62. DURAN PG, CRUZ LC. 2007. Full term delivery of river buffalo calves (2n=50) from in vitro-derived DISKIN MG, SREENAN JM. 1985. Production and vitrified embryos by swamp buffalo recipients management aspects of single-and twin-bearing cows. (2n=48). Livestock Sci 107:213-219. Anim Production 40: 553.
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