Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

THERIOGENOLOGY

EARLY EMBRYONIC IN THAI SWAMP BUFFALO

DEVELOPMENT (Bubalus bubalis) --

P. Chantaraprate$.p,' P. Sir~~~~"S:l~~~~~~'".' G. Kobayashi, and A. Limskul P. Prateep 1 Department of Obstetrics Gynaecolcgy and Reproduction Faculty of Veterinary Science , Chulalongkorn University, Bangkok, Thailand 2 of Livestock Surin Animal Breeding Station, Department Surin, Thailand Development, Received

for publication: Accepted:

January

19,

1989

April 4, 1989

ABSTRACT 33 nonsurgical embryo collections was carried out to A total of Collections investigate early embryo development in Thai swamp buffalo. Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages were performed on embryo development on these days were the 16-cell stage, compact of expanding blastocyst, morula, blastocyst, hatched blastocyst and hatched unfertilized some degenerating embryos and In addition, respectively. A higher recovery rate was obtained with single ova were also recovered. estrus than after induced estrus or embryo collection after natural superovulation, 78% (7/9) vs 46% (6/13) vs 54.5% (6/11), respectively. embryos also with single A higher percentage of normal was obtained after after either natural or induced estrus than embryo collection superovulation, 71% (5/7), 83% (5/6) and 38% (6/16), respectively. Key words

: embryo,

early

development,

swamp

buffalo

INTRODUCTION In most mammals, the embryo enters the uterus about 3 to 4 d after ovulation. the embryo Subsequently, reaches the morula stage and develops into a blastocyst which later becomes implanted. In cattle, this differentiation occurs about 6 to 7 d after the onset of estrus (estrus= Day 0). During Days 8 to 9, the bovine blastocyst expands and sheds its zona pellucida, and develoment continues implantation until (1,2). These events seem to occur earlier in Murrah buffalo (3) and in river buffalo (4).

Acknowledgements: The authors are grateful to the Institute of Health Research, Chulalongkorn University and the Royal Sports Club of Bangkok for their financial support. We also wish to thank the Department of Livestock Development and Mr. Udom Wangtal for their assistance in providing some experimental animals as well as Professor Maarten Drost, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA, for his help with the revision of the manuscript.

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embryonic development has not been reported for swamp buffalo, Early different from those of whose reproductive anatomy and physiology are Thus, the aim of our research was Murrah buffalo and of cattle (4, 5). to study swamp buffalo embryos at different stages of development.

MATERIALS

AND METHODS

Fourteen swamp buffalo (3 heifers and 11 cows, 4 to 8 yr old, Two mature bulls with weighing 315 to 480 kg) were used in this study. used as teaser animals for estrus surgically deviated penises were detection, while one fertile bull was used for natural mating. All animals were raised under similar management conditions,' were fed 1 kg of concentrate twice daily and were allowed to graze ad libitum in the field during the day. Three single ova collection types of embryo collection were used: after natural or induced estrus, or after superovulation. For induction of estrus, buffalo cows or heifers were palpated per rectum to identify a corpus luteum on the ovary. Only buffalo with prominent functional (i-m.1 with 15 mg of corpora lutea were injected intrsmusc#larly prostaglandin F 2 alpha (PGF 2 Prosolvin , Intervet, Boxmeer-Holland). in the morning and Estrus detection was carried out twice daily, animals showed the onset of evening, using teaser bulls.' Generally, remained in es&-us standing estrus (Day 01, and most donors for 12 h. at the onset of standing Donors were mated twice with a fertile bull, estrus and again 10 to 12 h later. were treated wigh In cases of superovulation, two and three donors Folligon ; 2500 and 2700 IU of pregnant mare serum gonadotropin (PMSG; Follicle stimulating hormoneIntervet, Boxmeer-Holland), respectively. pituitary (FSH-P; Armour, Burns-Biotec, Omaha, Nebraska, USA) was given in decreasing doses at 12 h intervals twice daily for 4 days (6-6, 5-5, and twice daily in 3-3 and 2-2 mg) for a total of 32 mg to 2 donors, equal doses for 5 days (5-5, 5-5, 5-5, 5-5 and 5-5) for a total of 50 mg to 4 donors. During the mid-cycle, between Days 8 and 12, donors were examined rectum for the presence of a prominent corpus luteum and treated with of four regimens. Prostaglandin F 2 alpha (15 mg) was administered the morning and evening at 48 and 60 h after PMSG and on the last day stimulating hormone (GnRH; 1.0 the FSHR treatment_ Gonadotropin Receptal , Hoechst, Munich, West Germany) was administered i-m. at time of the first mating.

per one in of mg; the

On Days 5.5, 6.0, 6.5, 7.0 and 7.5 after onset of standing estrus embryos were collected nonsurgically via the cervix as previously reported (8-11 ). Briefly, the donor was tranquilized with xylazine and epidural anesthesia was achieved with xylocaine (3 ml). (20 mg/i.m.), Embryos were collected with a two-way Foley catheter (French gauge 14 or 16) by flushing each uterine horn with 500 ml of isotonic lactated Ringer's solution to which 1,500,OOO IU penicillin had been added. Normally, the uterine horn ipsilateral to the corpus luteum was flushed first and embryos were collected into a graduated cylinder.

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after flushing at room Evaluation of embryos was performed 30 min (31°C) with a stereomicroscope at 10 to 40x magnification. temperature modification of The embryos were washed twice.in 2 ml of Whitttingham's Dulbecco's phosphate buffered saline (PBS; Flow Laboratories, North Ryde, serum (BFS; Gibco, N.S.W Australia) supplemented with 10% fetal bovine and their stage of development was recorded and New York, USA), a photograph was taken.

RESULTS A total of 19 (57.5%) normal and abnormal embryos was recovered in 33 embryo attempts. The highest success rate (78%) was obtained by single after while single embryo collection collection after natural estrus, resulted recovery and post superovulation induced estrus in 46% Two of 33 recovery attempts failed collection resulted in 55% (Table I). the because the cervix could not be passed. The quality of embryos and percentage of normal embryos was the same for natural and induced estrus when single collection was employed 71% (5/7) vs 83% (5/6). embryo the The lowest recovery rate of normal embryos was obtained in superovulated group (38%, b/lb; Table 1). Retarded and degenerating (abnormal) embryos were also observed in the superovulated group (38%, same approximately the b/lb). The percentage of unfertilized ova was among the three collection methods. Embryos recovered on Day 5.5 were at the lb-cell stage; morulas or early blastocysts were found when the collection was made on Day 6.0, and blastocysts were obtained on Day 6.5. When collections were carried out on Days 7.0 and 7.5, hatched (collapsed or expanding) blastocysts were found. In addition, degenerating embryos and unfertilized ova were recovered. The morphology of normal embryos, degenerating embryos and unfertilized ova is shown in Figures 1 and 2.

DISCUSSION The highest success rate of embryo recovery was 78% from single embryo collection following natural estrus. The recovery rate was 46% after induced estrus and 54.5% after superovulation. The reasons may partly be due to the fact that natural estrus does not disturb normal reproductive endocrine function. In addition, estrus sign in ==mP buffalo is difficult to detect (12) except by observing standing heat. Appropriate time for mating is especially critical after the induction of estrus, which resulted in conception rate of approximately 45% (13, 14). The quality of embryos obtained frcm the collection of single ova, either after natural or induced estrus , was higher than those of embryos collected after superovulation. The poor quality of embryos may have been due to inappropriate dosages or to the method of stimulating the ovaries. There have been few reports on the superovulatory treatment of buffalo. Existing studies, however, indicate a generally low response, with unfertilized ova and abnormal embryos obtained after superovulation (3, 7, 8).

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Figure

1134

I.

Various stages of swamp buffalo embryo development. A). A 5.5day 16-cell embryo. The contour of each blastomere is evident, some blastomeres are beginning to fuse, the perivitelline space is present and the zona pellucida is thick. B). A 6-day embryo, blastomeres are in close apposition, resulting in a dense mass called the compact morula. Perivitelline space is still present. Some vacuoles may be observed on surface of blastomere. C). A 6.0-day embryo, early blastocyst stage with intact zona pellucida. Blas'cocoele occupies 25% of the volume of the embryo; undifferentiated blastomeres are still present, no perivitelline space.

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THERIOGENOLOGY

Figure

1.

There is no stage. D). A 6.5-day embryo, expanding blastocyst cell line Trophoblastic the zona perivitelline space. the pellucida; the inner cell mass is well differentiated; blastocoele is about 75% of the volume of the embryo; the zona cells pellucida is considerably thinner, while the number of E). A -/-day embryo, a partially of the embryo has increased. the zona pellucida is gone. collapsed hatched blastocyst; Well-defined trophoblast cells and inner cell mass are present. (without a F). A 7.5-day embryo, hatched expanding blastocyst zona pellucida), increased diameter.

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Figur

1136

2.

Morphology of unfertilized and abnormal embryos superovulatad swamp buffalo recovered on Day 6 after ABN = Abnormal embryo; UNF = Unfertilized ova. magnification.

from a mating. 200 x

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Table

1.

Results estrus,

swamp buffalo of embryo recovery from induced estrus and superovulation

after

natural

______________________-____---_------_________-_---------~~~~~~~--~--__~~~ Embryo collection

NO. of collections

NO. of successful collections (%)

Recovered embryos/ova Total N ABN (%I (%I

Not found UNF (a)

Not able to collect

Single 1 7 5' 2 9 7 embryo (71) (29) collection (78) (natural) estrus ___________________-_--_-_-_---------------------------------------------6 6 5 I* 13 6 Single embryo (45) (80) (20) collection (induced estrus) Embryo collection 16 6 6 4 5 11 6 following (37.5)(37.5)(25) (54.5) superovulation -------------------------------------------------------------------------33 19 29 16 6 7 12 TOTAL (57.5) (55)(20.7)(24.1) ---_------_---------__I_________________~-~~~~~~~~~~~~~-~~~~~-~~~--~-----ABN = abnormal embryo; UNF = unfertilized N = normal embryo;

1

1

2

ova.

In our experiment, embryo recovery was carried out from Day 5.5 onwards, as a report on this species (Murrah breed) revealed that 4.5 d embryos can be found in the uterus after the onset of estrus (3). Our study demonstrates that the compaction of embryos occurred about Day 6.0 and the transition from morula to early blastocyst occurred after This chronology of development to the early blastocyst Day 6. stage is similar to that of cattle (15). A difference in rate of development between the swamp buffalo and cattle was observed from Days 7.0 to 7.5. The hatching of embryos from the zona pellucida in the swamp buffalo seemed to occur earlier than in cattle. Hatched blastocysts were found on Days 7.0 to 7.5, while they are found between Days 8.5 and 10 in cattle. Our results were also different from those reported by Karaivanov et al. (3) in Murrah buffalo. They reported that the hatched blastocyst was found on Day 5 after the onset of estrus. In one attempt, we could not find the embryo on Day 4.5 after mating, but repeat collection of the same animal on Day 6.5 yielded an embryo. The difference in results may be the fact that estrous behavior is difficult to observe in swamp buffalo. In our experience, the most practical way for detecting estrus in this species is to use a teaser bull (a bull with a deviated penis). The timing of embryo development from the onset of estrus might be inaccurate; a more reliable measure may

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be from the time of standing estrus. A comparative study of early embryonic development in Murrah and swamp buffalo needstobe conducted to explain the apparent differences. Our findings indicate that the chronology of embryo development in swamp buffalo is different from that of cattle and of Murrah buffalo.

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2,

The entry of Trounson, A-0. and L.E.A. Rowson, Newcomb, R., L.E.A.(ed). Egg In: Rowson, the uterus. into superovulated eggs Communities, European Commission of The Transfer in Cattle. Luxembourg, 1976, pp. 1-15.

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6.

Bodhipaksha, P. Physiology of female swamp buffalo reproduction. In: Chantaraprateep, P., Virakul, P., Lohachit, C. and Kunavongkrit, A,, (eds). Swamp Buffalo Reproduction. Chulalongkorn University Press, Bangkok, Thailand, 1987, pp. 117-124. Chantaraprateep, Lohachit, C., Virakul, P., Kobayashi, P., G., Techakumphu, M., Kunavongkrit, A. and Prateep, P. Ovarian responses to gonadotrophin stimulation in swamp buffalo. Buffalo Bulletin 7 : 82-86 (1988). Drost, M., Wright, J.M., Gripe, W.S. transfer in water buffalo (Bubalus ~~ 20 : 579-584 (1983). -

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Chantaraprateep, P. Control of estrous cycle of swamp buffaloes. In: Chantaraprateep, P., Virakul, P., Lohachit, C. and Kunavongkrit, Swamp Buffalo Reproduction. Chulalongkorn University A,, (eds). Press, Bangkok, Thailand, pp_ 187-209.

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Chantaraprateep, P., Lohachit, C., Bodhipaksha, P. and Virakul, P. Prostaglandin F2 alpha for oestrus control of 2nd buffaloes. Proc. Internat. World Buffalo Production Italy, Congress. Casserta, 29 Sept - 2 Ott pp. 385-393 (1982).

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Betteridge, K.J. Superovulation. In: Betteridge, Transfer in Farm Animals. A Reviewof Techniques Agriculture Canada. Monograph 16, 1977, pp. 1-9.

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