CSIRO PUBLISHING

Reproduction, Fertility and Development, 2019, 31, 68–81

https://doi.org/10.1071/RD18315

Effects of reproductive season on embryo development

in the buffalo

Bianca Gasparrini

Department of Veterinary Medicine and Animal Production, Federico II University,

Via F. Delpino 1, 80137 Naples, Italy. Email: bgasparr@unina.it

Abstract. Interest in buffalo farming is increasing worldwide due to the critical role played by buffaloes as sources of
animal protein in tropical and subtropical environments. However, reproductive seasonality negatively affects the
profitability of buffalo farming. Buffaloes tend to be short-day breeders, with seasonality patterns increasing with greater
distances from the Equator. Although ovarian cyclic activity may occur throughout the year, seasonal anoestrus and cycles
in calving and milk production are recorded. When buffaloes are forced to mate during the unfavourable season, to meet
market demand, they may undergo a higher incidence of embryo mortality. This review addresses the effects of the
reproductive season on embryo development in the buffalo, analysing the different factors involved in determining embryo
mortality during the unfavourable season, such as impaired luteal function, oocyte competence and sperm quality. The
review then focuses on strategies to control the photoperiod-dependent annual fluctuations in conception and embryo
mortality in the female buffalo.

Additional keywords: Bubalus bubalis, photoperiod, luteal function, oocyte competence, sperm quality.

Published online 3 December 2018

Introduction
The worldwide interest in buffalo farming has been steadily
increasing over the years, as indicated by the significant popu-
lation growth from 89 million to 199 million head (þ125%)
between 1961 and 2013 (Zicarelli 2016). This is primarily due to
the pivotal role played by this species as a source of animal
protein in tropical and subtropical developing countries. Else-
where, like in Italy, the buffalo is linked to the production of
mozzarella cheese, in great demand worldwide and a major
source of income for dairy farmers.
Water buffaloes are divided into two subspecies, the river
buffalo (chromosome number ¼ 50) and the swamp buffalo
(chromosome number ¼ 48), with the former, bred for milk
and meat production, primarily accounting for the increased
number of head. Indeed, swamp buffaloes, historically bred as
draught animals in South-east Asia, have been decreasing due
to the advent of mechanisation in agriculture, and are often
cross-bred with the river type to meet increased milk demand.
Several factors make buffalo an irreplaceable protein source
in low-input production systems, such as the high feed
conversion efficiency, relatively low maintenance require-
ments, capacity to exploit pastures restricting cattle produc-
tion, perfect interaction between seasonality and forage
availability in most countries, resistance to diseases, adapt-
ability to tropical environments, longevity and, hence, a lower
culling rate (Zicarelli 1994, 2016; Paul et al. 2002). Indeed,
river buffalo fits most climatic areas, from the Equator to
higher latitudes, being present in Asia, Europe, the Middle
East, northern Africa, North and South America and Australia
(Zicarelli 1994).
The reproductive activity of buffalo cows is strongly influ-
enced by environmental cues, with nutrition and photoperiod
contributing to different extents according to the areas con-
cerned. Around the equatorial belt, where daily light length is
similar across the year, feed scarcity is the major factor affecting
reproduction (Vale et al. 1996), resulting in delayed puberty and
long inter-calving intervals (Zicarelli 2007). In contrast, in
subtropical countries and at higher latitudes, where there are
annual cyclic variations in the light : dark ratio, photoperiod has
a major effect on ovarian cyclic activity, with increased fertility
recorded during months with decreased day length (Campanile
et al. 2010; Zicarelli 2016). In most countries where buffaloes
are bred, the breeding season coincides with the annual period of
greater forage availability, corresponding to months with
decreased daylight length (i.e. July–November in tropical coun-
tries north of the Equator; Singh and Desai 1979; Singh and Lal
1992; Qureshi et al. 1999). Indeed, seasonal breeding is a
survival strategy adopted by many wild mammals to ensure that
their progeny are born at the most favourable time of year, when
the weather is milder, food is available and infectious agents and
parasites are less aggressive. The biological programming of
births in accordance to the most appropriate environmental
conditions undoubtedly results in higher chances of offspring
survival (Wood et al. 2006). In contrast, in Italy, there is a
marked tendency for buffalo to give birth during a period when
forage availability is poor and the temperature is not beneficial

Journal compilation Ó IETS 2019 www.publish.csiro.au/journals/rfd

Reproductive seasonality in the buffalo Reproduction, Fertility and Development 69

(a) 25

20

15

10

0
J F M A M J J A S O N D

Italy India Venezuela Pakistan

(b) 35
30
25
20
15
10
5
0
J F M A M J J A S O N D

5 10 20 30

Fig. 1. (a) Monthly calving rates in Italy, India, Venezuela and Pakistan. (b) Monthly calving
rates in Brazil at different latitudes. Adapted from Zicarelli (2016).

16 13.0

15
12.5
Naples-Delhi-Lahore

14
12.0

Venezuela
13

12 11.5

11
11.0
10
10.5
9

8 10.0
J F M A M J J A S O N D

Naples Delhi Lahore Apure (Venezuela)

Fig. 2. Daylight hours in Naples (Italy), Delhi (India), Apure (Venezuela) and Lahore (Pakistan).
Adapted from Zicarelli (2016).

to offspring survival (Zicarelli 1997, 2016). In addition, because
a balanced diet is consistently provided throughout the year
under current production systems, the seasonal pattern of
reproduction clearly responds to an endogenous rhythm linked
to the photoperiod (Zicarelli 1994). This pattern of seasonality,
with reproductive events not synchronised with forage avail-
ability, indicates that buffaloes bred in Italy are not autochtho-
nous and that seasonality is a remnant of their place of origin
(i.e. tropical and subtropical areas corresponding to the large
valley of the Indus River in Pakistan). The pattern of seasonality
is similar in Asia, Italy, Venezuela and Colombia, with calving
concentrated between July and December (decreasing daylight
length), with the opposite pattern in the Southern Hemisphere
(i.e. in Brazil; Fig. 1).
Buffalo seasonal anoestrus in countries north of the Equator
has been attributed to either heat stress (India, Pakistan, Egypt),
low environmental humidity (Venezuela) or photoperiod (Italy).
Interestingly, despite differences in environmental temperature
and rainfall, all these countries share the same pattern of
daylight hours, confirming the importance of photoperiod as
the main factor affecting reproduction in this species (Fig. 2).
However, it is worth pointing out that in countries like India,
70 Reproduction, Fertility and Development
20
15
% 10
0
J F M A M J
Month
Control
Fig. 3. Monthly calving rates in farms where seasonality is not modified (control) and in farms where
the out-of-breeding mating strategy (OBMS) is used. Adapted from Zicarelli (2016).
Pakistan and Egypt, heat stress may further contribute to
seasonal anoestrus (Ahmad et al. 1981; Singh and Nanda
1993; Marai and Haeeb 2010).
The seasonal effect on reproductive activity is regulated by
melatonin, secreted by the pineal gland in response to darkness,
which acts on the hypothalamic–pituitary axis, increasing
gonadotrophin-releasing hormone (GnRH) and gonadotrophin
concentrations in short-day breeders, leading to follicular
growth and ovulation (Misztal et al. 2002). In Italian Mediterra-
nean buffaloes, higher plasma concentrations of melatonin were
detected 2 h after sunset in animals with a marked tendency to
seasonality (Zicarelli 2016). Furthermore, upon moving these
animals to farms with herds that were less sensitive to photope-
riod stimuli and had low plasma melatonin concentrations, this
pattern did not change (Di Palo et al. 1997). It was also reported
that the difference in plasma melatonin concentrations between
day and night is much lower in buffalo heifers, which are less
sensitive to photoperiod, than in buffalo cows (Borghese et al.
1995). The high repeatability (0.733) of plasma melatonin
concentrations (Di Palo et al. 1993) suggests that this parameter
be included in genetic selection programs.
Therefore, buffaloes tend to be short-day breeders, with the
seasonality pattern increasing at greater distances from the
Equator. Although ovarian cyclic activity may occur throughout
the year, seasonal anoestrus and cycles in calving and milk
production are observed, with a consequent negative economic
effect on the profitability of dairy buffalo farming (Campanile
et al. 2010; Zicarelli 2016). In Italy, where buffalo farming is
strictly linked to the production of mozzarella cheese, marketed
under the European Union’s Protected Designation of Origin
(PDO) label, the peak of lactation does not meet the milk market
demand. Hence, in order to distribute calving around the year to
ensure continuity of market supply, it is necessary to use the out-
of-breeding mating strategy (OBMS), which consists of inter-
rupting sexual promiscuity or the use of AI during the favourable
season (Zicarelli 1997). The OBMS strategy has been found
to be successful at modifying the calendar of calving according
to market demand (Fig. 3), but it results in decreased fertility.
Indeed, because buffaloes are forced to conceive during the
unfavourable season when ovarian activity is reduced, embryo
B. Gasparrini
J A S O N D
OBMS
mortality may be higher (Baruselli et al. 1997; Campanile et al.
2005; Campanile and Neglia 2007). In addition, buffaloes
calving during the unfavourable season show long postpartum
anoestrous periods, with a certain proportion resuming ovula-
tion in the following breeding season (Zicarelli 2007).
The present review focuses on the effects of the reproductive
season on embryo development in the buffalo, analysing the
different factors involved in determining embryo mortality
during the increased daylight length periods. Therefore, because
embryo development results from both a suitable environment
for embryo growth and gamete competence, a description of
corpus luteum (CL) function and progesterone secretion, as well
as oocyte competence and sperm quality, in different seasons is
provided. The review then addresses strategies that can be used
to control the photoperiod-dependent annual fluctuations in
conception and embryo mortality in female buffalo.
Embryonic mortality
Embryonic and fetal mortality (FM) are the main factors
affecting pregnancy outcome during months with increasing
daylight length (i.e. in the winter–spring transition at Italian
latitudes). Embryo loss in buffalo may occur between Days 15
and 24 (early embryonic mortality (EEM)), as well as between
Days 25 and 45 (late embryonic mortality (LEM)), whereas fetal
loss normally takes place between Days 46 and 90 (Vecchio
et al. 2010). However, the most critical window in the Medi-
terranean buffalo is 25–45 days after AI, with LEM accounting
for 20–40% of embryonic losses during the season characterised
by an increasing number of daylight hours (Campanile et al.
2005, 2007a, 2008a). A lower incidence of LEM (,7%) was
reported in Brazil during periods with decreasing daylight
length (Baruselli et al. 1997; i.e. summer–autumn transition),
whereas a LEM rate of 20% was recorded in river buffaloes in
equatorial areas (Vale et al. 1989). In a more recent trial con-
ducted in Italian Mediterranean buffalo, despite similar preg-
nancy rates on Day 25 across seasons (,60%), a higher
incidence of LEM was recorded in the increasing day length
period compared with the breeding season (23% vs 7%; Di
Francesco et al. 2012a). Seasonal effects on LEM were also
Reproductive seasonality in the buffalo
observed after natural mating (Vecchio et al. 2007). Indeed,
season is the main factor causing this phenomenon, which does
not seem to be affected by age, parity or lactation, with infec-
tious agents only accounting for 2–8% of embryonic losses
(Campanile et al. 2005). Therefore, embryo loss during the
unfavourable season is related, in part, to impaired luteal func-
tion, leading to reduced progesterone concentrations (Campa-
nile et al. 2005, 2007a), and, in part, to decreased oocyte
competence (Di Francesco et al. 2011, 2012b).
Seasonal effects on luteal function
Inadequate luteal function is involved in both EEM and LEM, as
well as in FM. Interestingly, the incidence of EEM is also higher
in the non-breeding season (Campanile et al. 2010, 2016), when
plasma progesterone concentrations during oestrous cycles and
early pregnancy are lower compared with the breeding season
(Di Francesco et al. 2012a; Campanile et al. 2016). A delayed
increase in progesterone concentrations was reported in river
buffaloes undergoing EEM, suggesting that adequate secretion
of progesterone early in the cycle is required to support buffalo
embryo development (Campanile et al. 2010, 2016). The critical
role of progesterone in EEM is also confirmed by the efficacy of
a prostaglandin (PG) F2a treatment at the time of AI to increase
circulating progesterone concentrations on Days 10 and 20,
which significantly improved pregnancy rates (Neglia et al.
2008). In addition, in buffaloes transitioning to seasonal
anoestrus, a higher pregnancy rate was recorded on Day 40 in
animals showing increased progesterone secretion in response
to GnRH treatment on Day 5 after AI (Campanile et al. 2008b).
Faster CL growth from Day 5 to Day 10 after AI, associated with
increased vascularisation and an earlier increase in progesterone
concentrations, was observed in pregnant buffaloes compared
with buffaloes that did not become pregnant (Vecchio et al.
2012). The importance of the early development and function of
the CL in supporting embryo development and pregnancy was
recently confirmed in buffalo (Neglia et al. 2015). Indeed, early
development of the CL on Day 5 was associated with increased
expression of vascular endothelial growth factor and Factor
VIII, a denser vasculature, increased blood flow and greater
secretion of progesterone. This confirms previous findings in
cattle and sheep, showing an association of vascularisation of
the CL and blood flow with progesterone synthesis and release
(Stormshak et al. 1963; Kobayashi et al. 2001). An early
increase in circulating progesterone is critical for appropriate
embryo growth and elongation in cattle (Carter et al. 2008;
Beltman et al. 2009). Embryo elongation in buffaloes is thought
to occur from around Day 13, with the preattachment phase
occurring from Day 17 to Day 24 and the transitory attachment
phase being initiated around Day 25 (Campanile and Neglia
2007). It is likely that EEM is caused by failure of maternal
recognition of pregnancy, due to inadequate progesterone
secretion, leading to a decreased capacity of the developing
embryo to secrete enough interferon-t (IFNT) to prevent
luteolysis (Wathes et al. 1998).
In contrast with other ruminants, EEM as a result of failure of
pregnancy recognition has relatively little effect on mating
outcomes in buffalo, with the main loss occurring between Days
Reproduction, Fertility and Development 71
25 and 45 after mating (Campanile and Neglia 2007; Campanile
et al. 2010; Vecchio et al. 2010). With regard to LEM, an earlier
trial recorded a high incidence of LEM (45%) during the non-
breeding season; as mentioned previously, the presence of
infectious agents in the uterine flushing only accounted for
8% of these losses (Campanile et al. 2005). In that study,
animals undergoing LEM had lower progesterone concentra-
tions on Days 10 and 20 after AI than those maintaining
pregnancy on Day 45, indicating that impaired luteal function
certainly plays a role in embryo mortality. However, decreased
progesterone concentrations were only found in approximately
50% of buffaloes with LEM, suggesting that other factors may
also contribute to embryo loss. Subsequently, it was observed
that the decline in progesterone between Days 10 and 20 plays
the main role in determining LEM (Russo et al. 2010). Using
colour flow Doppler ultrasonography it was possible to show
that increased blood flow to the CL on Days 10, 20 and 25 is
linked to a positive pregnancy outcome in buffalo (Russo et al.
2010; Vecchio et al. 2012; Neglia et al. 2015). This is likely due
to increased angiogenesis (Papa et al. 2007) and progesterone
secretion, known to be associated with blood flow in the buffalo
(Neglia et al. 2015). The decrease in progesterone after Day 10
may interfere with attachment and implantation processes by
affecting mucin 1 (MUC-1) expression in the uterine epithelium.
For proper embryo attachment, adequate concentrations of
progesterone are required because progesterone decreases the
expression of MUC-1, a transmembrane receptor present on the
apical surface of the epithelium (Perucatti et al. 2006), creating a
barrier to the developing blastocyst (Geisert and Malayer 2000).
A comparison between seasons (breeding vs transition
periods, characterised by decreasing and increasing daylight
length respectively) showed a seasonal effect on CL structure
and function, as well as on pregnancy outcome in the Mediter-
ranean buffalo (Di Francesco et al. 2012a). As expected, a
higher LEM was recorded in the transition period compared
with the breeding season. Interestingly, progesterone concen-
trations on Days 10 and 20 after AI decreased two- to threefold
in the transition compared with breeding season. This differ-
ence in progesterone concentrations between seasons was also
observed in animals that maintained pregnancy. The improved
steroidogenic activity of the CL was not due to the size and
growth of the CL, but rather to increased blood flow, as
indicated by the greater timed average medium velocity
(TAMV) recorded on Day 10 during the breeding season.
Regardless of season, pregnant buffaloes had greater CL
dimensions and area, progesterone concentrations and TAMV
than buffaloes undergoing LEM. In addition, in buffaloes
pregnant on Day 45, the CL increased in size (long axis) from
Day 10 to Day 20, whereas the size of the CL decreased in non-
pregnant buffaloes, with this trend more evident during the
breeding season (Di Francesco et al. 2012a).
The effectiveness of hormone treatments increasing proges-
terone secretion on Day 25 after mating or AI in reducing LEM
(Campanile et al. 2008a) confirms the importance of high levels
of progesterone around Day 27 (Days 25–32; corresponding to
the transition phase of embryo attachment in buffaloes) for the
maintenance of pregnancy. Interestingly, in buffaloes undergo-
ing LEM, embryo growth is retarded, as indicated by the smaller
72 Reproduction, Fertility and Development
embryo size (particularly width) on Day 25 compared with that
of pregnant animals (Neglia et al. 2012; Balestrieri et al. 2013).
The delayed development is likely due to decreased progester-
one, because progesterone regulates growth and elongation
(Carter et al. 2008; Beltman et al. 2009). Therefore, retarded
embryo development on Day 25 and reduced progesterone can
be considered indicators of imminent embryo mortality. Pro-
gesterone plays an important role in ensuring a suitable uterine
environment for the conceptus, and hence insufficient concen-
trations may alter the structure and function of the uterine
endometrium, which, in turn, affects the developing embryos
(Clemente et al. 2009; Forde et al. 2011). Indeed, retarded
buffalo embryos on Day 27 exhibit an altered transcriptome
profile compared with non-retarded embryos, with extensive
changes in the expression of genes involved in developmental
processes, cell metabolism and differentiation, complement
cascade and coagulation, signalling, ion transport, secretion,
cell junctions, adhesion and extracellular matrix components
(Strazzullo et al. 2014). In addition, the proteome profile of the
chorioamnion and adjacent caruncles of retarded embryos was
substantially altered, especially for proteins involved in protein
folding, protease inhibition, calcium binding, DNA and RNA
binding, chromosome segregation, cytoskeletal organisation,
cell redox homeostasis, coagulation and hemoglobin binding
(Balestrieri et al. 2013). Interestingly, transcripts for annexin A2
and annexin A3 were upregulated in retarded embryos (Straz-
zullo et al. 2014), whereas annexin A1 and annexin A2 proteins
were downregulated in corresponding chorioamnions (Bales-
trieri et al. 2013). Annexin A2 is involved in cellular growth and
adhesion and has been implicated in embryo attachment to the
endometrium (Garrido-Gomez et al. 2012) and placentation
(Talbot et al. 2010). Annexins A1 and A2 also affect the
synthesis of prostaglandins (Bennett et al. 1994), involved in
several reproductive events, including maternal recognition of
pregnancy, implantation and maintenance of pregnancy
(Weems et al. 2006). The deregulation of both transcripts in
retarded buffalo embryos and proteins of the serpin family in the
chorioamnions and caruncles ( Balestrieri et al. 2013; Strazzullo
et al. 2014) suggests a disequilibrium in proteolytic control
that has been reported in human pre-eclampsia (Stark 1978;
Blumenstein et al. 2009). In retarded chorioamnions, there was
also extensive downregulation of proteins involved in cell redox
homeostasis (disulfide isomerase A3, aldose reductase and
peroxiredoxin-1 (PRDX1)) and heat shock proteins (HSPs), a
family of stress-induced proteins upregulated by a variety of
stresses, including exposure to oxidative radicals. Retarded
chorioamnions showed upregulation of amine oxidase A, a
potent vasoactive mediator that regulates blood flow and capil-
lary permeability (Barkai and Kraicer 1996), and ezrin, a
membrane–cytoskeletal linking protein known to be regulated
by IFNT in the cow uterus during attachment (Gray et al. 2006;
Ledgard et al. 2009). Caruncles adjacent to retarded embryos
also showed upregulation of haptoglobin, known to modulate
the maternal immune system and hence with a possible role in
immunological tolerance during attachment (Herrler et al.
2003), and downregulation of a-chain fibronectin, a component
of the extracellular matrix involved in the development of fetal–
maternal vascular communication and the stabilisation of
B. Gasparrini
implantation in mice and humans (Iwaki et al. 2002; Blumen-
stein et al. 2009).
Therefore, what is unequivocal is that in the non-breeding
season the decreased progesterone concentration during early
embryonic development affects embryo growth, resulting in
delayed development of the conceptus, as well as marked
changes in transcriptome and proteome profiles of the embryos
and the chorioamnios/caruncles respectively, interfering with
the capacity of the embryo to attach to the uterine endometrium.
Seasonal effects on oocyte competence
Another important factor contributing to reproductive failure in
the non-breeding season is oocyte developmental competence,
normally acquired during the maturation process. Several
studies have investigated seasonal effects on the follicle popu-
lation and oocyte quality in buffalo at different latitudes, with
small discrepancies likely due to differences in breed, man-
agement and donor age. However, a common denominator of
these studies is evidence of a marked seasonal effect on ovarian
activity. Indian authors reported a seasonal effect on in vitro
embryo production (IVEP) efficiency in buffaloes undergoing
ovum pick-up (OPU), mainly related to the decreased follicle
population: the numbers of aspirated follicles, recovered
oocytes and blastocysts produced per animal per session were
reduced during the unfavourable season, whereas oocyte
developmental competence, assessed in terms of cleavage and
blastocyst rates, was unaffected (Manjunatha et al. 2009).
Recently, a decreased follicle and oocyte population was
observed in abattoir-derived ovaries during summer by Egyp-
tian authors who attributed the seasonal effect to heat stress
(Abdoon et al. 2014). However, in that study oocyte competence
was also impaired during the unfavourable season, as indicated
by poorer maturation, cleavage and blastocyst rates, as well as
altered gene expression. During the unfavourable period,
HSP70, known to act as a proapoptotic factor, was upregulated
in both cumulus–oocyte complexes (COCs) and IVM oocytes
(Abdoon et al. 2014). This may indicate a role for heat stress in
buffalo reproductive seasonality, because the expression of
HSP70 increases in cumulus cells of bovine COCs exposed to
high temperatures (Payton et al. 2011). IVM buffalo oocytes in
the non-breeding season also exhibited downregulation of
glyceraldehyde-3-phosphate dehydrogenase, involved in
energy metabolism, and upregulation of b-actin (ACTB), a
constituent of the actin cytoskeleton, implicated in cortical
granule translocation (Wessel et al. 2002). This is in line with
the disruption of cortical granule translocation (Payton et al.
2004) and premature aging resulting from hastened cytoplasmic
maturation observed in bovine oocytes exposed to 418C during
IVM (Edwards et al. 2005). In addition, it was reported previ-
ously that high environmental temperatures affect oocyte yield
and the competence of abattoir-derived buffalo oocytes (Nandi
et al. 2001).
Nevertheless, in temperate climates, like that of Italy, the
seasonality pattern is exactly the same as in tropical countries
north of the Equator, with coincident non-breeding and breeding
seasons, despite differences in the temperature humidity index
(THI), confirming the primary role played by photoperiod in the
Reproductive seasonality in the buffalo Reproduction, Fertility and Development 73

(a) 80 16 35 b 16
B 15 15
75 30
AB 14

TMBL/ BL adv BL
AB 14
Cleavage

70 13 25

Light
65 13
12 20 b

Light
A 11 a 12
60 15
10 a 11
55
9 10 10
50 8 a a
Jan-Mar Apr-Jun Jul-Sep Oct-Dec 5 9
Months 0 8
% cleavage Light (h) Transition Spring-summer Fall

(b) 30 % TMBL % adv BL Light (h)

16
b
28 ab ab 15
TMBL/ BL G 1&2

26 Fig. 5. Tight morulae–blastocysts (TMBL) and advanced blastocysts (adv

24
22
ab a
20
18 ab
16 a
14
12
10
Jan-Mar Apr-Jun Jul-Sep
Months
% TM  BL % G1&2 BL
A, B,
Values with different letters are significantly different; P  0.01
a, b
Values with different letters are significantly different; P  0.05
Fig. 4. (a) Cleavage rate of abattoir-derived buffalo cumulus–oocyte
complexes (COCs) in different seasons in relation to daylight hours. (b)
Tight morulae–blastocysts (TM þ BL) and good-quality blastocysts (BL G
1&2) as a percentage of all abattoir-derived COCs, in different seasons in
relation to daylight hours. Adapted from Di Francesco et al. (2011).
buffalo. A retrospective analysis of data collected from abattoir-
derived ovaries over a 3-year period showed an improvement of
oocyte developmental competence during decreasing daylight
months, with higher cleavage and blastocyst rates (Fig. 4), but
no effects on the total number of oocytes or on the number or
percentage of good-quality oocytes (i.e. Grade A þ B; Di
Francesco et al. 2011). Therefore, although oocyte quality
assessed by morphological criteria was unaffected, the ability
of buffalo oocytes to be fertilised and develop into viable
blastocysts improved significantly during the breeding season.
Subsequently, the seasonal effect on oocyte developmental
competence was confirmed in Italian Mediterranean buffaloes
undergoing OPU, with a twofold increase in blastocyst yields
recorded in autumn compared with mid-winter and spring–
summer (Fig. 5), despite similar follicular and oocyte popula-
tions (Di Francesco et al. 2012b). These data are in accord with
the seasonality pattern of the species, showing increased fertility
during short-day months. Reproductive activity in the buffalo is
similar to that in other short-day breeders, like small ruminants
in subtropical environments and at higher latitudes (Zicarelli
1994, 1997; Vale et al. 1996; Zicarelli et al. 1997). In sheep,
oocyte competence also decreases during the unfavourable
season, characterised by increasing daylight hours, as revealed
by the poorer response to multiple ovulation (Mitchell et al.
2002), the reduced cleavage rates after IVF of ovulated oocytes
14
b BL) as a percentage of all cumulus–oocyte complexes in different seasons in
13
relation to daylight hours. Adapted from Di Francesco et al. (2012b). Values
Light
12 with different letters differ significantly (P , 0.01).
11
10
9
(Stenbak et al. 2001) and decreased blastocyst yields obtained
8
Oct-Dec from abattoir-derived oocytes after IVEP (Mara et al. 2014).
In Italian Mediterranean buffalo, a seasonal effect was also
Light (h) recorded on embryo quality, indicated by poorer cryotolerance
of in vitro-produced (IVP) buffalo embryos during months with
increasing daylight hours. Indeed, both survival (94.6% and
74.0% in periods with decreasing and increasing day length
respectively; P , 0.01) and development rates (67.3 and 40.0%
in periods with decreasing and increasing day length respec-
tively; P , 0.01) of vitrified blastocysts decreased after 24 h
post-warming culture during the non-breeding season (B. Gas-
parrini, pers. comm.). These findings suggest that the reduced
developmental competence of buffalo oocytes during the non-
breeding season may also lead to lower blastocyst quality. This
is in contrast with the evidence in cattle that embryo quality is
primarily determined by culture conditions, whereas blastocyst
production depends on oocyte quality (Rizos et al. 2002; Lone-
rgan et al. 2003).
A seasonal effect on oocyte quality was also observed in
Murrah buffalo heifers maintained at constant liveweight and
body condition score (BCS) levels by a restricted diet (86%
recommended daily intake) in Brazil (A Salzano, B Gasparrini,
D Vecchio, V Longobardi, PS Baruselli, G Campanile, MJ
D’Occhio, G Neglia, unpubl. data). In that study, reduced
follicular population and deterioration of oocyte quality during
long day months were associated with decreased oestradiol
concentrations in both the plasma and follicular fluid, as
well as decreased intrafollicular insulin-like growth factor
(IGF) 1 concentrations (A Salzano, B Gasparrini, D Vecchio,
V Longobardi, PS Baruselli, G Campanile, MJ D’Occhio,
G Neglia, unpubl. data). Given the role of IGF1 in increasing
the proliferation and oestradiol production of granulosa cells
(Gong et al. 1994; Glister et al. 2001), the reduced oestradiol
concentration may well have been due to reduced intrafollicular
IGF1, although it cannot be ruled out that the decrease in
oestradiol also depended on decreased gonadotrophin levels.
The intrafollicular concentration of IGF1 was lower in the
increasing daylight hours period, despite similar plasma con-
centrations of IGF1, insulin and growth hormone. This may be
74 Reproduction, Fertility and Development
due to either decreased sequestration of IGF1 from the blood,
reduced local production or reduced bioavailability, known to
be regulated by IGF-binding proteins (Armstrong and Webb
1997; Mazerbourg et al. 2000). However, regardless of the
mechanism, increased intrafollicular IGF1 and increased oes-
tradiol production in the favourable season were associated with
improved oocyte quality, which would have been expected
given the importance of IGF1 in oocyte viability (Walters
et al. 2006) and maturation (Izadyar et al. 1997). In addition,
because IGF1 promotes follicular vascular endothelial growth
factor production in ruminants (Schams et al. 2001), we specu-
late that, in buffaloes, the increased angiogenesis of the follicle
during decreasing day length would result in higher oocyte
quality, as well as in the subsequent development of a vascu-
larised, functional CL (Shimizu 2016). Indeed, environmental
factors are known to interfere with follicular growth, with
potential carry-over effects on oocyte quality and CL function.
Recently, differences in microRNA (miRNA) profiles were
detected in Kazakh sheep ovaries during oestrus between the
breeding and non-breeding seasons (Zhai et al. 2018). Interest-
ingly, target gene expression and pathway enrichment analysis
revealed that the differentially expressed miRNAs identified
may affect ovarian activity by regulating hormone secretion,
follicular development and angiogenesis. To better understand
the molecular mechanism underlying the seasonal effects on
oocyte competence in buffalo, studies are underway to investi-
gate the transcriptome profile of oocytes and the miRNA profile
of preovulatory follicles in the two seasons.
Together, all the studies described above demonstrate a
seasonal effect on ovarian activity and oocyte quality, suggest-
ing that, when planning OPU trials, the use of reproductive
technologies, especially oocyte collection for IVEP, be
restricted to the favourable season in order to save resources
and improve the cost–benefit ratio. Yet, this constitutes a further
impediment for the use of advanced reproductive technologies
in this species, aggravating the main limitation, namely the
small number of oocytes that can be recovered per donor
(Gasparrini 2002, 2017). Indeed, poor oocyte recovery, respon-
sible for the higher costs of embryo production, is known to arise
from physiological features typical of the species, such as the
low number of primordial and antral follicles (Danell 1987;
Kumar et al. 1997), as well as a high rate of atresia (Ocampo
et al. 1994; Palta et al. 1998).
Seasonal effects on sperm quality
Male reproductive activity is also widely acknowledged to be
affected by season. A paternal contribution to embryo devel-
opment is also well recognised, because a high incidence of
EEM in various species is caused by genetic and centrosomal
defects of spermatozoa (Yanagimachi 2005). Therefore, under
natural mating conditions, season-dependent changes in sperm
quality may affect both fertilisation and embryo development.
The higher pregnancy rates recorded after AI compared with
natural mating in Italian Mediterranean buffalo during the
increasing daylight length period clearly indicate a seasonal
effect on buffalo bulls (Pelagalli et al. 2009). Photoperiod
affects seasonal male reproductive activity, because increased
B. Gasparrini
daily melatonin secretion acts on the hypothalamic–pituitary–
gonadal axis, improving gonad function and sperm quality
(Zicarelli 1997). Increases in the weight and size of both the
testes and epididymides in buffalo bulls of proven fertility were
found in the breeding compared with non-breeding season
(Arrighi et al. 2010). In addition, in the non-breeding season,
tubular diameter and epithelial cell height decreased in the caput
epididymidis and corpus epididymidis, but increased in the
cauda epididymidis, suggesting an accumulation of non-ejacu-
lated spermatozoa. Furthermore, decreased noradrenergic
innervation to the vas deferens and accessory sex glands was
observed during the non-breeding period (Mirabella et al. 2007).
The absence of aquaporin 9 in the epithelium of the cauda epi-
didymidis and vas deferens in the non-breeding season, in
contrast with marked expression found in the breeding season,
suggests an impairment of water absorption during transit in the
epididymis, affecting sperm viability (Arrighi et al. 2016).
Undoubtedly, although buffalo bulls can breed throughout
the year, a reduction in libido (Sansone et al. 2000) and reduced
semen quality are observed in the non-mating period (Lon-
gobardi 2015). Seasonal variations were recorded in plasma
membrane integrity and stability in Thai swamp buffalo sires
(Koonjaenak et al. 2007), and in post-thaw motility (Bahga and
Khokar 1991; Sagdeo et al. 1991; Younis et al. 1998; Andrabi
2009) and sperm abnormalities (Younis et al. 1998; Andrabi
2009) in Indian buffaloes. In addition, it was reported that
seminal plasma proteins undergo seasonal changes in buffalo
bulls (Sharma et al. 2014), suggesting an implication in fertility.
Furthermore, significant glycosylation changes of the sperm
glycocalyx in relation to season have been reported recently in
Italian Mediterranean buffalo (Accogli et al. 2017).
In Italian Mediterranean buffalo, no differences were
observed in ejaculate volume, sperm concentration or the
viability and motility of fresh spermatozoa between seasons
characterised by a negative and positive photoperiod (Lon-
gobardi 2015). Instead, seasonal differences were reported in
cryopreserved spermatozoa after thawing (Longobardi 2015;
Albero et al. 2016). In fact, post-thaw sperm motility increased
in the decreasing compared with increasing daylight length
period, despite comparable sperm viability. Furthermore, cryo-
preservation-induced capacitation-like changes decreased dur-
ing the favourable season. In fact, the proportion of spermatozoa
exhibiting chlortetracycline (CTC) Pattern F (non-capacitated
spermatozoa) increased in the breeding compared with non-
breeding season (40.5% and 27.3% respectively; P , 0.01),
whereas the percentage of spermatozoa with Pattern B (capaci-
tated spermatozoa; 57.9% and 66.6% respectively; P , 0.01)
and acrosome reacted (AR; 1.6% and 6.1% respectively; P
, 0.01) decreased. The importance of this finding is related to
the very high incidence of sperm capacitation-like changes
observed following cryopreservation in buffalo (Elkhawagah
et al. 2014) due to the membrane phospholipid composition and
particularly to the low cholesterol : phospholipid ratio (Rajoriya
et al. 2016). Furthermore, improved cleavage, total penetration
and normospermic penetration rates after IVF were recorded
during the breeding compared with non-breeding season,
whereas the incidence of polyspermy was unaffected (Lon-
gobardi 2015). Finally, a retrospective analysis of IVEP data
Reproductive seasonality in the buffalo
restricted to the breeding season (to reduce the oocyte source of
variability) in our laboratory revealed an improvement in
blastocyst rates with semen collected during months with
decreasing daylight length compared with months with increas-
ing daylight length (mean  s.e.m.: 38.5  2.2 vs 20.8  6.4%;
P , 0.01; B. Gasparrini, pers. comm.).
Strategies to improve embryonic development during the
non-breeding season
Several strategies have been developed to overcome the sea-
sonal fluctuations in ovarian function in buffalo. These can be
divided into four main groups according to the target: (1)
strategies designed to overcome the impaired luteal function
causing LEM; (2) strategies to improve oocyte developmental
competence; (3) strategies to modify the circannual rhythms of
ovarian activity; and (4) strategies to improve semen quality.
Treatments to improve luteal function
The efficacy of exogenous hormone treatments that increase
progesterone at specific time points after mating or AI has been
widely demonstrated in buffalo. Increased progesterone con-
centrations can be achieved by treating animals with exogenous
progesterone or with trophic hormones, such as human chori-
onic gonadotrophin (hCG) and GnRH agonists (Campanile et al.
2008a, 2008b; Vecchio et al. 2010). Treatment with hCG
increases progesterone secretion via a transient trophic effect on
the existing CL and induction of ovulation with subsequent
formation of an accessory CL, whereas the effectiveness of a
GnRH agonist is primarily related to the development of an
accessory CL. The perfect timing of hormone treatments is very
important to ensure an increase in progesterone at the most
critical time windows for embryo development. It is worth
pointing out that most of the hormone treatments have been
merely extrapolated from cattle, without taking into account
species-specific differences in the chronology of embryo
development (Drost and Elsden 1985; Galli et al. 2001).
In order to reduce EEM, treatments have been given at Day 4
or 5 after AI, with inconsistent results. Higher conception rates
were recorded in buffaloes treated with 17a-hydroxyprogester-
one caproate on Day 4 after AI (Kumar et al. 2003), whereas a
higher incidence of embryo mortality was recorded after treat-
ment with a progesterone internal drug-releasing device on Day
5 after AI (Campanile et al. 2007a). The latter unexpected result
could be due to a reduction in LH secretion by progesterone and
inhibition of endogenous progesterone production. Treatment
with a GnRH agonist on Day 5 after AI was effective in
increasing progesterone concentrations on Days 10, 15 and 20
and pregnancy rates on Day 40 in buffaloes that ovulated and
formed an accessory CL (Campanile et al. 2008b). The admin-
istration of a PGF analogue at the time of AI in Mediterranean
buffaloes synchronised by Ovsynch increased the size of the CL
and progesterone production on Day 11, resulting in a higher
pregnancy rate on Day 45 (Neglia et al. 2008). However, the
efficacy of this treatment was not consistent in commercial
buffalo dairies (Di Palo et al. 2007).
There is evidence that delayed treatments with progesterone
or trophic hormones are effective in reducing the incidence of
Reproduction, Fertility and Development 75
LEM, representing the main cause of embryo losses in buffalo.
Treatments with hCG and a GnRH agonist at Day 25 after AI
significantly decreased the incidence of LEM in a farm with
repeated pregnancy failure (,11% vs 41%) during the non-
breeding season (Campanile et al. 2008a). The beneficial effect
of hCG and the GnRH agonist on embryo development is due
primarily to the development of an accessory CL and increased
progesterone secretion, because both treatments triggered ovu-
lation in 60–70% of buffaloes with a dominant follicle (Campa-
nile et al. 2007b). However, reduced embryo losses were found
in buffaloes that ovulated after GnRH, whereas no differences
were found with hCG between animals that ovulated and those
that did not. This could be accounted for by the additional
trophic effect exerted by hCG on the CL. The efficacy of these
treatments at reducing the occurrence of LEM was confirmed in
another trial that also retrospectively demonstrated that the same
treatments fail to increase progesterone concentration to levels
required for pregnancy maintenance in animals showing a
decline of progesterone on Days 20 and 25 after AI (Vecchio
et al. 2010). In Mediterranean buffalo undergoing Ovsynch–
fixed time artificial insemination (FTAI) programs, a GnRH
agonist is routinely administered on Day 20 after AI. This
schedule allows both improvement in luteal function and pro-
gesterone secretion, hence decreasing the risks of LEM, and
resynchronisation of buffaloes that do not conceive (Rossi et al.
2014). In commercial programs, buffaloes are treated with a
GnRH agonist on Day 20 after TAI and then undergo ultrasound
examination on Day 27 for pregnancy diagnosis so that non-
pregnant animals can be treated with prostaglandin on Day 27, a
GnRH agonist on Day 29 and inseminated 20 h later (Rossi et al.
2014). Repeating this schedule a third time results in conception
rates around 95% after three services.
Treatments to improve oocyte competence
With regard to oocyte developmental competence, we recently
evaluated the effect of OPU priming with FSH treatment in the
presence of progesterone in different seasons. That preliminary
trial was performed in buffalo cows reared in two different
farms, during the breeding season (n ¼ 14) and non-breeding
season (n ¼ 10), over four runs. The priming consisted of
removal of dominant follicles and insertion of a progesterone-
releasing device on Day 0, followed by twice daily adminis-
tration of 40 mg FSH for 3 days and a coasting period of around
40–44 h, with OPU performed on Day 6. The treatment was
effective in increasing the number of follicles, the total number
of COCs, the number of good-quality COCs and the number of
blastocysts in both seasons (Table 1). Interestingly, the effect
was remarkable in the non-breeding season, when treatment
resulted in a higher number of follicles, COCs and good quality
COCs per animal, with a tendency for an improved blastocyst
rate (P ¼ 0.07), leading to increased blastocyst yield per donor
(i.e. comparable to that recorded in the breeding season in
treated animals). These encouraging results certainly suggest
that oocyte developmental competence can be improved during
the non-breeding season with hormone treatments, allowing
better exploitation of the germinal material, especially when
planning an OPU trial. However, due to the high individual
variability in the follicle and oocyte population (Gasparrini et al.
76 Reproduction, Fertility and Development
Table 1. Effect of ovum pick-up priming with FSH and progesterone (treated group) on reproductive parameters in different seasons
Data are the mean  s.e.m. *P , 0.05, **P , 0.001 compared with the control group in the same season. Abbreviation: COCs, cumulus–oocyte complexes
Breeding season
Control
Total no. follicles 3.42  0.34
No. small follicles 2.44  0.23
No. medium follicles 0.72  0.18
No. large follicles 0.28  0.10
Total no. COCs 2.42  0.36
No. Grade A, B and C COCs 1.22  0.21
No. blastocysts 0.32  0.10
2014), it is worth extending the trial to include a large number of
cases and to assess the ability of embryos to maintain pregnancy
after embryo transfer in the non-breeding season. Furthermore,
regardless of FSH priming, selection of buffalo donors for OPU
programs, based on the antral follicular count (Gasparrini et al.
2014) or anti-Müllerian hormone concentration (Baldrighi et al.
2014; Liang et al. 2016), is undoubtedly required to offset the
high embryo production costs in this species.
Treatments to manipulate photoperiod
In order to artificially manipulate the photoperiod and improve
fertility during the non-breeding season, melatonin implants
have been recently used in buffalo under tropical conditions, as
in sheep (Luridiana et al. 2015; Mura et al. 2017). Treatment of
summer anoestrous buffalo heifers with a melatonin implant
was effective in inducing ovulation of the dominant follicle,
resulting in the formation of a functional CL, as indicated by the
larger size and increased progesterone production (Ghuman
et al. 2010). In a subsequent trial, melatonin implant followed by
a progesterone-releasing device induced oestrus in 100% of
anoestrous buffalo heifers, increased the diameter of the domi-
nant follicle and improved CL maintenance on Day 21 after AI,
as well as conception rates (Ramadan et al. 2014). Improved CL
function and conception rates, associated with increased
superoxide dismutase activity, were also recorded with the same
treatment in lactating buffaloes (Ramadan et al. 2016). The
protective effect of exogenous melatonin against oxidative
stress was confirmed in another study, where anoestrous buf-
faloes that received a slow-release subcutaneous injection of
melatonin showed increased total antioxidant capacity (Kumar
et al. 2015). The same route of administration of melatonin also
proved effective in reactivating cyclic activity and improving
conception rates in anoestrous buffaloes (Kumar et al. 2016).
Finally, melatonin supplementation before Ovsynch in anoes-
trous heifers during the non-breeding season improved follicular
growth, CL development and function and conception rates
(Kavita et al. 2018).
Treatments to improve semen quality
To our knowledge, the effect of exogenous melatonin supple-
mentation on sperm quality has not yet been investigated in
buffalo bulls. However, evidence of beneficial effects on sem-
inal plasma composition and sperm fertilisation competence
B. Gasparrini
Non-breeding season
Treated Control Treated
7.45  1.27** 4.30  0.32 11.45  2.70**
3.53  1.10 2.03  0.31 6.70  2.98*
2.70  0.18** 1.41  0.14 3.35  0.46**
1.25  0.03** 0.87  0.12 1.40  0.76
4.53  0.43** 2.21  0.21 6.83  2.77*
3.40  0.35** 0.40  0.08 3.93  1.17**
1.43  0.31** 0.35  0.07 1.78  0.50**
comes from studies in rams (Satta et al. 2018), suggesting a
potential use of melatonin implants to correct semen quality in
the non-breeding season also in buffaloes. Modification of the
extender composition prior to semen cryopreservation may
represent an alternative strategy to improve semen quality in
buffalo. Enrichment of the extender prior to cryopreservation
with antioxidants, such as carnitine and resveratrol, improved
buffalo semen quality by decreasing capacitation-like changes
and oxidative stress (Longobardi et al. 2017a, 2017b). Extender
supplementation with cholesterol-loaded cyclodextrins also
proved effective at decreasing sperm cryocapacitation damage
(Longobardi et al. 2017c), known to increase in the non-
breeding season (Albero et al. 2016). However, the improved
semen quality did not always translate to improved fertility. It
remains to be determined whether these treatments improve the
fertility of semen collected during the non-breeding season.
Conclusions
Reproductive seasonality, resulting in cycles in calving and milk
production, is one of the main factors affecting the profitability
of buffalo farming. When buffaloes are forced to mate during the
non-breeding season (i.e. months with increasing daylight
length) to meet the market demand, the incidence of embryo
mortality is higher. The most critical window for embryo loss in
buffalo is between 25 and 45 days after AI. There is evidence
that embryo mortality is caused, in part, by impaired luteal
function, leading to reduced progesterone concentrations and
retarded embryo growth, and, in part, to decreased oocyte
developmental competence. Furthermore, under natural mating
conditions, season-dependent changes in sperm quality may also
play a role in embryo losses. Corrective strategies have been
developed to improve overall fertility and embryo development
in the non-breeding season in order to ensure milk production
throughout the year and speed up genetic progress. Undoubt-
edly, improved management, including water availability for
bathing, can further improve fertility (Neglia et al. 2009),
counteracting the negative effects of heat stress, particularly in
tropical countries. Furthermore, it is worth recalling that
selecting animals that are less sensitive to the light : dark ratio,
empirically carried out in Italy at culling, has resulted in a
reduced seasonality of the species over the years to a certain
extent in some farms. The possibility of including genetic
markers of seasonality in selection programs, together with a
Reproductive seasonality in the buffalo
wider use of reproductive technologies, such as AI and OPU-
IVEP, could undoubtedly affect the future economy of buffalo
farming.
Conflicts of interest
The author declares no conflict of interest.
Acknowledgements
Most of the results on seasonal effects on buffalo oocyte competence and
luteal function here reported were obtained within the project ‘Strategies
aimed to improve the efficiency of reproductive biotechnologies in buffalo
species’, funded by Programma di Ricerca scientifica di Rilevante Interesse
Nazionale (PRIN) 2008. The semen capacitation/quality trials were sup-
ported by project ‘PIFEMARU’ Misura 1.2.4. PSR Umbria 2007/2013.
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