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Twin Calf Production in Water Buffaloes Following Non-Surgical Transfer of in Vitro-Produced-Vitrified Embryos

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Twin Calf Production in Water Buffaloes Following Non-Surgical Transfer of in Vitro-Produced-Vitrified Embryos 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) 99 Philippine Journal of Science Hufana-Duran et al.: Twin Calf Vol. 137 No. 2, December 2008 Production in Water Buffaloes 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. 100 Philippine Journal of Science Hufana-Duran et al.: Twin Calf Vol. 137 No. 2, Decmber 2008 Production in Water Buffaloes 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.
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