Buffalo Follicular Dynamics

1. Buffalo follicular dynamics: Basic & applied concepts
RK Sharma, SK Phulia, Jerome A, AK Balhara
&
Inderjeet Singh
(c) JEROME A

2. Ovarian follicular dynamics
Follicular dynamics cattle (Ginther et al. 1989), buffaloes (Taneja
et al. 1996)
Numerous work on follicular dynamics of buffalo.
(Baruselli et al.1997; Singh et al.2000; Manik et al.2002; Ali et
al.2003; Presicce et al.2005; Azawi et al.2009; Yindee et
al.2011; Sharma et al. 2012)
Follicular growth in buffalo occurs in a wave-like pattern.
A follicular wave involves synchronous growth of a group of
follicles in both ovaries, from which one follicle attains dominance
over others to become the dominant follicle
Follicles failing to ovulate – undergo atresia
(c) JEROME A

3. Follicle development stages:
Recruitment, Selection, Growth, Dominance, Regression &
atresia
Dominance Regression
Growth
Selection
Next
Recruitment Recruitment
Usually 2 to 3 follicular waves occur during the estrous cycle in
buffaloes
(c) JEROME A

4. Endocrinology of the Estrous Cycle w.r.t
follicular dynamics
Basic endocrine mechanism leading to the occurrence of the
wave pattern - similar between cattle and buffalo in terms of
follicular dynamics
In buffalo that a transient rise in serum concentrations of
FSH begins each follicular wave (Presicce et al. 2003)
Decreased episodic secretion of LH is associated with loss of
dominance and with the end of a non-ovulatory follicular wave.
Several intra-follicular growth factors: endocrine, autocrine
or paracrine actions that modify gonadotropin stimulated
follicular growth and differentiation
(c) JEROME A

5. Hormones controlling follicular development
FSH, LH, Estrogen, Progesterone (Direct)
PG (Indirect)
Other intra-follicular factors
IGF-1, BMP1
EGF, FGF, PDGF ,
IGF (including IGF-binding protein)
TGFβ - (inhibin & activin)
HGF - (cytokines)
(Bruno et al.2009)
(c) JEROME A

6. BUFFALO FOLLICULAR
DYNAMICS
(c) JEROME A

7. Fetal period
During fetal development, germinal cells is formed in a series of stages
such as migration of primordial germinative cells to the gonadal ridge,
(proliferation and initiation of prophase I, blocking at diplotene stage, and
finally, the formation of primordial follicles)
Transition in morphological features of follicles, - presence of oogones and
primordial follicles from 0 to 3 months of pregnancy, followed by preantral
follicles (primary and secondary follicles) between 4 to 6 months, and
preantral follicles and in some cases, antral follicles from 7th month to the
end of pregnancy (Carvalho et al. 2007)
(c) JEROME A

8. Increasing fetal age: Marked decrease in the proportion of primordial and
primary follicles, and a concomitant increase in secondary follicles.
Mean number of follicles isolated at different stages of development, and
at different fetal ages is generally characterized by considerable
individual variation (Santos et al.
2006)
Buffalo primordial follicles: 10,000–20,000 compared with over 100,000 in
cattle (Danell, 1987)
(c) JEROME A

9. Pre-pubertal period
Follicular development in prepubertal buffalo heifers, crucial to initiation
of puberty that is often delayed in the species (Cockrill, 1980), has not
been studied in detail.
Delayed puberty leads to higher age at first calving and lower life-time
milk production and net calf crop.
CIRB studies suggest that follicular growth in prepubertal buffalo heifers
occurs in a wave-like pattern. (Sharma et al. 2010, 2012)
The largest DF of each wave attains a diameter of 7-11 mm and
subsequently undergoes atresia without ovulation. The regression of DF is
followed by emergence of a new follicular wave.
(c) JEROME A

10. Studies revealed growing dominant follicles respond to exogenous
GnRH either with atresia, ovulation or formation of luteinized follicles.
GnRH induced corpus luteum is smaller and short-lived and these
heifers failed to resume ovarian cyclicity following CL regression.
These findings suggest that although the pituitary of prepubertal
buffalo heifers is responsive to exogenous GnRH, the H-P-O axis
remains immature and does not maintain cyclicity.
(c) JEROME A

11. Puberty
Puberty when they reach about 55–60% of their adult body
weight, but the age at puberty widely ranges from 18 to 46
months (Jainudeen and Hafez, 1993) and body weight of 250 to
400 kg for river buffaloes,
Influenced by factors: genotype, nutrition, management, climate,
year or season of birth and diseases.
As puberty approaches, the growth rate and maximum size of
dominant follicle increases until a sufficient size
(Sharma et al. 2010)
Circulating estradiol to produce LH surge and ovulation are
achieved. As puberty approaches, continued estradiol becomes
less inhibitory to LH pulses and circulating LH rises.
(c) JEROME A

12. On puberty, the number of estradiol receptors in hypothalamus
decreases, resulting in a possible decrease in negative feedback
of estradiol on release of LH pulses
(Adams et al. 1994)
The subsequent increase in frequency of LH pulses allows the
dominant follicle to grow to ovulatory size and produce enough
estradiol to induce an LH surge and ovulation.
buffalo heifers do experience follicular development in wave
like pattern, but largest dominant follicle size and its growth
rate remain lower than in adult buffaloes.
(c) JEROME A

13. POST PUBERTY
Ovarian follicular growth during the estrous cycle in buffalo - similar to
cattle, characterized by primarily two and less commonly three-waves of
follicular recruitment, growth and regression. (Taneja , 1996)
Buffaloes have estrous cycles with 1-wave (3.3%), 2-waves (63.3%) or 3
waves (33.3%) of follicular growth
The first wave begins on day 1, the second around day 9-11 while the third
wave appears on day 17 of the estrous cycle.
Only the DF of the final wave ovulates, whereas the DFs of the preceding
waves undergo atresia.
The wave-like pattern of ovarian follicular growth continues even during
anoestrus period and diameter of the largest DF may attain a size
equivalent to that of the pre-ovulatory follicle . (Manik et al. 2002)
(c) JEROME A

14. Number of follicular waves per cycle and their characteristics have been
reported in buffalo from different countries.
In Brazil, Baruselli et al. (1997) showed that the number of follicular waves
during an estrous cycle was one in 3% of animals, two in 63% and three in
33%.
A study on Egyptian buffalo, however, indicated the majority of estrous
cycles (54%) had three waves of follicular development
(Barkawi et al. 2009).
A study on suckled Thai swamp buffalo has shown that the first postpartum
ovulation was followed by a short estrous cycle (10.2±0.38 days) in 84% of
the animals, but the prevalence of these decreased thereafter.
The mean diameters of ovulatory follicles increased between the first and
second ovulation (13.50±0.52–14.31±0.38 mm). (Yindee et al. 2010)
(c) JEROME A

15. CIRB studies revealed the incidence of two and three wave cycles in
two-third and one-third proportion, respectively, of adult buffaloes.
The first wave usually commences on day 1 (day 0 = ovulation), while the
second wave emerges earlier in buffaloes with three wave cycles. The
third wave emerges on day 16.8 in the latter group.
The estrous cycles with two and three follicular waves of follicular
development differed in the mean length of the luteal phase (10.4
compared to 12.7 days) and the interval between ovulations (22.3
compared to 24.5 days).
Our studies in cyclic Murrah buffaloes during summer season indicated
that there was a predominance of 2 wave compared to 3 wave cycles with
the diameter of the ovulatory DF at estrus being 14.32±0.43 mm.
(Sharma et al. 2009)
(c) JEROME A

16. The anovulatory DF also attained maximum diameter of 13.46±0.58 mm on day
11.14±0.96 of the cycle. In three wave buffaloes, the average durations of the
first, second and the ovulatory waves were 23.14±1.14, 13.0±1.0 and 12.85±1.06
days, respectively.
Buffaloes with two-waves of follicular development during estrous cycles, the
growth rate and diameter of the largest follicle are significantly smaller in
heifers than cows. (Sharma et al. 2009)
(c) JEROME A

17. Anoestrus
Continuous waves of follicular growth
In each wave a variable number of small sized follicles emerged
together, one of which subsequently developed to a size of 7.1-19.2 mm in
diameter.
Remaining follicles in the wave ceased to grow further and started
regressing.
Largest DF fails to ovulate and undergo atresia
Mean maximum diameter of dominant follicle (DF) was 12.52±0.22 mm and
that of SF was 7.58±0.19 mm. This DF diameter: comparable to DF
diameter of ovulatory follicles.
The growth phase and regression phase duration of DF did not differ
significantly. (10.96±0.41 and 10.64±0.29 days).
(c) JEROME A
(Sharma et al. 2009)

18. The wave persisted for a period of 21.18±0.53 days, whereas
average duration of subordinate follicle (SF) was 9.86±0.36 days
In anoestrus buffaloes, the DF undergoes atresia rather than
ovulation, possibly due to failure of appropriate pre-ovulatory LH
surge..
This study revealed that the anestrus condition is due to failure
of the DF to ovulate rather than failure in follicular
development.
Anoestrus condition is due to failure of DF to ovulate rather
than to their absence (Savio et al. 1990)
Insufficient release of LH from the pituitary (Roche et al. 2000)
(c) JEROME A

19. Effect of Superovulation on follicular dynamics
Superovulation associated with embryo-recovery and embryo-transfer to
synchronized recipient females is considered an effective means of
increasing the contribution of high quality females to the gene pool of the
population.
Superovulatory effect of PMSG and FSH have been used to increase
ovulation rates in buffaloes and have been applied in conjunction with
progestagen and/or prostaglandin F2α treatments to regulate the oestrus
cycle.
superovulation by 3000 IU of PMSG in buffaloes, rapidly induced LH surges
of low magnitude, causing unovulated follicles to become endocrinologically
active (Shallenberger et al. 1990)
FSH reported as effective as the multiple dose regimen (Kasiraj et al.
1992) or to produce a lower superovulatory response compared to a multiple
injection regimen (Misra, 1997)
(c) JEROME A

20. Buffalo- poor superovulatory response: poor follicular response to
exogenous gonadotrophins, inherent endocrine patterns as well as to the
characteristics of the follicular population and ovarian folliculogenesis.
Nine to 14 ovulatory size follicles were consistently observed in
buffaloes stimulated with FSH (Baruselli et al. 1999). ovulation rates of
62.8 percent, a value similar to that found in cattle (Desaulniers et al.
1995; Shaw et al. 1995; Stock et al. 1996)
Number of ovulations presented a high correlation (0.86) with the
number of corpora lutea found on the day of embryo collection, but only
one to three ova/embryos were recovered (average recovery rate/CL =
30 percent)
(c) JEROME A

21. Increase in the number of ovulations has been reported when
superstimulatory treatments were initiated in the absence of a
dominant follicle or when the dominant follicle was in a regressing or
plateau phase
(Taneja et al. 1995).
Low number of embryos: failure of oocytes to enter the fallopian
tubes and/or impaired transport of ova/embryos
(c) JEROME A

22. Research at CIRB
In superovulated buffaloes, follicles of over 10 mm diameter may ovulate
following FSH treatment as the preparation does contain some LH
contamination.
This mid cycle ovulation is not desirable as this new CL does not respond
to PG and continues to grow at the time of expected SOV heat – disturbs
hormonal synchrony, ovulation, fertilization, embryo development.
Ovulatory response: low, CL developed following SOV treatment were of
short duration and showed regression by day 10 of superovulatory cycle.
Synchronize the superovulatory treatment with the emergence of a new
follicular wave via ultrasound guided DF aspiration / ablation was
attempted prior to initiation of superovulatory treatment.
With ablation, improved results were obtained vis-à-vis mean number of
follicles greater than 9 mm at AI as well as greater mean number of CLs
and viable embryo recovery (Sharma et al. 2011)
(c) JEROME A

23. Hormonally intervened cycle
Follicular dynamics changes according to the hormonal
treatment
Synchronization of oestrus with Ovsynch protocol resulted in Most
ovulations were synchronized and recorded at AI and the following day in
nulliparous (24/30; 80%) and pluriparous (12/14; 85.7%) buffaloes
respectively.
A follicle shift was recorded in 14 of 30 (46.6%) and 11 of 14 (78.5%) in
nulliparous and pluriparous buffaloes respectively .
Pregnancy rate following Ovsynch protocol was 40% (12/30) and 42.8%
(6/14) in nulliparous and pluriparous buffaloes respectively.
(c) JEROME A

24. PRID-PMSG protocol: Majority of ovulations were synchronized and
recorded at first, second AI and following day (76.4%). A follicle
shift occurred in 88.2% animals and among the 12 recorded
pregnancies, 11 derived from follicle shift (91.6%). Pregnancy rate:
70.5% .
Progestagen treatment on acyclic buffaloes resulted in significantly
higher pregnancy rate compared with Ovsynch protocol
Presicce et al. 2005
(c) JEROME A

25. Gnrh Treatemnt
Post GnRh treatment ovarian dominant follicle became persistent in all
females given 100 µg ovulated within 48 h, subsequent with an emergence
of a new follicular wave - 100 µg of Gonadorelin seems to be the most
effective dose to induce ovulation followed by an emergence of a
new follicular wave in river buffalo
Rastegarnia et al. 2004
Replacing GnRH with LH in the ovulation synchronization protocol
in buffaloes in ovsynch protocol: Ovulation rate after the first GnRH was
86.6% ;Ovulation rates 93.3% after the second dose of GnRH and 93.3%
after LH .
Ovulation occurred 36.4+/-10.4 h after the first GnRH. The interval for
treatment to ovulation was 26.5+/-9.6 h for buffaloes treated with GnRH
and 24.4+/-7.9 h for buffaloes treated with LH ; the time of ovulation
did not differ statistically between the two groups (GnRH versus LH)
Conception rates: 56.5% (GnRH) and 64.2% (LH). Therefore, the
exogenous injection of LH can substitute the GnRH injections in the
Ovsynch program in buffaloes.
(c) JEROME A de Araujo Berber et al. 2002

26. PGF2alpha treatment
The dynamics of ovulatory follicular growth before oestrus were similar
in buffaloes undergoing spontaneous and PGF(2alpha)-induced luteolysis
and majority of buffaloes had a two wave pattern of follicular growth
and emergence of a third wave was associated with a longer luteal phase.
Warriach and Ahmad, 2007
Efficacy of PGF(2alpha) for causing luteolysis and synchronizing estrus
and ovulation in buffalo cows : dependent upon plasma progesterone
concentration, CL size and ovarian follicular status before treatment.
Brito et al. 2002
Studies by Sharma et al. 2009 using PG in subestrus Murrah heifers
All heifers, receiving 750 µg tiaprost, returned to estrus after mean
interval of 4.13±0.69 days.
Majority of heifers (66.7%) ovulated the largest dominant follicle,
present at the time of PG injection; while remaining 33.3% ovulated the
second largest follicle.
Ovulatory follicle grew @0.88±0.14 mm/day and reached a diameter of
11.25±0.35mm on the day of estrus.
(c) JEROME A

27. CL regressed @ 46.9±4.9 mm2/day and declined from 237.6±12.5 mm
at treatment to 79.9±7.9 mm2 on the day of estrus. Pregnancy rate at
estrus following treatment was 46.7% (7/15).
Sharma et al. 2009
Ovsynch: There is high degree of synchrony for estrus and ovulation in
cyclic buffaloes but developing CL fails to sustain its viability for full
cycle in 33% buffaloes
There is reduced diameter of ovulatory DF at induced estrus (13.45±0.82
mm vs 15.08±0.64 mm at subsequent spontaneous estrus).
Only one follicular ovulatory wave was recorded during short estrous cycle
in contrast to two waves during normal length cycle.
Malik et al. 2010.
(c) JEROME A

28. Ovarian response to Ovsynch: Buffaloes
Largest DF present at the time of 1st GnRH, ovulated or luteinized only
in 33% buffaloes.
In response to the 2nd GnRH, however, all buffaloes responded with
ovulations. largest DF which ovulated.
In 78% buffaloes, the largest DF ovulated but it was the 2nd largest DF
which ovulated in the remaining 22% buffaloes.
Ovulation, characterized by sudden disappearance of the largest or the
2nd largest ovarian follicle, occurred between 24-48 h after the 2nd
GnRH in majority of treated buffaloes, with rare exceptions in which
ovulation was recorded 24 h before the 2nd GnRH.
These findings suggest greater synchrony of estrus and ovulation
following Ovsynch treatment of cyclic Murrah buffaloes.
(Sharma et al. 2012)
(c) JEROME A

29. Conclusion
Ovarian follicular dynamics in the buffalo species :
similar to cattle in terms of follicular dynamics and basic
endocrine mechanism - leading to the occurrence of the
wave pattern
Waves seen from fetal and continue throughout the life
span even during anestrus, pregnancy
Hormonal treatment, follicular ablation, presence of
corpus luteum affects follicular dynamics
Several intra-follicular growth factors exert
endocrine, autocrine or paracrine actions for follicular
growth and differentiation needed for oocyte
competence
(c) JEROME A

30. Thank you
(c) JEROME A