application of embryo biotechnology to augment

Transcription

AKMisra :Application of embryo biotechnology to augment reproduction andproduction in buffaloes
APPLICATION OF EMBRYO BIOTECHNOLOGY TO AUGMENT
REPRODUCTION AND PRODUCTION IN BUFFALOES : CURRENT
STATUS AND FUTURE POSSIBILITIES
A.K. Misra
Project Directorate on Cattle (ICAR)
Post Box No. 17, Grass farm Road, Meerut Cantt-250 001 (UP), India .
INTRODUCTION
The buffalo occupies an important place in livestock economy of Asia, several
European and American countries, benefiting nearly half of the humanity by providing milk,
meat and work power. The world buffalo population is approximately 172 .6 million spreads
over in some 129 countries (including traditionally non-buffalo regions), of which 167 .5
million (97 .1%) are in Asia (FAO, 2004) . In the last four decades the buffalo population has
increased from 89.95 million to 170 .66 million (nearly doubled) whereas the milk production
has increased from 18 .67 to 72 .69 million metric tons (nearly four times ; FAO, 2004) . The
developing world contains over 77% of the world's domestic animals and the potential for
improving their productivity is amply illustrated by the fact that about 76% of the world's cattle
and nearly all (99 .6%) the buffaloes are in the developing countries and yet they produce only
.about 41 per cent of the world's milk and 52% of the world's beef and buffalo meat (FAO,
2004) . Among Asian countries, India is the harbinger of some of the best breeds of buffaloes
such as Murrah, Nili-Ravi, Bhadawari, Surti, Mehsana, Jaffarabadi (Misra, 2005) and buffalo is
the mainstay of Indian dairy industry contributing about 52 per cent of the 91 .94 million MT
milk produced annually (FAO, 2005), to make this country the largest producer of milk .
Buffaloes suffer from lower reproductive efficiency due to delayed puberty, higher
age at first calving, long postpartum anoestrus period, long inter-calving period, lack of overt
signs of heat and low conception rate . Also, buffaloes have fewer primordial follicles and a
high rate of follicular atresia . Biotechnology has given us the unprecedented opportunities to
improve reproductive efficiency of buffaloes and penetrate their genetic ceiling to enhance
their productivity. The most important application of embryo biotechnology is for the
production of Al bulls from the best proven bulls and buffaloes available . Importance of this
technology can be gauged from the fact that in USA in 2003, embryo transfer produced 66 of
the top 100 Total Production Index (TPI) international Holstein bulls . The MOET (Multiple
Ovulation Embryo Transfer) schemes have more relevance to developing countries where
conventional progeny testing is not possible due to lack of milk recording, .The genetic gain
per year from the nucleus herd of 100 buffaloes and 5-10 numbers'of bulls for ONBS in
buffaloes is expected to be 1 .84-1 .50% over-the herd average (Sethi, 2003) .In vitro embryo
production (IVEP) using oocytes collected from slaughtered buffaloes provides an excellent
source of low cost embryos for basic research on developmental physiology, farm animal
breeding and for commercial application of biotechnologies like embryo sexing, infra
cytoplasmic sperm injection (ICSI), nuclear transfer and transgenic animal production (Nandi
et al., 2003 ; Misra, 2005) . However, IVEP by repeated ultrasound guided trans-vaginal
aspiration of oocytes (ovum pick-up, OPU) from live females and their subsequent in vitro,
maturation (IVM), fertilization (IVF) and culture (IVC) offers an opportunity to produce
embryos more efficiently and economically (Misra, 2005) .
36
AKMirra :Application of embgo biotechnology to augment reproduction and production in buffaloes
This paper reviews available reproductive biotechnologies, which are either being
used or are currently under development and have potential to enhance reproductive
efficiency and productivity of buffaloes .
The birth of first buffalo calf through embryo transfer was reported at University of Florida,
USA (Drost et al. 1983). Subsequently, Drost in collaboration with Bulgarians and reported the birth
of the second calf using ETT (Drost et al, 1988) . In Asia/ India the birth of the first calf through
surgical (Misra et al, 1988b) and non-surgical embryo transfer (Misra et al., 1988a) was reported at
the National Dairy Development Board, Anand . Subsequently several reports on work on embryo
transfer emerged from buffalo rearing countries including Bulgaria (Alexieve et al., 1988 ;
Karaivanov et al., 1990), India (Kurup, 1988 ; Madan et al. 1988; Singh et ad. 1988a,b ; Taneja et al.
1988 ; Misra et al., 1994 ; Misra et al., 1999a), Malaysia (Jainudeen, 1989), Pakistan (Mahmood et
al, 1988; Rahil et al., 1988a,b ; Ullah et al., 1992), Thailand (Parnpai et al., 1985 ; Sophon et al.,
1987 ; Vanitkul, 1989 ; Chantaraprateep et al., 1989 ; Techakumphu et al., 1989), Japan (Ocampo et
al., 1988), Italy (Zicarelli et al., 1994 ; Schallenberger et al., 1990; Campanile et al., 1995),
Phillipines (Cruz et al., 1991), Vietnam (Uoc et al., 1992 a, b), Egypt (Ismail et al., 1993 ; Osman
and Shehata, 2002), China (Wang et al., 1994 ; Hesheng et al., 2006 ; Jixian et al., 2006 ; Quin-yang
et al., 2006), Brazil (Baruselli et al., 2000), Iran (Ardebili et al., 1999) etc .
The first calf following in-vitro fertilization using abattoir derived oocytes was born
at National Dairy Research Institute, Karnal (Madan et al., 1991), however, the first buffalo
calf following oocyte collection from live buffaloes was produced by Galli et al., (1998) in
Italy. The first calf (Kasiraj et al., 1993) following cryopreservation of buffalo embryos was
produced in India, and Neglia et al .,(2004), reported the birth of the first calves (Neglia et al .,
2004) in Italy following the successful freezing of the in-vitro generated embryos. Transfer of
in-vitro generated and vitrified blastocysts using oocytes of the slaughtered buffaloes resulted
in birth of several calves in Philippines (Duran et al., 2004) . Lu et al., (2006) reported the
birth of the first buffalo calves using sexed semen for IVF and Shi et al, (2006) reported the
birth of the cloned buffalo calves in China .
IN-VIVO EMBRYO PRODUCTION
Induction of multiple ovulations in elite donor buffaloes, their breeding with top sires
and subsequent embryo collection, embryo cryopreservation and their transfer to estrus
synchronized surrogate mothers has been used commercially since about two decades .
Progress of ET work in buffalo betweenl988-89 to 1992-93 is given in Table 1 .
Table 1 . Embryo recovery in superovulated buffaloes during 1988-1992
Parameter
Donors flushed
Embryos collected
(Mean)
Viable embryos
(mean)
Year
88-89
70
188
(2 .68)
102
(1 .45)
89-90
113
201
(1 .77)
121
(0 .92)
90-91
117
320
(2 .73)
144
(1 .23)
91-92
136
369
(2 .63)
215
(1 .58)
92-93
61
234
(3 .83)
130
(2 .13)
Total
497
1302
(2.61)
712
(1 .43)
(Misra et al., 1994) .
37
AK Misra : Application of embryo biotechnology to augment reproduction andproduction in buffaloes
In India, NDDB Labs superovulated perhaps the largest number of buffaloes (about
1100) between 1987-1998 and reported recovery of about 2 .5 to 3 .0 viable embryos (VE) per
collection and - 30% conception following transfer of low quality embryos and 60% with
grade I embryos (Misra et al., 1999b), which are better than the results reported by many
other buffalo rearing countries . In isolated trials, however, up to mean 5 .9 transferable
embryos (Table 2) have been recovered (Misra et al ., 1999a) . Recently Hesheng et al., (2006)
also reported recovery of over 4 transferable embryos in buffaloes in China .
,Table 2 . Embryo transfer work in the in the field (Punjab state)
Attributes
No. of buffaloes superovulated
No. of buffaloes flushed
No. of ovulations(CL)
Embryos recovered (mean)
Total embryos (%)
Transferable embryos (mean)
Embryos frozen
Fresh embryos transferred*
Recipients pregnant (%)
Ludhiana
10
10
40
22(2 .2)
22 (55 .0)
15(1 .5)
15/12
4(33 .3)
Patiala
10
10
96
75(7 .5)
75 (78 .1)
59(5 .9)
34
25/20
7(35 .0)
District
Bhatinda
26
2$
148
94(3 .6)
94 (63 .5)
56 (2 .15)
32
24/19
4(21 .0)
Total
46
45
284
191 (4 .15)
191 (67 .2)
130 (2 .82)
66
64/51
15 (29 .4)
(Misra et al., 1999a)
* No . of embryos/no . of (some recipients received two embryos)
The serious limitations of this technology are the highly variable and unpredictable
superovulatory response (nearly half of the buffaloes had poor superovulatory response ;
Misra, 1996), high cost of the FSH"and its unavailability (needs to be imported) .
Initial reviews on the embryo transfer in buffaloes suggested low ovulation rate, low
fertility, low embryo recovery and poor conception rate (Ocampo et al., 1988 ; Jainudeen,
1989, Kamonpatana, 1990). Nonsurgical embryo collection from 189 (Alexiev et al., 1988)
and 145 (Kurup, 1988) buffaloes yielded 0 .7 or less viable embryo and less than 10%
buffaloes became pregnant following transfer of fresh embryos . However, subsequent work
(Misra et al., 1990; Misra et al., 1991 ; Rao et al., 1994 ; Wang et al. 1994; Misra et al.,1999a)
reported improved results and up to 3 .4 (Rao et al., 1994) and 2 .9 (Misra et al., 1999a) viable
embryos were recovered in farm and field conditions, respectively . Also, the large-scale
transfer of mostly lower grade nonfreezable embryos resulted in 25-30% conception on farm
(Misra et al., 1992 ; Misra et al., 1999b) and field (Misra et al., 1999a) . However, transfer of
Grade I fresh embryos lead to pregnancy rate of 60% (Misra et al., 1999b) . Recently,
Hesheng et al., (2006) reported 67 .5% conception following transfer of fresh buffalo
embryos . These results suggest that the technique of embryo transfer in buffalo has improved
significantly over the years ; however, there is need to improve it further .
A. Superovulation and Embryo Recovery
Superovulation is the most critical step in the embryo transfer program, which
influences the yield of the embryos per donor. Some of the factors that affect the
superovulatory response and embryo recovery in buffalo are :
38
AK Misra : Application ofembryo biotechnology to augment reproduction and production in buffaloes
1. Nature of Gonadotrophin
Both, pituitary (follicle stimulating hormone, FSH) and placental (equine chorionic
gonadotrophin, eCG) gonadotrophins have been used to induce superovulation in buffalo .
2. Follicle Stimulating Hormone (FSH)
The short biological half-life of this pituitary gonadotrophin, necessitates its use twice
daily for 3 .5 (Songasen et al., 1999), 4 (Drost et al., 1988 ; Singla and Madan,1990 ; Quin-yang
et al., 2006)) or 5 days (Drost et al., 1983 ; Misra et al., 1990 ; Ullah et al, 1992 ; Hesheng et al.,
2006 ; Quin-yang et al., 2006) in decreasing (Drost et al., 1988 ; Karaivanov et al,1990; Misra
et al, 1990 ; Misra et al., 1991 ; Hesheng et al., 2006 ; Quin-Yang et al., 2006) or constant
dosages (Drost et al., 1983 ; Yadav et al., 1988b ; Ullah et al., 1992) with varying degree of
success . ,
I
Initial work to superovulate buffaloes with FSH-P® (Shering, USA)(Drost et al.
1983 ; Kurup, 1988 ; Singla and Madan, 1990 ; Misra, 1993) resulted in poor superovulatory
response and embryo recovery . Subsequent work to standardize the dose of Folltropin
(purified FSH ; Vetrepharm, Canada) for the first time in buffaloes lead to average 3 .7
ovulations and recovery of 2 .7 total and 1 .5 VE per buffalo (Misra et al., 1990). Use of the
best dose.(30 .0 mg PPFE) of Folltropin on 16 buffaloes which responded to superovulatory
treatment during previous trial lead to the mean 6 .8 ovulations and recovery of 4 .4 total and
3.1 VE (Misra et al., 1991) . Later use of 600 mg NIH-FSH-P1 on over 300 buffaloes induced
average > 6 .0 ovulations and recovery of 2 .0 to 2 .9 VE on the farm (Misra, 1996 ; Misra et
al., 1999a, 2002) and 6 .2 ovulations and 2 .8 VE in the field (Misra et al., 1999a).
Interestingly, during field trial at one place, 10 superovulated buffaloes yielded 75 total (mean
7.5) and 59 VE (mean 5 .9; Misra et al., 1999a). With the use of same superovulatory protocol, the
repeated superovulation of buffaloes lead to an average 8 .2 ovulations and recovery of 3 .4 VE
from 41 buffaloes during the first flushing, however, corresponding values declined significantly
to only 6.3 ovulations and 2 .2 VE during repeated superovulations up to six times (Rao et al,
1994). Use of another purified pituitary extracts 'Super-ov' on 7 buffaloes induced 20 ovulations
and produced 8 total and 2 VE (Singla and Madan, 1990) . Interestingly, in buffalo, superovulation
of 17 donors with 40 mg equine FSH produced only 2 unfertilized ovum (Jain et al., 1992). In a
study involving superovulation of only 6 buffaloes, Chinese workers (Wang et al., 1994) reported
recovery of up to 8 .0 totals and 7 .0 VE after sacrifice. Recently, in the same country, using the
FSH produced by the Chinese Academy of Science, over 4 mean VE were collected (Hesheng et
al., 2006), however, with the same FSH, Quin-yang et aL,(2006) could induce only 1 .22±0.97 to
4.10±1 .20 ovulation and concluded that 4 days dosage regimen leads to development of ovarian
cysts and 5 days regimen is better. A comparative study (Baruselli et al., 1999) of use of FSH-P
(25 mg), Folltropin (14 mg) and Pluset (500 or 333 IU) revealed no significant difference in the
ovulation rate (5 .7 to 6 .5 CL) and the embryo recovery (1 .0-2 .5). A study to use GnRH agonist to
control ovulation subsequent to superovulation (using 200 mg Folltropin) produced only 4 .3±1 .2
ovulations and 2 .0±0 .9 embryos (Carvalho et al., 2000) .
Initial comparison of superovulation of buffaloes with Constant/Tapering dosage of
FSH administration revealed no significant difference in the ovulation rate (Yadav et al.,
1988b ; Ambrose et al., 1991) or embryo recovery (Ambrose et al., 1991), however, subsequent
trials involving large number of buffaloes, indicated better superovulatory response and higher
yield of embryos with tapering dosage of FSH (Karaivanov et al., 1990 ; Misra et al., 1991,
1999a,b,c, 2002 ; Mutha Rao et al ., 1994) .
39
AK Misra : Application of embryo biotechnology to augment reproduction and production in buffaloes
3. Route of Administration of FSH
To reduce stress because of repeated handling and avoid frequent visits to administer
FSH to donors, especially if they are located in field, attempt have been made to regulate
slow absorption of FSH by injecting total dose of FSH as single dose subcutaneously (s .c)
Initially, we observed no significant difference between single s .c. administration of FSH
diluted in normal saline and multiple i.m . (intramuscular) treatments (Kasiraj et al., 1992) .
Subsequently, however, contrary to these observations, s .c. administration of FSH resulted in
the poor superovulatory response and embryo recovery (Misra, 1996) .
4. Equine Chorionic Gonadotrophin (eCG)
To superovulate buffaloes, eCG has been used at the dose rate of 2500 to 40001U (Jain
Misra, 1993), however, 3000 IU of eCG by intramuscular route is commonly
practiced (Karaivano4 et al., 1990; Schallenberger et al., 1990; Cruz et al., 1991 ; Misra, 1996)'.
The superovulatory response and mean viable embryo recovery following eCG treatment was
reported to be low compared to FSH, as eCG continue to stimulate growth of follicles even after
the ovulations which in-turn adversely affects endocrine profile, gamete transport and embryo
quality. (Alexieve et al., 1988 ; Misra et al., 1994 ; Misra, 1996).
et al., 1992;
Table 3 . Superovulatory response of buffaloes to FSH or PMSG
Hormone
n
Total CL (mean)
Folltropin
(600 mg NIH-FSH-P1)
eCG (3000 IU)
97
680
(7 .01)
94 (3 .76)
25
Total embryos
(mean)
398
(4 .1)
40(1 .6)
Viable embryos
(mean)
203
(2 .09)
14 (0 .56)
(Misra et al., 1994)
5. Progesterone Levels at the Time of Superovulation Treatment
Progesterone level at the time of superovulation treatment is reported to have significant
effect on subsequent ovulation in buffalo (Madan et al., 1988 ; Misra et al., 2000), however, an
attempt to improve CL function by administration of the GnRH on day 5 of the estrus cycle
before superovulation on day 10 failed to improve superovulatory response (Misra et al., 2002) .
On the contrary, Ullah et al., (1992) reported no difference in ovulation rate and embryo recovery
when buffaloes were superovulated in the presence or absence of functional CL .
6. Stage of Estrus Cycle
As the numbers of follicles responsive to exogenous gonadotrophin vary during
different stages of estrus cycle, the stage of cycle influences the superovulatory response . In
the buffalo, most superovulations have been performed during mid-luteal phase .
Superovulation of buffaloes during mid (8-12 days) or late (13-15 days) luteal phase using
FSH (Folltropin) had no significant difference in the ovulation rate (Misra et al., 1990 ; Beg et
al., 1997) . Workers in Bulgaria obtained better superovulation rate when 3000 IU eCG was
administered on Day 10 compared to Day 6 or Day 14 of cycle (Karaivanov et al., 1990) . On
the other hand, using 3000 IU eCG, Mehmood et aL(1989) observed better ovulation rate
when superovulation was initiated on Day 14, compared to Day 9 or 12 . Quin-yang et al. ,
(2006) reported that initiation of superovulation treatment on Days 7-8 induced significantly
better superovulatory response than treatment on Days 9-10 .
40
AK Mitra : Application ofemb9o biotechnology to augment reproduction andproduction in buffaloes
7. Effect of Season
Ambient temperature also affects superovulatory response in buffalo . We observed that,
the superovulatory response to gonadotrophin is poor in summer (April to June) than other
seasons (Rao et al., 1994). Taneja et al., (1995) observed no difference in the ovulation rate and
embryo recovery when the buffaloes were superovulated during April to June (dry hot) or August
to February (wet cool), however, the embryos recovered during dry hot period were not
transferable . Poor expression of behavioural signs of estrus during summer (heat stress) limits
estrus detection and affects embryo transfer programs .
8 . Repeated Superovulation
During the lone study on repeated superovulations, we observed that the repeated
superovulations, of buffaloes at an average interval of 77 days over a period of 20 months
produced 8 .2/5 .1/3 .4 (n=40), 6.6/3 .7/2 .2 (n=35), 5 .8/2 .7/1 .7 (n=27), 5.1/2 .4/1 .7 (n=24),
5 .1/2 .5/1 .6 (n=19) and 3 .9/2.4/1 .4 (n=8) mean ovulations/ total embryos/ viable embryos during
1st to 6th superovulation, respectively, suggesting a drop in embryo production after first and
second superovulation . However, these parameters were not affected after third superovulation,
as the total/ viable embryo recovery was already low . Interestingly, the repetitive
superovulatory treatments up to the 6th time did not significantly affect the fertility of the donors
as 83 .3% buffaloes (25/30) became pregnant with only 1 .8 services per conception (Rao et al.,
1994) .
Table 4. Superovulatory response and embryo recovery following repeated superovulations
Supeovulation No.
1
II
III
IV
V
VI
Total
n
41
35
27
24
19
.9
158
Ovulations (CL)
8 .2±0 .50
6 .7±0 .60
5 .8±0 .62
5.1±0 .52
5 .2±0 .62
3.9±0 .59
6 .3±0 .26
Viable embryos
3 .4±0 .37
2.2±0 .35
1 .7±0 .29
1 .6±0 .41
1 .6±0.38
2.2±0 .16
2.2±0 .16
(Rao et al., 1994).
9. Priming Before Superovulation
In order to increase the follicular development and superovulatory response in
buffaloes, attempts were made to inject 4-5 mg FSH on Day 3 and 4 of the estrus before their
superovulation on Day 10 or 11, however, the treatment failed to increase ovulation rate and
embryo recovery (Jailkhani et al ., 1990 ; Joshi et al ., 1992)
10 . Effect of Parity on Superovulatory Response
A study involving 107 buffaloes (22 nulliparous, 28 upto 3rd para and 57 calved for
more than 3 times) revealed that the mean ovulation rate in the nulliparous (6 .59 ± 0 .60), up to 3rd
para (6 .82± 0 .69) and more than 3rd para (5 .98 ± 0 .53) was not significantly different with each
other (Misra, 1996) .
41
AKMisra :Application ofembgo biotechnology to augment reproduction andproduction in buffaloes
Table 5. Effect of parity on superovulatory response and embryo recovery
Attributes
Parity
Upto 3rd para
28
27(96.4)
6.82±0 .69
Nullipara
22
21(95 .4)
6.59±0 .60
No. of animals
No. responded (%)
No.of ovulations (Mean-S .E)
> 3`d para
57
48(84.2)
5.98±0 .53
11 . Superovulatory Response in Buffaloes
The frequency distribution of total number of ovulations (CL) in 107 superovulated
buffaloes in one of our studies indicated that overall 10 .3% buffaloes failed to respond (0-2 CL) to
superovulatory treatment and 38 .3% had only 3-5 CL; together, about half (48 .6%) of the
buffaloes dad poor superovulatory response. Other 39 .3% buffaloes had average response (6-10
CL) and the remaining 12 .1% buffaloes had well (>10 CL) response including 3 (2 .8%) buffaloes
having excellent superovulatory response (>15 CL) . In this study less than 1/3 (29 .9%) buffaloes,
responding with 8 or more ovulations, can be considered as good donors (Misra, 1996) .
Table 6. Relative distribution of estimated CL in superovulated buffaloes
Superovulatory response
per donor
No. responded (n= 107)
of donors
0-5 CL
0-2 CL
3-5 CL
11
41
38 .3
10 .3
48.6
6-10 CL
6-7 CL
8-10 CL
23
19
21 .5
17 .8
39 .3
> 10 CL
11-15 CL > 15 CL
10
3
9.3
2.8
12 .1
12. Time of Non-Surgical Embryo Collecton
Optimum time for nonsurgical collection of embryos is decided based on the development
and transport rate of embryos in superovulated buffaloes . Our study (Misra et al., 1998), suggested
that nearly all the ovatembryos reached uterus around 5 .25 days after the onset of superovulatory
estrus (estrus=Day 0) . These observations were similar to Bulgarian (Karaivanov et al., 1990) and
Chinese reports (Wang et al. 1994), however, other researchers reported the descent of the bubaline
embryo into the uterus around Day 4 (Alexiev et al., 1988 ; Drost 1991) or between Days 5 .5 to 7
(Chantaraprateep et al. 1988) of the superovulated cycle . Accordingly, the ideal time for nonsurgical
collection of embryos in buffalo is around Day 5 .5 to 6 .0.
4.5
4 .75
5
525
5 .5
5 .75
After Sov estrus
Transport rate of ovalembryos in the superovulated
buffaloes (Misra et aI .,I 998)
42
6
AK Mirra : Application ofembryo biotechnology to augment reproduction andproduction in buffaloes
Embryo development in superovulated buffalo varies considerably and various stages
of embryos (8 cell to expanded blastocyst) may be recovered from the same donor buffalo
We observed that the circulating concentration of E2 and duration of E2 rise were
considerably higher in the superovulated cycle (Misra et al., 2000), and this may have
affected the development of buffalo embryos (Schallenberger . et al., 1990) .
0
L
E0 La,
0 0
U
>
0
4 .5
5
5 .5
6
After Sov estrus
Development rate of embryos in the superovulated buffalo
(M is ra et al ., 1998)
Our study (Misra et al., 1998) suggested that most of the early stage (4 cell ) embryos
developed to 8 to 16-cell stage/morula stage by Day 5 . Further, by Day 5 .5, most of the 8
tol6-cell embryos grew to morula or compact morula stage . Subsequent recovery of blastocysts
by Day 6 indicated further development of morula to the blastocyst stage by this time . Recovery
of 11 hatched blastocysts (of total 184 embryos recovered from 56 buffaloes) following nonsurgical embryo collection between Day 6 .0 to 6 .5 following Al in our earlier study (Misra et al.,
1990) clearly indicated that the buffalo embryos develop very fast after reaching the uterus (. Day
5.25) and hatch (-. Day 6.0-6 .5), leaving only about half a day to collect embryos prior to
hatching . This pattern of development of buffalo embryos closely resembles with other reports,
indicating 24 to 36 h faster growth of buffalo embryos than cattle (Chantatraprateep et at., 1989 ;
Drost and Elsden, 1985 ; Misra et al., 1990 ; Osman and Shehata, 2002).
13 . Embryo Recovery Rate
Embryo recovery in buffalo is reportedly poor (-50%) like cattle . Even after sacrifice
recovery of 20.5 to 32 .8% (9/44 to 22/67: Karaivanov et al., 1990), 56 .5% (26/46 : Wang et al.,
1994), 54% (Osman and Shehata, 2002) ; 46 .8% (74/158 : Misra et al., 1998) and 34.8% (Baruselli
et al., 2000) ova/embryos in buffalo and 22 to 63% in cattle (Gonzales et al., 1990 ; Mc Gowan et
al., 1985) has been reported . Loss of nearly half of the ova/embryos after superovulation in buffalo
may be due to high estrogen levels/ prolonged duration of rise in estrogen / high estrogen :
progesterone ratio during superovulatory estrus might not be allowing oocytes to be either picked
up or they may have been lost in the peritoneal cavity as a consequence of reversed peristalsis in the
oviduct (Misra et al., 1998). This conclusion is further substantiated by significant negative
correlations between the estrogen concentration and between the estrogens : progesterone ratio on
Day I after superovulatory estrus and total embryo recovery (Misra et al., 2000) .
43
AKMisra :Application of embryo biotechnology to augment reproduction andproduction in buffaloes
E2 :P4 RATIO IN THE SUPEROVULATED BUFFALOES IN
RELATION TO EMBRYO RECOVERY (MISRA et al ., 1998)
250 -
-.-More-VE
-a-Less-VE
0
Ca
L
aC14
W
-72
24 32
40
48
56
64
72
84
96
108 120 132 144
Hours post PG3
14. Effect of Bull on the Quality of Embryos
The comparison of frozen semen of five bulls, which were used to breed
superovulated buffaloes, suggested that the per cent viable embryo recovery varied
considerably as it ranged from 32 .6 to 84 .4% (32 .6, 35 .1, 43 .7, 65 .1, and 84 .4). Also, the
mean number of unfertilized ovum and per cent unfertilized ovum recovered varied
significantly between bulls (Misra et al., 1999c) .
EFFECT OF BULL ON THE FERTILZATION & VE RECOVERY
(Misra et al ., 1999c)
90 80 70
Cd
60
50
d
40 30 -
a
84 .4
o Viable embryos
a cgeneratea er -,ryca
a Unfertilised Ovum
36
46.5
-
43.7 '
41 .3
35 .1
65.1
37.7
36 .9
222
20
10
9_3
6 .3
0
Bull 42
' Bull 44
Bull 47
Bull 43
Bull 46
15 . Distribution of Embryos Collected Non-Surgically From Donor Buffaloes
In this study, out of 107 buffaloes used for embryo collection following superovulation, 6
(5 .65%) buffaloes which were having only 1 CL were not flushed and another 9 (8 .9%) buffaloes
did not produce any embryo ; thus from 107 superovulated buffaloes 15 (14 .02%) did not
produce any embryo . Another 25 (24 .8%) buffaloes produced only 1 to 2 embryos and
together with the above 9 buffaloes which did not produce any embryo, a total of 33 .7%
(34/101) of buffaloes flushed were observed to be very poor donors . Another 39 .6% (40/101)
buffaloes were also poor donors producing only 3-5 embryos . Remaining 16 .8% (17/101) and
9.9% (10/101) buffaloes produced 6-8 and > 8 embryos respectively ; thus together 26 .7%
buffaloes can be considered as good donors (Misra, 1996) .
44
AKMirra : Application ofembryo Giotecbrro/ogy to augment reproduction andproduction in bnffa/oes
Table 7. Distribution of total ova/embryos collected nonsurgically in buffaloes
Total ova / embryos
0
1-23-56-8>8
Total
9
25
40
17
10
Number of flushings
8.9
24.8
16 .8
39 .6
9.9
* Six buffaloes having only I CL were not flushed
Attributes
16. Embryo Recovery in Relation to Superovulatory Response
The study involving 107 buffaloes showed that the mean embryo recovery in relation
to superovulatory response, 0-5 (mean 3 .40 ± 0.17), 6-10 (mean 7 .67 ± 0.21) and > 10 CL
(mean 13 .69 ± 0.56) was 2.33 f 0.21 to 4 .86 ± 0 .40 and 8 .46 ± 1 .16, respectively . As the CL
number increased the mean number of total embryo recovery increased significantly .
However, the per cent total embryo recovery (62 .6%) with 0-5 ovulations was similar to
63.4% and 61 .8% when total ovulation increased to 6-10 and > 10 respectively ; suggesting
that the superovulatory response did not influence the per cent total embryo recovery .
Interestingly, however, when the total number of ovulations were > 15, only 33 .1% embryos
were recovered which were significantly (P < 0 .05) less than the recovery of 66 .4 and 72 .0
embryos when total ovulations were 6-7 and 11-15, respectively (Misra, 1996) .
Table 8 . Embryo recovery in relation to superovulatory response in buffaloes
Attributes
No. of animals
No. flushed (%)
No. of ovulations (Mean±S .E.)
Total ova/embryos recovered (Mean ±S .E.)
Per cent total ova/embryos recovered
Ovulatory response per donor
0-5CL
6-10CL
>10 CL
42
52
13
46
42
13
13 .69±0 .56
3.40±0 .17
7.67±0 .21
2.33±0 .21 a
4.86±0 .40b
8.46±1 .16`
62 .6
63 .4
61 .8
* 6 animals with only one CL were not flushed
Figures within a row with different superscripts differ significantly (P< 0 .005)
17. Effect of Interval From PGF2oc Treatment to Superovulatory Estrus on Embryo
Recovery
In one of the experiment involving superovulation of 54 buffaloes (Misra et al., 2003),
out of 83 buffaloes, 33 (39.8%) exhibited estrus within 36 h (Group 1), 45 (54 .2%) between 37 to
48 h (Group 11) and 5 (6 .0%) after 48 h (Group III) of PG3 treatment (Table 5.2). The number of
ovulations in the three groups were 5 .91 ± 0 .61, 6 .40 ± 0 .60 and 7.60 ± 2 .53, respectively;
however, the differences in ovulation rates among these groups were not significant .
The total number of mean (%) ova/embryos recovered was 3.77±0 .44 (60 .0%),
4.04±0.47 (57 .6%) and 5 .20±2 .82 (68.4%) for Groups I, II and III respectively and were not
statistically different . The fertilization rate in this study was significantly (P < 0 .005) higher when
the buffaloes exhibited oestrus between 24-36 h (72 .6%) or 37-48 h (77.1%) after PG3 treatment
compared to only 26 .9% when estrus was expressed after 48 h of PG3 treatment .
45
Table 9 . Effect of interval from PG3 treatment to superovulatory estrus on ovulatory response
and embryo recovery
Interval (h) from PG3 treatment to standing oestrus
24-36
37-48
> 48
(Group I)
(Group II)
(Group III)
33
45
No . of animals
5
30 (90 .9)
40 (88 .9)
3(60.0)
No . responded (%)
No. of ovulations (Mean±S .E .)
5.91±0 .61
6.40±0 .60
7.60±2.53
31 (93 .9)
41 (91 .1)
No . flushed (%)
5(100 .0)
Mean ova/embryos recovered total
3 .77±0 .44
4.04±0 .47
5 .20±2 .82
viable
2.25±0 .34a
2.43±0 .39a
0.20±0 .206
degenerated
0.48±0 .13
0.68±0 .18
1 .20±0 .58
unfertilized
0.84±0 .3 l a
0.70±0 .22a
3 .80±2.48 6
Fertilization rate (%)
72 .6a
77 .1 a
26.9 6
Degeneration rate (%)
17 .6a
21 .9a
85 .7 6
Attributes
18. Return to Estrus Time Following Pg5 Treatment
Early return-to-estrus after embryo collection would shorten the time interval between
consecutive superovulations and improve efficiency of embryo production . Following
superovulation and embryo collection, of 80 buffaloes treated with 15 .0 mg Luprostiol (PGF2(X
analogue) 67 .5% donors returned to estrus on average 11 .8±0.84 days after the PGF2a treatment.
The number of ovulations (5 5 or >5 CL) had no significant effect on per cent donors returning to
estrus within 30 days . However, an increase in the number of ovulations significantly delayed the
return to estrus as this duration was 9 .7 ± 0.93 days in the buffaloes with <_ 5 CL compared to
14 .1 ± 1 .29 days in those having > 5 CL (Misra and Pant, 2003) . The days to return to estrus
reported in this study (11 .8±0 .84) were significantly less than the 23 .39±3.99 days reported earlier
(Sarvaiya et al., 1993), but are in agreement with our earlier reports (Yadav et al., 1988a ;
Rajeshwaran et al., 1993).
Table 10. Return to estrus time following PGF 2 a treatment on the day of embryo collection
Attributes
No. of animals
No.responded (in oestrus) within 30 days (%)
Standing (%)
Non-standing (%)
Duration (days) of return to oestrus (Mean±S .E.)
Number of ovulations (CL)
<5
>5
40
28 (70 .0)
27 (96.4)
1(3 .6)
9.7 ± 0 .93
40
26 (65 .0)
26 (100.0)
14 .1 ± 1 .29
Total
80
54 (67 .5)
53 (98 .1)
1(1 .9)
11 .8 ± 0 .84
B . Effect of Superovulation and Embryo Collection on Milk Production.
The data on daily milk production of 36 buffaloes between 72 and 175 days post-partum
were analyzed. Overall mean values for daily milk production were 7 .56±0.21 kg during 15 days
before treatment (BT), 6.65±0 .23 kg during 12 days of superovulatory treatment and embryo
collection (DT) and 6 .25±0.18 kg during 15 days after embryo collection (AT). These results
indicate that in the buffalo despite a transient drop in milk production at 24 h after flushing, milk
production, in general, was not affected by superovulation and embryo recovery . To our
knowledge, this is the first such report in the buffalo ; however, these results seem to support
earlier reports in cattle (Greve, 1981, Haaland et al., 1983) Bak et al., 1989).
46
AK Mirra : Application ofembryo biotechnology to augment reproduction andproduction in buffaloes
• s 7 0 11 Isis iviss1 a•
078041
Os
d
in mBk
Fig 1 : oonVwb a
nd
Mon r»s l Jo*iC rab d dwkw
and aAriC.' xa~pwtil~lo
s
rn
It
rp•atd dope w~r+a+c raMnMnt ~aoh
VWQ
Table 11 . Estimated linear regression equations for daily milk production before, during and
after superovulation and embryo collection
Treatment periods
Before treatment
During treatment
After treatment
Regression equations
Y= 7 .555 - 0.0589 X
Y= 6 .648 - 0.0028 X
Y= 6 .247 - 0.0056 X
R2
0.0123
-0 .0023
-0 .0017
F
7.69**
0.01
0.06
Y= Milk yield, X= Days in milk, ** (P< 0 .005)
IN-VITRO EMBRYO PRODUCTION
The serious limitations of in-vivo embryo production include poor superovulatory
response of nearly half of the donors following superovulation treatment and high cost of
FSH used for superovulation . -On the contrary, in-vitro embryo production is the most
economic and efficient technique to produce embryos in bulk to meet the demand of elite
animal production as well as newer reproductive technology research . Merton et al., (2003)
estimated that approximately 50 freezable embryos can be produced per cow per year using
superovulation compared to 150 embryos per year using ovum pick-up (OPU) and in-vitro
embryo production (IVEP) . In this technique, oocytes may be aspirated from live cows /
buffaloes using ultrasound guided trans-vaginal technique, popularly called as ovum pick-up
(OPU) or oocytes may be collected from the ovaries collected from the slaughtered animals .
Following aspiration, oocytes are evaluated and subjected to In-vitro maturation (IVM), Invitro fertilization (IVF) and in-vitro culture (IVC)) . This technique to produce embryos has
become so popular that over 3,30,848 embryos were produced in-vitro in the year 2004 . Now
47
AK Misra :Application
offembryo
bidtecbnology to augment reproduction andproduction in buffaloes
OPU-IVEP is used commercially for the large scale embryo production in several countries
(Merton et al., 2003) . Recently OPU-IVEP technique has been used for the collection of
oocytes from buffaloes (Boni et al., 1997 ; Galli et al., 1998, 2001 ; Manik et al., 2002 : Huang
Youjun et al., 2004 ; Yadav et al., 2006 ; Gasparrini, 2006 : Xianwei et al., 2006), leading to
the birth of several calves (Galli et al., 1998 ; Huang Youjun et al ., 2004) .
Oocyte Recovery
From the slaughterhouse ovaries, oocytes are harvested by slicing of ovary/ follicular
aspiration or follicular dissection (Boni et al., 1994b ; Das et al., 1996a). Oocytes recovered by
slicing are reported to be more than the other two methods as the follicles embedded deep in the
ovarian cortex are also recovered, however, this method of oocyte recovery is more time
consuming 4nd less practical (Das et al., 1996a) and is not used routinely . Oocyte recovery by
follicular ablation method, which takes more time than follicular aspiration, is also not efficient .
Therefore, oocyte recovery by aspiration of follicles is considered to be the most practical
method.
Compared to cattle, recovery rate of immature oocytes in buffalo is poor . The recovery
of acceptable quality oocytes in buffalo is reported to be 0 .4 (Totey et al., 1992 ; Madan et
at.,1994b), 0 .6 (Das et al., 1996a; Nandi et' al., 2000), 1 .76 (Samad et al., 1998) and 2 .4
(Gasparrini et al., 2000), which is mainly attributed to very low primordial follicle reserve
pool in Surd (12000, Danell, 1987), Nili-Ravi (-19000, Samad and Nasseri, 1979) and other
buffaloes compared to - 150000 in cows (Erickson, 1966) and high incidences of follicular atresia
in swamp (Ocampo et al., 1994) and riverine (Palta et al., 1998) buffaloes .
The average yield of oocytes per ovary per OPU session in buffaloes was reported to
be 1 .33 (Boni et al., 1994a) and only 31 .3% were suitable for in-vitro embryo production .
However, in gonadotropin (FSH) stimulated animals, oocyte yield increased to an average of
3 .0 per ovary per OPU (Boni et al., 1994b) . Maturation of these is reported to range from
44.5% to 58.3% (Tavares et al. 1997; Boni et al., . 1997) leading to the development of 0 .15
to 0 .33 embryos per session or of about 15 .7 to 34 .6 transferable embryos per buffalo, if the
oocytes are collected bi-weekly . Yadav et al., (2006) reported an average recovery of 1 .82
(without ovarian stimulation) and 2 .27 (with 1500 IU eCG stimulation) oocytes foliwing
biweekly collection . Contrarily, however, Kityanant (1995) reported recovery of mean
number of total, usable and grade I oocytes as 12 .4, 5 .5 and 4 .0, respectively and obtained
87 .5% maturation, 67 .8% cleavage and 26 .7% blastocyst development . In China, Huang
Youjun et al., (2004) has also reported recovery of mean 5 .34 total and 3 .18 usable oocytes
of which 45 .2-70.8% matured and 18 .8-30 .6% developed to biastocyst stage.
In -Vitro Maturation (IVM) of Oocytes
Various types of defined and undefined media have been used for in-vitro maturation
of buffalo oocytes . Buffalo oocytes are generally cultured for 24 hours in complex medium
such as TCM-199 (Singh et al ., 1989 ; Totey et al., 1991, 92 . 93 ; Madan et al., 1994a ; Dhanda
et al., 1996 ; Das et al., 1996b ; Chauhan et al., 1996, 1997b), Ham's F-10 (Totey et at.,1993),
Ham's F-12 (Singh et al., 1989; Chauhan et al., 1991 ; Totey et al ., 1993), minimum essential
medium and way mouth medium (Ravindranatha et al., 2001) . However, these media have
48
AKMisra :Application of embryo biotechnology too augment reproductiot, and production in buffaloes
been developed for general cell culture and are not tailor-made for the culture of oocytes, hence
cannot support high level of oocytes maturation and needs to be supplemented with serum .
Hormones like FSH, LH, estradiol and growth factors like EGF, IGF are reported to
be beneficial for maturation of oocytes (Chauhan et al., 1998c, 1999) . LH in estrus serum
initiates resumption of meiosis in dictyate oocytes, which are arrested in M-I1 stage (OcanoQuero et al., 1994) . Higher cytoplasmic maturation (60-80%) had been achieved by addition
(10-20%) of fetal calf serum (FCS ; Totey et al., 1991 . 1992, 1993a,b, 1996), fetal bovine
serum (FBS ; Chauhan et al., 1996, 1997a,b, 1998a-e, 1999 ; Nandi et al., 1998), oestrus
buffalo serum (OBS ; Totey et al ., 1992 ; Madan et al., 1994a,b ; Chauhan et al., 1998a) proestrus buffalo serum (PrBS ; Samad et al ., 1998), Oestrus cow serum (OCS ; Samad et al.,
1998), human serum (HS ; Chuangsoongneon & Kamonpatana 1991), steer serum (SS ;
C~auhan et al., . 1998a) or superovulated buffalo serum (SBS ; Chauhan et cd., 1998a) to the IVM
media. Hormones like human chorionic gonadotropin (Chuangsoongneon and Kamonpatana,
1991), follicle stimulating hormone (FSH) (Chauhan et at., 1996), pregnant mare serum
gonadotropin (PMSG) (Gupta et al., 2001 b), luteinizing hormone (LH) and estradiol either alone
or in combination (Totey et al., 1992 ; Nandi et al., 2002) have also been used to induce higher
maturation rates . Additionally, various growth factors such as epidermal growth factor (EGF)
(Chauhan et al., 1999 ; Raghu et al., 2002), EGF plus fibroblast growth factor (FGF) (Gupta et al.
, 2002), insulin like growth factor - II (IGF-I1) (Chauhan et al., 1998c) have been used to increase
oocyte maturation and post fertilization cleavage rates .
Addition of LH to maturation medium (TCM-199+20% OBS) improved maturation
rate from 47% to up to 77% (Totey et al., 1992) . Contrarily, however, Madan et al.,
(1994a,b) reported 80% maturation rate in TCM-199+20% OBS, without the use of any
hormones . On the other hand, Totey et al., (1992) reported higher maturation rate (81 .7%) in
TCM-199 and BES as compared to Ham's F-10 (71 .7%) . A maturation rates of 70-80% has
been achieved by using TCM-199 supplemented with FBS & 5 .ig/ml FSH-P (Chauhan et al.,
1996, 1997a, 1998a, c, e, 1999) . Defined and semi defined media has also been used
successfully for IVM of buffalo oocytes by others (Abdoon et al., 2001 ; Gupta et al., 2002 ;
Raghu et al., 2002) .
To develop cost effective maturation media, partial or complete replacement of basic
medium with follicular fluid lead to 65 and 100% maturation, respectively (Chauhan et al.,
1997c; Gupta et al., 2001 a). Taziki et al., (2000) obtained higher maturation rate in medium
containing 10 or 20% follicular fluid in comparison to the TCM-199 alone. Choi et al.,
(1997) got higher maturation rate (98%) when bovine oocytes were matured in large
follicular fluid than that in small follicular fluid (69%) . Regardless of higher maturation rate,
low blastocyst yield was reported when follicular fluid was used during IVF and culture of
embryos (Nandi et al ., 2004) . While using rabbit peritoneal fluid, Singh and Dhanda (1997)
recorded higher maturation and cleavage than the control .
C. In -Vitro Fertilization (IVF) of Oocytes
Buffalo oocytes matured in-vitro is generally fertilized with frozen-thawed in-vitro
capacitated spermatozoa in TALP medium (Totey et al., 1996) or BO medium (Nandi et al .,
1998). In buffalo, low fertilization rate (19 .6%) was recorded when capacitated frozen semen
was used in HEPES-TALP medium (Totey et al., 1991). Subsequently, this improved to
29 .8% by using BO medium (Chauhan et al ., 1997a) . A higher fertilization rate of 78 .15%
49
was reported in Murrah buffaloes by Madan et al., (1994b). An in-vitrofertilization rate of
60-80% has been reported in both the media . In-vitro capacitation of buffalo spermatozoa is
carried out by incubating motile spermatozoa separately by swim up (Nandi et al., 1998) or
Percoll gradient technique (Totey et al., 1996) . Sperm motility enhancers commonly used
includes caffeine (Chauhan et al., 1998a ; Nandi et al., 1998), theophylline (Chauhan et al.,
1998e), pentoxifyline (Ramesha et al., 2000) and a mixture of penicillamine, hypotaurine and
epinephrine (PRE) (Totey et al., 1996) . A fertilization rate of 55% and 40% were reported
with caffeine and theophylline, respectively . Considerable variations have been reported
among different buffalo bulls in terms of the ability of their spermatozoa to fertilize oocytes
in-vitro (Totey et al., 1996 ; Chauhan et al., 1998d) . Different authors have reported different
sperm concentration and sperm oocyte co-incubation period optimum for buffalo IVF . A
sperm concentration of 4-5 millions with a co-incubation period of 20-24 h resulted in 6075% fertilization rate (Totey et al ., 1996 ; Nandi et al., 1998) . There was a high occurrence of
polyspermy observed among IVF buffalo oocytes and increasing sperm concentration from 1,
5 and 10 x 106 increased polyspermy from 24.0%, 43 .2% and 64 .0%, respectively (Ocampo et
al ., 1996) . We observed that the cleavage rate following the use of mSOF supplemented with 5
tg Heparin and 8 mg BSA ml-1 was better (50 .33%) than the FERT TALP supplemented with 5
pg Heparin and 6 mg BSA ml-I (Misra and Sharma, 2004-unpublished) .
D . Embryo Culture
The in vitro culture of embryo requires an appropriate environment so that the early
embryos could undergo several cleavage divisions to enable it to reach the blastocyst stage of
development. In vitro embryonic development is critical in order to increase the IVEP efficiency
in buffalo (Gasparrini, 2002). Some of the factors which affect the in vitro culture in buffalo are
as follows.
To optimize the culture conditions for the development of majority of cleaved
zygotes to blastocysts, buffalo embryos have been cultured in vitro in, (i) complex medium
comprised TCM-199 with cumulus cells (Madan et al., 1994b), oviductal epithelial cells
(Nandi et al., 2003), (ii) semi defined media (Raghu et al., 2002) or (iii) defined media, viz.
CRlaa (Totey et al., 1996) and mSOF (Nandi et al., 2003 ; Misra et al., 2004), mCR2aa
(Kumar et al., 2006) . The yield of blastocyst was lower in serum free semi defined media
(P<0 .05) than in complex co-culture system . The most commonly used media for in vitro
embryo culture were the co-culture of fertilized oocytes with oviductal cells, granulosa cell
monolayer, BRL/VERO cell monolayer in defined media .
More recently, buffalo zygotes have been successfully cultured in either SOF or in
another defined cell-free system, termed potassium simplex optimized medium (KSOM)
(Zicarelli et a1.,2003) . Culture of the cleaved embryos in SOF supplemented with BSA and
essential and non-essential amino acids resulted in a higher cleavage rate (60-70 %) with
production of up to 45 % blastocysts (Galli et al., 2001) . Some authors suggested better
cleavage rate in culture media TCM-199 supplemented with 10% fetal calf serum and BRL
cells as compared to SOF media (60 vs 55 ..1%) although post cleavage development was
better in SOF media (13 .5 vs 17%) (Boni et al., 1999). The proportion of cleaved embryos
that developed to morula + blastocyst stage was significantly (P<0 .05) higher for mSOFaa
and mSOFaa + 10% FBS (62 .3 and 61 .5%, respectively) than that for mCR2aa and mCR2aa
+ 10% FBS (54.2 and 58 .6%, respectively) which in turn, was significantly (P<0 .05) higher
50
than that for TCM-199 + 10% FBS + BOEC (48.6%) in OPU derived buffalo oocytes
subjected to IVMFC (Kumar et al., 2006) .
The bovine oviductal cells provide the ideal microenvironment for the final
maturation of oocytes, fertilization and early embryonic development . Madan et al. (1994a)
and Singh and Dhanda (1997) also reported low blastocyst development (6 .10 and 8 .10%)
from buffalo oocytes co-cultured with cumulus + oviductal cells . On the contrary, Chauhan et
al., (1997a) recorded a high rate of morula (41%) and blastocyst (31%) development from
buffalo oocytes using same co-culture system . Nandi et al., (2003) also reported significantly
higher embryo development and blastocyst yield in the co-culture system than in defined
medium . Using oviductal cell co-culture with mSOF, it was observed that on an average 33%
(maximum 54%) of the cleaved oocytes became morula and average 7% (maximum 9 .4%)
became, blastocyst and majority hatched during subsequent culture (Misra, ,2004) . For the
purpose of in vitro culture, BRL (buffalo rat liver) cell line (Boni et al., 1999 ; Kruip et al., 1994)
and Vero cell line (kidney epithelial cell line of green monkey) (Lacaze et al., 1997; Duszeuska et
al., 2000) have also been successfully used and culturing of oocytes on BRL cells monolayer
resulted in the development of 28 % blastocyts (Gibbons et al., 1994). It has been reported that the
use of cumulus cell monolayer and buffalo oviductal epithelial cell (BOEC) for culture improved
the development of morulae and blastocysts (Totey et al., 1992 ; Madan et al., 1994a) .
Supplementation of the embryo culture medium with IGF-1 (Narula et al., 1996) or
insulin (Chauhan et al., 1997c) was found to increase the blastocyst production.
Supplementation of culture medium with 3-ME and cysteamine had beneficial effect on
development of 6-8 cells stage bovine embryos to the blastocyst stage (Takahashi et al.,
1993) and Feugang et al., (2004) confirmed that when (3-mercaptoethanol is added to culture
system, it increases GSH levels and reduces the basal level of apoptosis in IVP blastocysts . It
was reported that addition of cysteamine in maturation medium had beneficial effect on post
cleavage embryo development (Gasparrini et al., 2000) and cysteemine induced glutathione
synthesis significantly enhanced the buffalo embryo development by protecting the embryo
from oxidative stress (Zicarelli and Gasparrini, 2004) . Use of insulin in culture media
(TCM199) supplemented with 10 % FBS and co-cultured with oviductal cells also improved
the blastocyst production up to 34 % (Chauhan et al., 1997c).
It was observed that buffalo embryos those complete first cleavage before 30 hr post
insemination are more likely to develop to blastocyst than those completing first cleavage
after 30 hr and the quality and viability of former were superior (Totey et al., 1996). Hatching
is a good indicator of embryo quality and supplementation of IVC medium with FBS, BSA,
and insulin had been reported to increase the hatching rate of buffalo embryos (Chauhan et
al., 1998b) .
E. Embryo Cryopreservation
Cryopreservation of embryo is an essential component in the commercial embryo
transfer program as not only the surplus embryos (more than the available recipients) can be
frozen; it facilitates global movement of the complete animal as embryo . The first successful
freezing and transfer of the post thaw good quality buffalo embryos in to 8 recipients reported
3 confirmed pregnancies (37 .5% conception), however, subsequently all the 3 pregnancies
resulted in abortion at > 5 months gestation (Misra et al., 1990) . Soon after, we reported for
the first time successful culmination of pregnancy and birth of 9 calves following transfer of
51
AK Mum :Application
of embryo biotechnology to augment reproduction and production in buffaloes
frozen-thawed buffalo embryos (Kasiraj et al., 1993) . In this experiment, transfer of total 39
frozen/thawed buffalo embryos resulted in 11 pregnancies (28% conception) . During phase I
and the phase II of the Department of Biotechnology sponsored project, about 700 buffalo
embryos were frozen (Misra et al., 1999a). In general, the conception rate following transfer of
frozen buffalo embryos appeared to be similar to that of lower grade fresh embryos ; however, it is
lower than the frozen cattle embryos . Campanile et al., (1995) observed no difference in the
average pregnancy rate of the fresh or frozen-thawed embryos (31 .4% vs. 28 .0%) . Neglia et al., .
(2003), reported the conception in buffaloes following the successful freezing of the in-vitro
generated embryos, leading to the birth of the first calves (Neglia et al., 2004) in Italy. The
transfer of in-vitro generated and vitrified blastocysts using oocytes of the slaughtered buffaloes,
however, resulted in birth of several calves in Philippines (Duran et al., 2004) . Attempts are being
made to vitrify immature buffalo oocytes (Wani et al., 2003, 2004) to create 'Oocyte Bank' for
the in-vitro production of embryos .
EMBRYO TRANSFER AND CONCEPTION
Compared to cattle, there are limited reports regarding the pregnancy and birth of
buffalo calves following embryo transfer (Campanile et al., 1995 ; Misra et al., 1999b) .
Initially, for obvious reasons including training period, the pregnancy rate following embryo
transfer was reported to be only 10-18 % (Alexiev et al., 1988 ; Kurup, 1988). Subsequently,
following transfer of mostly non-freezable embryos (grade II & III) the pregnancy rate improved
significantly to 25-26 .4% on the farm (Misra et al., 1992, 1999b) and 29.4 % in the field (Misra et
al., 1999a) . A higher conception rate of 60 % was also observed following transfer of only Grade
I embryos, however, the pregnancy rate declined to 25 and 14% when grade II and III embryos
were used (Misra et al., 1999b). Other workers (Campanile et al., 1995) reported 30 .2%
pregnancy rate following transfer of 76 grade *1 and 2 embryos . Poor conception in buffalo has
been attributed to the lack of overt signs of oestrus, seasonality in breeding, difficulty in per rectal
palpation of CL, subclinical metritis and generally low fertility in this species because of lack of
selection of breeding stock (Jainudeen, 1989 ; Drost, 1991).
The factors which may influence pregnancy rate following embryo transfer are listed
below (Misra et al., 1999b) :
A. Season
Embryo transfer during autumn and winter months had no significant effect on the
pregnancy rate (20 .7 vs 29 .0%) .
B. Natural/ Induced Estrus
Pregnancy rate of recipients that came into estrus naturally (25 .0%) was similar to
those in which estrus was induced (28 .2%) with administration of PGF2a (Misra et al.,
1999b) . Use of in-vitro produced vitrified embryos, however, indicated better conception rate
during natural than induced estrus (Duran et al., 2004) .
C. Synchrony of Donor/Recipient Estrus
The pregnancy rate was highest (40 .7%) following transfers when donors and
recipients were closely synchronized (Misra et al., 1999b) . This was in agreement with earlier
report (Misra and Joshi, 1991) . Interestingly, however, pregnancy rates were compromised
52
AK Mrsra :Application of embryo biotechnology to augment rep rodue*on and production in buffaloes
when recipients were in estrus as much as 12 h before (pregnancy rate 14 .3%) or 12 h after
the donor (pregnancy rate 18 .5%), although the observed decline was not significant . Asynchrony
beyond 12 h on either side resulted into total conception failure (Misra et al., 1999b) . In our
earlier study in buffalo, pregnancies occurred even when recipients were in oestrus as much as 40
h after the donor (Misra and Joshi, 1991). More studies are therefore required to ascertain if
precise synchrony is indeed essential for obtaining higher pregnancy rate in buffalo .
D. Size of Recipient CL
The pregnancy rate nearly doubled with an increase in CL size from -5 mm (15 .8%)
to > 10 mm (31 .8%) . It remained to be seen if this increase was due to higher circulating
progesterone levels in recipients with bigger CL . Interestingly, however, Campanile et al .,
(1995) observed significantly higher conception in the recipients with smaller CL (<1 .0 cm)
at the time of embryo transfer than those with a large (1 .3 cm) CL .
E. Side of Ovulation and Pregnancy Rate
In our study (Misra et al., 1999b), 48 .3% of the CL were on the right side and 51 .6%
on the left side of the ovary of the recipients indicating no difference on the incidence of right
versus . left ovulations, which is in contrast to cattle in which ovulation on right side
predominates (Reece and Turner, 1938 ; Hasler et al., 1987) . There was no significant
difference in pregnancy rate following embryo transfer to the right (31 .8%) or left uterine
horn (21 .3%) of the buffalo as it is in cattle (Greve, 1981 ; Wright, 1981) .
F. Stage of Embryo
The average pregnancy rate follwing transfer of morula or blastocyst (29 .4% vs .
30 .5%) was not very different (Campanile et al., 1995). We too observed no difference in the
conception rate following transfer of different stages of buffalo embryos (Misra et al.,
1999b) . Interestingly, however, we observed (Misra et al., 1999b) that the nonsurgical
transfer of one 8-cell stage embryo along with a 16-cell stage embryo lead to twin pregnancy
in a recipient which culminated in the birth of a male and a female calf indicating that 8-cell
embryos recovered on Day 6 are capable of establishing pregnancy although they are
considered retarded by about two days (Drost, 1991) .
G. Embryo Quality
There was a significant effect of embryo quality on conception rate following
transfer in synchronized recipients . The grade I embryos resulted into a significantly higher
(60.0%) pregnancy rate than grade 11(25 .0%) or III embryos (13 .9%) . We are not aware of
any such earlier study in the buffalo but these results are similar to earlier reports in cattle
(Markette et al., 1985 ; Hasler et al., 1987).
ESTRUS INDUCTION FOLLOWING PGF2a TREATMENT IN THE SUPEROVULATED
BUFFALO
Early return-to-estrus after embryo collection would shorten the time interval
between consecutive superovulations and improve efficiency of embryo production .
53
Following superovulation and embryo collection, of 80 buffaloes treated with 15 .0 mg
Luprostiol (PGF2a analogue) 67 .5% donors returned to estrus on average 11 .8±0 .84 days
after the PGF2a treatment . The number of ovulations (<_ 5 or >5 CL) had no significant effect
on per cent donors returning to estrus within 30 days . However, an increase in the number of
ovulations significantly delayed the return to estrus as this duration was 9 .7 ± 0 .93 days in the
buffaloes with _< 5 CL compared to 14 .1 ± 1 .29 days in those having > 5 CL (Misra and Pant,
2003). The days to return to estrus reported in this study (11 .8±0 .84) were significantly less
than the 23 .39±3 .99 days reported earlier (Sarvaiya et al., 1993), but are in agreement with
our earlier reports (Yadav et al., 1988a ; Rajeshwaran et al., 1993) .
SEXING AND CLONING
In order to have the desired sex of the progeny, sexing of buffalo embryos has been
I
demonstrated successfully by workers in India (Appa Rao and Totey, 1999) and Italy (Manna
et al., 2003), using polymerase chain reaction , however, there is need to verify the technique
following transfer of the sexed embryos and resultant pregnancy . However, recently Lu et al .,
(2006) reported the birth of the first buffalo calves using sexed semen for IVF, which is the
more practical method of producing sexed embryos .
The cloning technology has been used to multiply the desired animals using
embryonic, fetal and somatic cells in several species including sheep, cattle, goat, and pigs .
Cloned buffalo embryos have been produced using embryonic (Singla et al., 1997), fetal
(Saikhun et al., 2004 ; Meena and Das, 2005) and adult somatic cells (Parnpai et al., 2004) . It
has also been demonstrated that vitrified swamp buffalo oocytes (Parnpai et al., 2004) and
bovine oocytes cytoplasm (Kitiyanant et al., 2001) can be used for the production of cloned
buffalo blastocysts . Pregnancy following transfer of cloned buffalo embryos have been
reported in Thailand (Saikhum et a1.,2004), however, pregnancy could not continue up to
term and the claf following transfer of cloned embryo is yet awaited . Recently (Shi et al.,
2006) reported the birth of 6 female cloned swamp buffalo calves in the year 2004-2005
using either fetal fibroblast or granulosa cells and demonstrated that . this technology can be
effectively used to produce buffalo clones.
PARTHENOGENESIS
Considering the fact that cleavage rate following IVF in buffalo is poor compared to
cattle and its application in somatic cell cloning and stem cell technology, we (Mishra et al, .
2006) compared cleavage and embryo development rate follwing either chemical activation
or IVF (natural activation). Abattoir derived good quality oocytes collected by aspiration
from 3-10 mm follicles were matured for 22-24 h and subsequently subjected to either IVF
(control) or chemical activation (treatment) : For IVF, in vitro matured oocytes were coincubated with in-vitro capacitated _1X106 frozen-thawed sperm of Murrah bull and fertilized
in mSOF medium . Chemicals for oocytes activation comprised of a) .7% ethanol (ET) for 7
min + 2 .5 mM 6-Di methyl amino purine (6-DMAP) for 4 h, b) .7% ET for 7 min + 10 .tg/ml
Cycloheximide (CHX) for 6 h and c) . 7% ET for 7 min + 2 .5 mM 6-DMAP + 10 p.glml CHX
for 6 h.Fertilized and chemically activated oocytes were cultured in mSOF medium for 8
days to study embryo development .
54
AK Misra :Application of embryo biotechnology to augment reproduction andproduction in buffaloes
The cleavage rate was significantly high following ET+DMAP, ET+CHX and
ET+CHX+DMAP activation (52 .5, 52 .5 and 44 .4%) than IVF (36.5, 23 .4 and 26 .8%),
respectively . Blastocyst development (30 .9 vs 15 .2%) was also significantly high following
ET+CHX+DMAP activation than IVF . The results of parthenogenesis revealed that buffalo
oocytes had better inherent developmental competence and the poor cleavage and embryo
development following IVF, in our study, may partly be due to the poor quality of frozenthawed sperm, improper sperm capacitation and/ or fertilization .
CONCLUSIONS & FUTURE PROSPECTS
1 . In the past two decades an excellent infrastructure of embryo biotechnology labs and
skilled manpower has been created to adopt embryo biotechnology in buffaloes p an
important tool for the faster multiplications of elite buffaloes and their genetic
improvement . In spite of limited oocyte pool and . restricted recruitment of follicles
following superovulation, even at the present level of success in terms of transferable
embryo recovery per superovulation (2 .5-3 .0) and conception rate (35-40%),
conventional in-vivo embryo production technology can be effectively used for
production of breeding stock of buffaloes . Simultaneously more research should be
conducted to improve efficiency of ET further, especially in respect of superovulatory
response, embryo recovery, embryo freezing and conception following embryo transfer .
Also there is need to study the reasons for the loss of 50% or more embryos after
superovulation and ways to recover these embryos . Recovery of even half of these lost
embryos may add substantially to the overall embryo yield per donor .
2. To meet the acute shortage of breeding bulls of various breeds, concerted efforts needs to
be made to mount this technology at the Regional/ State level with sufficient elite donors
and recipients and best available manpower to produce breeding bulls to cater the need of
that region.
3. Except limited studies on OPU-IVEP, most of the in-vitro buffalo embryos have been
produced from oocytes harvested from abattoir derived ovaries . Although blastocyst yield
has improved significantly in the last few years, conception rate following transfer of
these embryos is still poor and only limited number of calves has been produced from invitro generated embryos . Therefore, despite these encouraging results more studies are
required to improve efficiency if in-vitro embryo and calf production so that the same
could be used for research in the areas of sexing, cloning, transgenesis, stem cell
techniques and in the breeding programs for the genetic improvement of buffalo .
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61
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'
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Question :
How long the time between repeated SO?
In my experience, the difference between first SO and the second consecutive SO was
3 months . If we do, the embryo collected is not significance difference that showed
number of decreased significant after second SO .
OPU in cattle is very difficult because we don't expect to collect oocytes . What we did
in our USG for OPU was with juvenile that aged 5-6 months old, it's very young and
small and then we induced GnRH . In cattle we had got good quality of oocytes . May
be you have experience in buffalo .
Answer :
In case of buffalo, animal should come back normally to estrus after 21 days . So my
data on this, it's about 10 days from the day of collecting, and leaved for 21 days . So,
all of it was 31 days . Then you start the treatment . Regarding the availability of
oocytes, we got recovery embryo of 50% .
67

application of embryo biotechnology to augment

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