Production of prolific microsheep by embryo transfer into large non-prolific sheep

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Papers & Articles Production of prolific microsheep by embryo transfer into large non-prolific sheep S. M. K. Naqvi, A. Joshi, R. Gulyani, D. Kumar, A. P. Kolte, S. Kumar, V. P. Maurya, S. Saha, J. P. Mittal, V. K. Singh The Garole is a prolific breed of microsheep that possesses the FecB gene, which increases ovulation rate. The purpose of this study was to compare embryo production by multiple ovulation in seven Garole ewes with that in seven normal size, non-prolific Malpura ewes, and assess the influence of the large body size of Awassi crossbred recipient ewes on the birthweight of Garole lambs. Oestrus was synchronised with two intramuscular injections of 7·5 mg prostaglandin F2α administered 10 days apart. The donor ewes were superovulated by the use of pregnant mare serum gonadotrophin and follicle-stimulating hormone. The onset and duration of oestrus were similar in both breeds. The Garole donors had higher total mean (se) ovarian responses (15·6 [3·6] v 9·1 [2·3]), ovulation rate (13·6 [3·1] v 8·4 [2·2]) and produced more transferable embryos (6·0 [3·5] v 4·0 [0·9]) than the Malpura donors, but the differences were not statistically significant. The Garole lambs produced by embryo transfer were on average 57·8 per cent heavier at birth than contemporary Garole lambs produced by natural mating.

THERE has been increasing interest in the identification and utilisation of major genes affecting the ovulation rate of sheep (Davis 2004, 2005). Embryo transfer has played a significant role in the spread of prolific sheep breeds from their country of origin (Fahmy 1996). The Garole sheep is India’s most valuable ovine germplasm, due to its high prolificacy and its ability to thrive well under harsh and adverse climatic conditions (Sharma and others 1999); it is a rare breed of microsheep found in the hot and humid Sunderban region of West Bengal. Garole sheep weigh 10 to 14 kg at maturity and have an average litter size of 2·27 lambs (Fahmy and Mason 1996), and could provide a means of incorporating prolificacy traits into monotocous sheep breeds (Naqvi and others 2002, Nimbkar and others 2003, Sharma and others 2004). The conservation of ovine genetic resources in the form of spermatozoa, ova and embryos becomes important if a gene with a major effect of interest is detected (Ponzoni 1997). Garole rams produce good-quality semen (Joshi and others 2003), which can be stored frozen (Joshi and others 2001, 2005). An earlier study provided evidence that a large number of embryos can be produced from a single flush of a Garole ewe (Naqvi and others 2006). DNA testing has shown that the major gene for prolificacy (FecB) in the Booroola merino strain was introduced from the Garole sheep (Davis and others 2002). The FecB gene, located on chromosome 6 of Booroola sheep (Montgomery and others 1993), has an additive effect on ovulation rate (Piper and others 1985). The high prolificacy of sheep carrying the FecB gene is the result of a mutation in the receptor (BMPR-1B) located in the region containing the FecB locus (Wilson and others 2001). Despite the small population of this original carrier of the FecB gene, no attention has been given to its conservation, faster multiplication and propagation through embryo transfer technology. The small size of Garole ewes makes them suitable only as embryo donors, but provides ample scope for the transfer of in vivo-derived Garole embryos into larger size recipients. It has been reported that lambs (Hunter 1956) and foals (Tischner 1987, Allen and others 2004) of small size breeds are born heavier if gestated in the uterus of larger recipient breeds. The aims of this study were, therefore, first, to compare the oestrous response, superovulatory response, embryo recovery and lamb production from the small, prolific Garole and normal size, non-prolific Malpura donor ewes, and secondly, to investigate whether rearing Garole embryos in ewes of the large Awassi breed would affect their birthweight.

MATERIALS AND METHODS Animals and feeding regimen Seven non-pregnant and cycling ewes of each of the Garole and Malpura breeds, aged four to seven years, were used as embryo donors. The donor Garole ewes were selected from offspring born at the Central Sheep and Wool Research Institute’s farm from the original flock procured from the Sunderban. The Malpura sheep is a hardy breed adapted to the semiarid hot environment of the country, and is known for mutton and coarse wool production (Acharya 1982). The bodyweights of the Garole and Malpura ewes ranged from 13 to 16 kg (mean [se] 14·2 [0·54] kg) and from 29·5 to 38 kg (32·8 [1·27] kg), respectively. Awassi x Malpura crossbred ewes weighing between 31 and 46·5 kg (mean [se] 38·7 [0·91] kg) were used as recipients. All the animals were grazed for eight to 10 hours daily on natural vegetation interspersed with seasonal shrubs and herbs. In addition to the grazing, the Garole ewes were provided with 150 g per ewe per day of a concentrate mixture, which was offered at the rate of 300 g per ewe per day to the Malpura and Awassi crossbred ewes. The concentrate mixture contained 18 per cent crude protein, 65 per cent total digestible nutrients, 1 per cent of a mineral mixture, 1 per cent common salt and vitamins A and D3. Oestrus synchronisation and superovulation Oestrus was synchronised in the embryo donor and recipient ewes by administering two intramuscular injections of 7·5 mg prostaglandin F2α (PGF2α) (Lutalyse; Pharmacia) 10 days apart. Superovulation was induced in the donor ewes by the use of pregnant mare serum gonadotrophin (PMSG) and follicle-stimulating hormone (FSH) of ovine origin (Ovagen; ICP); the superovulatory treatment was the same for all the ewes and commenced three days before the second injection of PGF2α. Each ewe received a total dose of 5·4 mg FSH (NIADDKO FSH-17) twice a day (morning and evening) at a constant dose over a period of four days, and 200 iu PMSG (Folligon; Intervet) intramuscularly at the beginning of the superovulatory treatment. Oestrus detection and mating Oestrus was detected in the ewes by exposing them to an aproned ram of high sexual vigour at six-hourly intervals for up to four days after the second dose of PGF2α. Donor ewes showing behavioural oestrus were mated twice a day (morning and evening) with a ram of proven fertility. The Veterinary Record, October 14, 2006

Veterinary Record (2006) 159, 522-526 S. M. K. Naqvi, MSc, PhD, A. Joshi, MSc, PhD, R. Gulyani, MSc, PhD, D. Kumar, MVSc, PhD, V. P. Maurya, MSc, PhD, S. Saha, MVSc, PhD, J. P. Mittal, MVSc, PhD, Division of Animal Physiology and Biochemistry, A. P. Kolte, MVSc, S. Kumar, MSc, Animal Biotechnology Section, V. K. Singh, MVSc, PhD, Central Sheep and Wool Research Institute, Avikanagar, via Jaipur, Rajasthan 304501, India

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Papers & Articles

FIG 1: Typical specimens of the Garole donor ewe (right) and the Awassi crossbred recipient ewes (left)

Ovarian examination and embryo recovery The ovaries of the ewes were examined with the aid of a laparoscope (Karl Storz), followed by a laparotomy to recover the embryos (Naqvi and others 2000, 2001) between three and six days after mating. The animals were fasted for at least 24 hours before the laparoscopy and/or laparotomy. The ewes were sedated with xylazine hydrochloride (Indian Immulogicals) and the abdominal area anterior to the udder was shaved, sprayed with 70 per cent alcohol and locally anaesthetised by the infiltration of lignocaine hydrochloride (Xylocaine; AstraZeneca). All the ewes treated for superovulation were examined by laparoscopy to determine whether it was worth attempting surgical embryo collection. Ewes with three or more corpora lutea were regarded as having a superovulatory response, and the embryos were collected from these ewes through a midventral laparotomy. The fallopian tubes were flushed with 20 ml Dulbecco’s phosphatebuffered saline (DPBS), pH 7·5 (0·3), supplemented with 2 per cent bovine serum albumin (Sigma) at room temperature, introduced near the tubal junction. The tubal washings were collected in sterile petri dishes and examined for ova or embryos under a Stereozoom microscope (Nikon) equipped with a warm stage platform (37°C) at x 50 magnification. The numbers of recent ovulations (corpora lutea) and persistent large follicles were recorded and the ewes’ ovarian responses were estimated by adding the numbers of corpora lutea and large follicles. Embryo grading and evaluation The embryos were transferred into sterile petri dishes containing DPBS for evaluation and gradation under an inverted microscope (Olympus) with a warm (37°C) stage platform at x 400 magnification. The fertilisation of ova was confirmed by the occurrence of cleavage. The quality of the embryos was assessed according to morphological criteria based on the symmetry of the cells, shrinkage, vacuolisation or lysis (Robertson and Nelson 1998); poor-quality embryos that had partially degenerated and vacuolated cells were not transferred. Embryo transfer and lambing The embryos were transferred by the laparoscope-aided procedure described by McMillan and Hall (1994), as modified

TABLE 1: Mean (se) body measurements of the donor and recipient ewes Breed Garole Malpura Awassi crossbred a, b, c,

Number 7 7 7

Heart girth (cm) Pin-shoulder length (cm) Height at withers (cm) 63·8 (0·8)c 78·5 (1·4)b 83·3 (1·6)a

45·9 (1·1)b 62·8 (0·9)a 65·9 (0·6)a

46·9 (0·7)b 65·8 (0·6)a 67·4 (0·6)a

Values with different superscripts in the same column differ significantly (P