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Effect of Environmental Factors on Buffalo Reproduction
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Wild animals are usually seasonal, although with their use by man, evolution and domestication, the seasonal characteristic has been declining. Nevertheless, taking as an example some European cattle breeds (Bos taurus taurus), this phenomenon still occurs in freely raised breeds such as the Podolica and Highlands breeds raised in the mountains in Italy or in some Indian/zebu breeds (Bos taurus indicus) raised in ultra-extensive regime in tropical countries. In a similar way, the seasonal reproduction in equine, ovine, caprine and even buffalo, has only been partially influenced by domestication and change in their breeding areas. Another very peculiar example is the fact that selection and better nutrition have decoupled the dairy cow – Holstein-Friesian cattle, from strict seasonal reproduction. However, it does not mean that the same will happen with other species, in a shorter interval between generations, as a consequence of environment cues or a more favorable condition to fix characteristics in very short periods of time. Also the nutritional requirements of other species must be better controlled as in the highly productive milk cows . A comparison between some mammalian species brings up some important questions from the physiological, ethological and genetic point of view [2,3]. The site of origin together with the fertility index (for ex; pregnancy rates) undoubtedly has a marked influence on the way in which the reproductive seasonality occurs with the results being peculiar to each species. On the other hand, the natural pattern needs to match delivery and weaning with more favorable conditions of the season to meet breeding and nutritional requirements. This needs to take into account periods in which etiological agents such as bacteria and parasites are less aggressive or present in lesser quantity. This is part of the adaptation process . The pathological load acquired under the most unfavorable conditions result in a natural selection of the subjects associated with an ideal reproduction during the season regardless of the possibility of survival of the species [5-7]. The reproductive characteristics of the animal have probably been determined by stimuli received from the Central Nervous System (CNS) during gestation or during the first days of the animal’s life. Thus the site of origin along with the duration of gestation certainly influences seasonal reproduction taking into account that animal species, such as sheep, goats and buffaloes are mostly used in different production systems .
Some wild buffaloes that are still found in tropical and subtropical areas are: Anoa (Bubalus depressicornis) in the Islands Cebeles in Indonesia; Tamarao, (Bubalus mindorensis) in the Mindoro island in the Philippines, Arnee buffalo (Bubalus arnee), in India, Sri Lanka and south western Asia, wild buffaloes (Syncerus caffer caffer) in South and East Africa and (Syncerus caffer nanus) in West Africa. For those species calving in spring and from March to June, a large amount of fodder is guaranteed for the calf. In temperate zones to the north of the equator, when the reproduction takes place in autumn, calving occurs from September to November for animals with 5-month gestations (sheep and goats) or, in the next spring for animals with 11- and 12-month gestations (horses and donkeys) [1-3].
Thus, the period of puberty is conditioned by the sensitivity of the neuroendocrine system to the activation of the reproductive cycle. In some tropical areas the availability of fodder is usually found after the rainy season usually from August to December, or is conditioned by providing better fodder in flood areas. In species such as the domestic buffalo where gestation lasts approximately 10 months, it appears that the sensitivity to the decrease in light stimulus and reproductive season coincide in the northern hemisphere between September and January [3,7]. However, with new generations, light stimulation has been a phenomenon that prevails over the reproductive conditions of the photosensitive species even after being moved from their place of origin. This means that when animals are transferred to areas nearby the equator, the sensitivity to the light stimulus does not influence the reproductive activity due to a constant length in the relationship of day-night throughout the year. In some cases, the optimization of nutritional requirements (buffalo horses and sheep in North and South America such as in the Amazon Valley) override the stimulus of light . It can therefore be assumed that there is a seasonal trend in buffalo species even if these have been relocated to Equatorial regions.
1. Some Aspects of Environment Affecting Animal Reproduction
1. 1. Effect of Environment on Buffalo Reproduction
According to Jöchle and Lamond , environmental factors that influence or interfere with reproduction are expressed below:
A. Inanimate factors:
1. Biometereological factors:
(a) Climate and its seasonal changes in photoperiod, temperature, humidity and precipitation;
(b) Significant short-term changes in climate=weather- heat stress
(c) Microclimate imposed by man;
B. Animate factors:
1. Other forms of life interacting in an undesirable or fatal manner: infections, endoparasites, ectoparasites;
2. Interaction with members of the same species: social factors
3. Interaction with man;
C. These and other factors affecting the "milieu intern", the homeostatic capability of the organism having repercussions on reproductive functions.
The same authors mentioned that climate and its seasonal changes play an important role on the phenomena of reproduction. Regardless of the type of photoperiod some species shows sensitivity to the length and intensity of light which influences the hypothalamic-pituitary-gonadal axis, transmitted by a neural pathway of many steps that returns to the retina, the suprachiasmatic nucleus of the central nervous system (CNS), through the superior cervical ganglia and finally the pineal gland. In-depth studies conducted on sheep have shown that the CNS is an internal biological clock that regulates the endogenous circadian rhythm (Fig. 1).
Figure 1. Regulation of pineal gland melatonin secretion by photoperiod: schematic sagittal view of a sheep’s eye and brain. Light is perceived by the eye’s retina (i. e., photic information is converted into neural information). Neural information generated in the retina is transmitted to the suprachiasmatic nuclei (SCN). Connections from the suprachiasmatic ganglia (SCG) reach the spinal cord. Spinal neurons send the information to the anterior cervical ganglion (ACG). Postganglionic SCG neurons send neural paths to the pineal gland, where neural information is converted into hormonal information (high melatonin secretion levels during dark hours) (image from: Maeda KI, Lincoln GA. Phase shifts in the circadian rhythm in plasma concentration of melatonin in rams induced by 1-hour light impulse. J Biol Rhythms 1990; 5:97-106).
The duration of high melatonin blood levels indicate the duration of dark hours to the whole animal’s body. Melatonin is a small indole molecule . In goats, this response is not influenced by the time of day in which the administration of melatonin begins, similarly melatonin micro implants placed in the basal mean hypothalamus determine whether the goat has the same responses with a short day cycle .
The processed stimuli are carried by the superior cervical ganglion to the pineal which operates as a translator by converting the neuronal information that comes from the alternation of day and night cycles. In the pineal gland neuronal information stimulates the rhythm of melatonin secretion that regulates the hypothalamic-pituitary activity and consequently the gonad function, then, the light suppresses and the darkness favors the secretion of melatonin. In sheep the effect of the pineal gland has been demonstrated. Sheep treated with melatonin showed differential effects depending upon the total time of infusion. Daily 8-h infusions of melatonin induced long-day responses, while 16-h infusions induced short-day responses. The time of day when these treatments are given has no influence on the response [9-12].
1. 2. Reproductive Seasonality of Buffalo - Light Effect
Seasonality is the phenomenon related to the occurrence of a biological phenomenon in a particular phase or season of the year. Although some authors have considered the domestic buffalo as a seasonal breeding animal, in the humid tropical regions of the world, buffaloes are continuous polyestrual animals [3,7,11]. However, in temperate zones of the world, buffaloes have a marked seasonality. Thus the absence of seasonality and the possibility of looking for high quality pastures, allows buffaloes in the region of the Amazon Valley to have short calving intervals and thus increase their reproductive efficiency . That being so, climate and its seasonal changes plays an important role on the phenomena of reproduction in buffaloes. Taking in account that most of the reproductive phenomena are related to nutrition, it is expected that without accentuated day-light variation and constant offering of rich native pasture as occurs in the Amazon Valley, buffaloes can show estrus activity year round. As was explained before, studies conducted on sheep have shown that the CNS is an internal biological clock that regulates the endogenous circadian rhythm (Fig. 1) which is also similar for buffalo species and melatonin is the key hormone to trigger the reproductive signal for the onset of estrus cycles [9,13]. Several authors [1,3] have proposed two putative models for the action of melatonin, a dopaminergic and opioidergic neural pathway. In the first model, perhaps through the middle basal hypothalamus (MBH) which favors the exit of peptides and neurotransmitters that regulate the GnRH secretion that could stimulate the anterior pituitary to secrete FSH and LH directly affecting the gonad activity.
The second hypothesis is that the stimulation is indirect  mediated by the adjacent pars tuberalis (PT) which is particularly rich in melatonin receptors that will induce many types of responses. The second hypothesis is contradicted by the fact that melatonin implants placed in the pre-optic area determine an increase in LH in lesser amount than implants placed in the basal mean hypothalamus or in the areas of the third ventricle. For this reason it has been suggested that the mean basal hypothalamus is the target of melatonin whereas pars tuberalis may be at least one site to transmit the effects of melatonin which are responsible for seasonal variations in prolactin secretion levels (PRL) . Thus, it was postulated by some authors [15-20] that seasonal breeder animals are categorized as either long-day or short-day breeders (Fig. 2).
Figure 2. Effect of the photoperiod on the short-day breeders. During long period of light, melatonin causes release of GnRH and during short photoperiods, the excitatory pathways are less active (image from: Senger PL. Pathways to pregnancy and parturition Current Conceptions, Inc.; 2nd edition (2003); 368; ISBN-10: 0965764818).
Two primary factors can influence the onset of the breeding season: photoperiod and temperature . However, it seems that the production of GnRH does not depend directly on the action of melatonin on neurons . It appears that there is a complex inter-neuronal circuit involved, within which the hypothalamic cells of group A15 (a group of cells fluorescent for dopamine in a few species, such as sheep, and immunoreactive for tyrosine hydroxylase, a precursor of dopamine, in many other species including rodents and primates) located in the middle eminence, rich in dopamino-energetic terminals are seen. These are part of a complex mechanism that regulates GnRH secretion. The dopaminergic activity within the mean eminence and the presence of the activity of the enzyme tyrosine hydroxylase (regulator of dopamine synthesis) markedly decreases this phenomenon, if it is subject to a regime of short days. Melatonin administration decreases tyrosine hydroxylase activity and increases LH secretion. This modulation is independent of the action of estradiol .
It is interesting to note that the inhibition of tyrosine hydroxylase through the systemic injection of dopaminergic antagonists (pimozide) during seasonal anestrus produces temporary increases in LH secretion. It is also known that prolactin levels decrease after dopamine administration. Thus, the decrease in prolactin levels is essential for the resumption and regularization of the ovarian cycle in many domestic animals such as sheep, rodents, pigs, as well as in humans. This has led to the hypothesis that the role of dopamine varies between animal species and is probably related to its sensitivity to day length. The precise role of prolactin, and therefore dopamine, in the secretion of LH in buffaloes is difficult to define [22-24]. For example, it has been observed that higher levels of prolactin and lower levels of thyroid hormone (triiodothyronine and thyroxine) in the spring and summer months do not interfere with the estrous cycle or conception in buffaloes . On the other hand, Madan  has demonstrated high levels of prostaglandin in buffaloes in anestrus and their decrease after the administration of bromocriptine.
Different studies [26-28] found high levels of prolactin with low thyroid function. Low levels of thyroid hormones are physiological in spring and summer and evoke those elevations of Thyroid releasing hormone (TRH) that increase prolactin levels. Thus, there is evidence of serotonergic inhibition of LH secretion during seasonal anestrus, although the chemical structure of the substances involved is not fully identified.
Excitatory amino acids such as aspartic acid and glutamic acid cause LHRH output but do not modify the response of the pituitary to the action of LHRH . These substances and other neuronal systems that transmit steroidogenic negative feedback act as mediators of the effects of melatonin and produce LHRH secretion. They have a characteristic that makes the secretion of melatonin vary by minutes and vice versa. The estradiol treatment of ovariectomized sheep with melatonin implants and exposed to long days determines an increase in LH pulses (10 pulses every 6 hours versus 1 pulse every 5 hours), after 40 or 50 days of treatment and not within a few minutes as with the afore mentioned amino acids. This delay can be attributed to the effect of structural modifications within the CNS due to the presence of melatonin.
These modifications can be seen when evaluating the synaptic density of LHRH neurons in the preoptic area during the breeding season compared to anestrus . Similar differences related to the receptor 5 Hydroxytryptamine (5HT2) can be observed with photostimulated and photoinhibited sheep .
The identification and characterization of the neuronal circuits responsible for the decoding of melatonin signal duration can be a critical step in understanding the regulatory effect of photoperiod in reproduction . However, a cooperative study of the animal breeding department of the Veterinary College of Naples and the Veterinary Institute of Bologna found that the melatonin of the buffalo is an endocrine signal that marks the alternation of day and night as occurs in other species such as sheep . Plasma levels of this hormone are maintained at higher levels during more dark hours in autumn and winter as compared to summer and spring. Meanwhile, this pattern changes when the animals were kept on farms where out-of-season breeding systems were used, a technique that has been used for years with high efficiency (more than 90% of the animals conceived during the first seven months of the year) although in some farms this technique has been used without good results. Ignoring the effect of the farm, animals that usually give birth in spring show a different circadian pattern of melatonin compared to those animals born in autumn. During winter and especially in the spring, after two hours the sun has set, buffaloes most receptive to the stimulus of the photoperiod, have less response to out-of-season mating protocol showing higher levels of plasma melatonin than those animals that are less receptive to the effect of photoperiod and are born in the spring period .
Melatonin levels were measured in rams every two hours after sunset and showed a repeated value of 0.733 . In sheep lower values of 0.5 were found. There are no differences between animals sensitive and insensitive to light stimulation (this study was carried out during three breeding seasons in Italy). Based on this high repeatability, plasma melatonin levels can be used as a tool to measure the sensitivity of each animal to seasonal effects . If the heritability trait of this character can be changed to high, this characteristic can be used in genetic selection programs for these species. The natural determination of this characteristic was of great economic importance, especially in Italy and other countries in which buffalo production has a reproductive seasonal problem. For other geographic regions, these procedures could be useful in deciding when buffaloes should be bred where seasonality would not be a concern. This idea has not yet been tested because there have been no genetic studies conducted to determine this trait. Furthermore, in a one year study, buffaloes with a tendency to seasonality were tested. Buffaloes showed high levels of plasma melatonin two hours after sunset, even when they were moved to another farm with other groups of females that showed less sensitivity to the stimulus of light. The role of genetics in melatonin secretion patterns or the difference in sensitivity to light stimulation cannot be excluded. Some sheep breeds (Roman breed in 58° N, Karakul 41° N, White Face 51° N) show a continuous cyclicity throughout the year still living in latitudes where other genotypes are sensitive to the light/dark ratio .
It can be emphasized that spring buffaloes with lower melatonin secretion after sunset are more adaptable to off-season mating systems. Borghese et al.,  have shown in a study conducted in adult heifers and buffaloes that differences in plasma melatonin (values day and night) from March to early spring were lower in heifers (20.32 pg/ml in the evening vs 4.5 pg/ml in the day) with an increase of 5.02 times, compared with adult buffaloes (90.38 pg/ml at night vs 3.8 pg/ml in the day) with an increase of 28.3 times.
Finally, spring-bred buffaloes seem to be more adaptable to off-season breeding mating (OSBM). Similarly buffalo heifers (that are less sensitive to photoperiod), also show the same pattern of breeding as in adult buffaloes. The results lead us to assume that efficiency of the use of melatonin to resolve spring anestrus is not sufficient, since some individuals that show low plasma levels of melatonin, are able to reproduce in months in which photoperiod increases .
It has been known for a long time that several species need to time birth and weaning to favorable environmental conditions. These are the two main reasons for reproductive seasonality. The generations that are born in these favorable conditions, continue the line of the species and pass their particular reproductive characteristics. It is possible that the stimulation of the light perceived by the CNS during gestation and/or during the first states of life can determine the sensitivity to long and/or short day. Many authors attribute reproductive seasonality to nutritional factors in areas where 90% of calvings occur in the months with the greatest amount of forage [32-34].
Figure 3. Annual distribution of calvings in different regions of the world. The blue line denotes the light hours.
Figure 4. Annual distribution of buffalo calvings in the middle Amazon region in Brazil. The last two months, Nov. and Dec., correspond to the period when more fodder is available in the floodplain areas (n=2539) .
Therefore, around the equatorial belt, where the daylight relationship varies very little throughout the year, seasonal reproduction is highly conditioned to the availability of forage [18,20,35,36].
One could think that the tendency of buffaloes to negative photoperiod, depends on the environmental characteristics of their site of origin (subtropical areas to the north and south of the equator), coinciding with nutritional variations during the year ((Fig. 3 and Fig. 4).
Because forage availability is minimal during the dry season, the nutritional status of buffaloes is not optimal at the time of calving and during the first months of lactation. However, production does not seem to be affected if forage availability was favored by a previous humid season. On the other hand, weaning takes place during the dry season.
By transferring these species to other areas, the sensitivity of the hypothalamus axis to the low daylight relationship has not been modified.
In Italy, for herds where the out-of-season breeding technique has not been used on most farms ,the resumption of reproductive cycles takes place in September (under photoperiod) until June (when the period of light begins to increase) . This phenomenon has been observed in the southern part of Italy in grazing herds, as well as in stabled herds where 90% of the buffalo population is maintained with very poor pasture during September; sometimes good pasture may occasionally be available after a wet summer. Usually after mid-June the pastures are poor and the nutritional status of the herds is generally poor in autumn at the beginning of lactation and at the onset of the ovarian cycle. On the other hand, in equatorial zones during the same months there is usually abundance of forage  thus female buffaloes in these areas cycle throughout the year and buffaloes that have calved also resume ovarian cyclicity early and become pregnant .
To date, sensitivity to negative photoperiod has also been found on farms where a balanced diet is provided constantly throughout the year in places where animals are grazed [6,18]. This type of seasonality where the reproductive effects are not synchronized with the availability of forage, indicate that buffaloes raised in Italy are not native, meaning that sometimes some animals manage to give birth during low forage periods, and low temperature that endanger the survival of the calves. In this case, weaning occurs between the end of winter and the beginning of spring. As a consequence, the daily exposure to oscillating temperatures increases the sensitivity to Pasteurela bubaliseptica, an etiological agent of a disease known in Italy as "barbone" that can seriously affect the health of the buffalo.
In Italy, the findings are sufficient to define the buffalo as an animal that mates on short days and then can be considered as a short-day breeder. Seasonality similar to that found in Italy and the subtropical areas of Asia has also been reported in Venezuela, Egypt, India, Argentina and Brazil . However, in the tropical areas the buffalo is a continuous polyestrual breeder with an interestrus interval of 18 to 32 days, but more frequently between 21-23 days, estrus lasting 8 to 16 hours and ovulation occurring after the onset of estrus signs about 16 hours later .
1. 3. Effect of Thermal Stress
The interrelation between temperature and the productive performance of animals has been described by many authors [35,37,38]. The environmental temperature is one of the most important climatic factors in the adaptation systems of the domestic animals and the deleterious effect on Bos taurus species raised in subtropical and tropical regions has been observed in many countries . The temperature control is fundamental to run the metabolic functions in mammals, the maintenance of the internal temperature being one of the physiological priorities of homoeothermic animals. In domestic mammals, the physiological internal temperature varies between 37. 5° and 39°C, when the referred value is lower than the minimum value of 37. 5°C, it is called hypothermia and a value higher than 39°C is called hyperthermia [39-40]. Heat stress is the most important factor among the innumerable environmental factors that influence buffalo production and reproduction . Buffalo skin is covered with a thick epidermis, the basal cells of which contain many melanin particles that give the skin surface its characteristic black color .
According to Balhara et al.,  it is generally believed that buffaloes are sensitive to heat stress, owing to:
- Thick black skin color that absorbs more solar radiation, which is high in the region.
- Sparse hair coat, considered inadequate to insulate the buffalo from high temperatures.
- Buffalo skin has fewer sweat glands (almost 1/6th less) in the skin than Zebu cattle, situated deep in the skin, compromising heat dissipation through evaporative heat loss.
According to several authors, because buffaloes are found mostly in tropical areas, they thrive in hot and humid conditions. Environmental temperature seems to be the most important factor in determining the adaptation stress of animals in tropical regions. Among the circumstances that would increase the influence of ambient temperature, two should be highlighted:
- Sudden and significant changes in the climate, with an increase in temperature;
- Artificially imposed microclimates, especially without controls, that stabilize temperature at ideal levels.
Figure 5. Hormones affected by the effect of heat stress on buffaloes and effect on the negative energy balance.
This has been noted in numerous farms, where mating outside the season is practiced, in those that have the same macroclimate, it has been suggested to set up an improved management in animal, handling nutrition, hygiene and veterinary practices. These peculiar morphological and anatomical characteristics make buffaloes poor thermoregulators, with several deleterious aspects on the metabolism of the affected animal by heat stress. There is a decreased nutrient intake and decreased nutrient uptake by the viscera that drain into the portal vein of the buffalo, with a direct impairment on milk production. Moreover, a shift of blood flow to the peripheral tissues for cooling purpose alters the nutrient metabolism and contributes to lower milk yield in hot weather. Also, heat stress causes hormonal alterations including a lower secretion and lower levels of plasma STH and TSH concentration.
Furthermore, among the buffaloes there are animals unable to adjust to heat stress and this inability may be genetically controlled . In Fig. 5 and Fig. 6 the pathophysiology of the effects of thermal stress on the metabolism of buffaloes submitted to inadequate management in tropical regions is summarized. The blood acid-base chemistry is altered as a result of the impairment in thermoregulation from conduction, convection and radiation mediated heat-loss to evaporative cooling. Also, the rectal temperature, respiration and cardiac rate increase followed by diurnal variations in blood pH and bicarbonate values and the acid-base chemistry, from alkalosis to compensated acidosis, resulting in metabolic acidosis at a fairly low night temperature. Similarly, the buffering capacity due to the alteration of bicarbonate levels in blood, generally changes on hot summer days. Further there is a decrease in dry matter intake and in the ratio of forage to concentrate intake. This phenomenon is more marked in a buffalo that produces more milk. There is a significant correlation between the level of milk yield and the decline associated with the daily mean environmental temperature increases [42-44].
In India, the buffalo’s sexual vigor declines during summer and improves with the onset of the colder season . In Egypt, the reproductive behavior of buffaloes, in particular during estrus, is weak during the hot season. A herd of buffaloes had almost equal incidences of ovulation in the hot (May-October) and the cold (November-April) seasons. The observed signs of estrus, however, were more frequent and stronger in the colder season, accompanied by changes in the seasonal pattern of sex hormones [46,47]. The number and amplitude of the luteinizing hormone pulses were greater in the colder season .
Table 1. Physiological Effects of Heat Stress on Buffalo 
Increased blood flow to skin and peripheral tissue resulting in:
-increased hydrostatic pressure
-increased capillary permeability
-leukocytic and antibody infiltration
Increased nerve conduction velocity, decreased firing rate of motor neurons resulting in muscle relaxation and increased pain threshold
Stimulation of hypothalamus resulting in increased metabolic rate, oxygen uptake and accelerated healing
Soft Tissue Extensibility
Increased collagen extensibility for maintaining greater length after stretching for:
-less force required to increase length
-decreased risk of tissue tearing
Figure 6. Pathophysiology of the effects of thermal stress on the metabolism of buffaloes submitted to inadequate management in tropical regions .
The first reaction of the buffalo is to look for water or puddles of water to cool off. This seems like the natural instinct of the species, which really helps to reduce heat load accumulated in the body (Fig. 6). The use of natural sources of water in farms such as ponds, streams, corregos, pools, showers and sprinklers are all recommended sources to mitigate body heat in the buffalo (Fig. 7,8,9,10,11,12). The use of trees in pastures is also recommended as well as the use of artificial shade shrubs, have been widely used in buffalo dairy farms.
A study  was conducted to verify the influence of temperature and humidity on pregnancy rate of Murrah buffaloes maintained at the Livestock Research Centre, National Dairy Research Institute (NDRI), Karnal, Haryana, India, located at 292° 42' N latitude and 72° 02' E longitudes, at an altitude of 250 m above the mean sea level, in the bed of the Indo-Gangetic alluvial plain. The climate of Karnal is subtropical in nature. The average annual rainfall is approximately 760 to 960 mm and maximum rainfall occurs during July and August. The month wise average dry bulb temperature (The dry-bulb temperature is the temperature of air measured by a thermometer freely exposed to the air, but shielded from radiation and moisture) varied from 12. 432°C in the month of January to 32. 542°C in May while relative humidity ranged between 42. 01% in April to 80.72% in August. These data were gathered during a twenty year period (1993 through 2012). The direct effect of temperature humidity index (THI) on pregnancy rate of Murrah buffaloes in subtropical climatic conditions of India, is not available in the literature. This study indicated that both first parity pregnancy rate and overall pregnancy rate started to decline above THI 75. Therefore, the THI value of 75 was identified as the threshold THI for optimum pregnancy rate of Murrah buffaloes. A negative association was found between THI and pregnancy rate. The first parity pregnancy rate was decreased by 6% for each unit change in monthly average THI value under the heat stress zone. However, there was a maximum decline (-11%) in first parity pregnancy rate with the increase per unit in THI value for the months of May and June. The negative association was also found between THI and overall pregnancy rate under non-heat stress zone and heat stress zone with -1% and -4% changes in pregnancy rate for each unit change in monthly average THI value. There was maximum decrease (-7%) in overall pregnancy rate with the increase per unit in THI for the months of May and June. Therefore, May and June were identified as critical heat stress period in India for both the first parity pregnancy rate and overall pregnancy rate of Murrah buffaloes.
The average pregnancy rate in the 1st parity of Murrah buffaloes was estimated to be 0.40 in the non-heat stress period with an average THI of 64. 1 (56.7 to 73.2). In the heat stress period, the average pregnancy rate was 0.26 with an average THI of 79.4 (75.4 to 81.6). In the critical heat stress period (the months of May and June), the average pregnancy rate decreased to 0.22 with an average THI of 80.9 (80.3 to 81.6). First parity pregnancy rate was found to be significant (p<0.01) between the non-heat stress period and the heat stress period and significant (p<0.05) between the non-heat stress period and the critical heat stress period. Fewer buffaloes showed estrus in the critical heat stress period compared to the heat stress and non-heat stress periods. The highest overall pregnancy rate was estimated as 0.45 in the non-heat stress period which decreased to 0.28 in the heat stress period. The lowest pregnancy rate was 0.27 in the critical heat stress period. A highly significant difference (p< 0.001) for overall pregnancy rate was observed between non-heat stress and heat stress periods and between the non-heat stress period and the critical heat stress period .
A study in buffaloes in Belém, in the Brazilian Amazon, showed that rainfall, between 70 mm and 170 mm, with heavy rains followed by summer, raised the rectal temperature of buffaloes to 38. 9°C and contributed to the discomfort of the animal. The higher humidity of the environment, which is directly related to precipitation, had an influence on the daily weight gain of the animals, and their productive performance was higher when the relative humidity of the air was close to 100%, and in times of rain. Thus, rainfall was linked to greater animal comfort and better performance .
Buffaloes undergo thermal stress when environmental temperature is above 36°C and their thermoregulatory system reaches a critical point . At this temperature, buffaloes have to use their airways to eliminate excess heat. However, according to Baccari Júnior , buffaloes up to one year of age are more susceptible to higher environmental temperatures because their thermoregulation mechanism is not fully developed at this time. When under stress, body temperature increases, the respiratory rate rises, rumination stops and there are signs of discomfort . Approaches to reduce thermal stress in buffaloes include provision of natural shades from trees (Fig. 7), artificial shades (Fig. 8), natural (Fig. 9) and artificially constructed ponds for wallowing (Fig. 10) and use of showers or sprinklers to lower the body temperatures.
Figure 7. Buffaloes taking shelter under the tree during hot summer months.
Figure 8. The use of artificial shading is a very positive action in the reproductive management of dairy buffalo herds in tropical regions.
Figure 9. The best way to relieve heat stress in buffaloes is to provide controlled free access to natural sources of water.
Figure 10. In some cases, the use of artificial swimming pools should be recommended and used to attenuate the thermal stress of dairy buffaloes in southern hemisphere countries.
Figure 11. The use of showers or sprinkles to mitigate thermal stress is an important measure to relieve the effects of heat on buffaloes, improving reproductive performance.
The use of silvopastoral systems (SPS) (Fig. 12) combined with animal welfare and higher production values are suggested mainly in tropical regions of South America, including Los Llanos in Venezuela, the Northeast and the Amazon Valley in Brazil, where the heat stress is permanent.
Figure 12. The use of silvopastoral systems should be recommended to mitigate the thermal stress of dairy buffaloes in southern hemisphere countries.
Garcia et al.,  compared the heart rate (HR), respiratory rate (FR) and rectal temperature (TR) of buffaloes maintained on a pastoral system with (CS) or without shade (SS). The meteorological data (air temperature, relative air humidity, rainfall, velocity wind, brightness and solar radiation) were also recorded. The FR was higher during the rainfall, both in SS-managed animals, and in CS. This elevation was directly related to the variation of ambient temperature. TR values were lower in shade animals. The authors concluded that a silvopastoral system, which makes available shade in the pastures, provides maintenance of the physiological parameters of dairy buffaloes close to normal. da Silva et al.,  studying hormonal responses for cortisol, triiodothyronine (T3), and thyroxine (T4), compared parameters of dry bulb temperature (DBT), relative humidity (RH), and black-globe temperature in order to calculate the globe temperature and humidity index (GTHI), in different seasons of the year: rainy season (January-April), transition season (May-July), and less rainy season (August-December). It was shown that relative humidity was significantly different across seasons and particularly high in the rainy season. The seasons influenced cortisol such that higher values were observed in the transition season. The highest mean cortisol levels were recorded during the rainy and less rainy seasons, whereas the highest T3 and T4 levels were recorded only during the rainy season. T3 and T4 were negatively correlated with DBT and GTHI and positively correlated with RH. The authors recommended that in an silvopastoral system thermal comfort to buffaloes should be provided by shades.
Conditions under two different SPSs were compared. Both SPSs provided shaded areas and a lake where animals could immerse themselves. Physiological variables, morphometric data, THI and BCI indexes were calculated in two phases for further comparison by F test (P<0.05), for animals in both SSPs. THI indicated "alert level" during both experimental phases (Phase 1: 78.9±3.7 and Phase 2: 77.5±3.5). The RF was above normal levels, with variation of 32.2±9.2 to 56.5±19.0 mov/min. The RT (38. 3±0.26 to 39.3±0.38°C) and CF (64.6±15.2 to 76.6±13.9 beats/min) were within normal standards for buffaloes. The skin temperature ranged between 23.6±8.3 and 31.7±5.4°C. The BCI in SSP1 animals ranged from 2.46±0.33 to 3.31±0.62 and in SSP2 animals BCI ranged from 2.42±0.30 to 3.45±0.66 (P>0.05). The growth rate and the weight gain of calves in both SPSs was considered excellent (0.917±0.4 to 1.052±0.5 kg/day). The welfare of calves was improved by the shaded areas on pastures and the availability of water for swimming.
1. 4. Effect of Nutrition
Nutrition and reproduction are two aspects of the biophysiology of domestic mammals that have close correlations with animal production and reproduction systems . Buffaloes with normal or excellent management seem to achieve puberty early and are more fertile . Figure 13 shows the positive effect of nutrition on energy balance for cell metabolism.
Figure 13. Energy balance as it relates to the priorities of various physiological functions in mammals (image from: US National Library of Medicine, National Center for Biotechnology Information NCBI, https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=2781850_rstb20090140f01.jpg).
The great variation in the availability of food during the different periods of the year made it necessary for primitive species of domestic animals to develop mechanisms to adapt to food shortage conditions, perhaps by replacing fundamental elements of their food.
Some species have preserved these mechanisms or part of them until now, but the intensification of domestication and the implantation of the technification of the intensive or extensive management of the animals, eliminated totally or partially, the atavistic strategies, overloading the artificial actions to adapt the animals to the new norms and conditions and environmental impacts of breeding. The loss of these characteristics of adaptation to unfavorable situations makes the individual more sensitive to nutritional variations. In fact, with the advent of genetic selection for intensive production, there was a gradual decrease in the adaptability conditions and the animals lost their attributes related to tolerance to situations created by the lower availability of food, resulting in serious problems in the reproduction of animals affected by the restriction to feed .
For example, in Brazilian tropical forages, particularly those disseminated through several geo-ecological regions, present quantitative and qualitative variations depending on the season [56,57]. Below are some detailed characteristics highlighted by the authors:
- The dry matter (DM) and protein content of these forages was considered as average during the rainy season and, deficient during the dry season. But, under both circumstances, they were insufficient for the ruminal microbiota to multiply and provide sufficient nutrients for the maintenance of the animals;
- The digestibility of these forages was considered reasonable in the rainy season and deficient during the dry season.
- Depending on the quality and quantity of the food ingested by the animals, imbalances and mineral deficiencies would occur, impeding the perfect functioning of organic enzymatic systems, fundamental to life, both for fetal and newborn development, as well as for animal production.
In most cases, buffaloes would maintain the body condition score (BCS-scale 1 to 5, where 1=meagre and 5=fat; poor=2, reasonable=3, more than 3=good and then more than 4=very good). Animals with a BCS between 1 to 3, would remain in anestrus, until better food conditions were restored (Fig. 14).
Figure 14. Effect of the rainy season with high pasture supply and the dry season, on body condition score (BCS) in cattle and buffaloes .
Taking into account that nutrition in buffaloes is a very important factor for production and reproduction, it is erroneous to think that buffaloes in the tropics can survive and reproduce under minimal food availability conditions when compared to other large ruminants species.
In tropical areas, there are two favorable periods regarding the availability of natural grass, (Fig. 14). During the rainy period, animals reach an excellent BCS, resulting in an anabolic condition due to the high content of dry matter and protein. During the dry period, animals become thin and this results in a catabolic condition. These aspects have a direct effect on the metabolic routes related to reproduction. Regardless of the metabolic pathway involved, the regulation exerted by nutrition on the reproduction of male and female animals occurs mainly by the action of hypothalamic brain factors, altering IGF-1, insulin, GnRH, FSH/LH and steroid hormone secretion (Fig. 15).
Figure 15. Negative energetic balance due to undernutrition – Lipolysis.
It is not possible to dissociate the interrelation between nutrition and reproduction, when referring to the minimum needs of energy/dry matter (DM)/crude protein (CP) and reproductive performance (puberty and fertility). In general, food, in the form of fodder or supplements, provides carbohydrates, which will meet the energy needs, a certain amount of protein derivatives presented as crude protein are degraded and resynthesized by ruminal fermentation (action of microfauna in the rumen) . The most striking effects of dietary deficiency or energy and protein supplementation occur during puberty in females in most domestic mammalian animals. This was studied in detail in cows and buffaloes. In cattle, depending on whether the animal is Bos taurus taurus or Bos taurus indicus, puberty occurs between 6 to 18 months of age. Factors such as reproductive management, body development, age, seasonality and race may all influence with the onset of puberty. In general, dairy and beef heifers from European breeds need to reach 35-45% and 50% (respectively) of the body weight of an adult female when reaching puberty .
The effect of dietary supplementation on the time of onset and manifestation of puberty in heifers was established by Asdell  when he demonstrated that Dutch Holstein heifers submitted to a food management plan with diets containing small, medium and large energy values, reached puberty at 9, 11 and 15 months of age, respectively . This has clearly demonstrated that as the energy level increases, the time to puberty is reduced. Similar results were also observed in beef cattle breeds in the USA and in Nelore zebu and Murrah buffaloes reared in similar conditions in northern Brazil [8,20,60].
Another example that supports the low dependence of buffaloes on negative photoperiod is provided by reports of reproductive seasonality in Brazil, especially in the state of Sao Paulo, that is crossed by the tropic of Capricorn . Here the wet season and consequently the availability of fodder begin around October and November, and continues until March and April while the availability of pasture declines from May and June until October and November.
The calving period of the buffalo in these conditions is concentrated from February to May, the mating period from April to July and the weaning period under free grazing conditions from September to December. These events allow a coincidence in forage availability between the first two to four months of lactation and most of the dry period, October to April, depending on the month of calving.
In addition, the amount of milk production in the first stage of lactation and fertility is influenced by the BCS  at the time of delivery. Generally, some of the animals benefit from the availability of forage in the first 2/3 of lactation and perhaps throughout the dry period. Weaning of buffaloes occurs from September to December in animals born from February to May. Only buffaloes weaned from September to October suffer from low availability of forage and a experience a transitory blocking period in their growth at 60 days (Fig. 3 and Fig. 4).
During the second year of life, forage availability limits the growth by five months in buffaloes. This does not prevent buffaloes from using the next 6-7 months of good pasture to reach their slaughter weight (approximately 400 kg) at around the first two years of life, . After calving, females usually re-establish their body losses during the forage availability period (November through May) before continuing with their own growth.
The reproductive season for cattle under these conditions does not allow the restoration of BCS during the dry period (July to October). The normal production will have to wait until the following lactation when it can be satisfied exclusively through food consumption. In other words, it is confirmed that cattle experience the same effects as buffaloes in tropical zones north of the equator. During the first lactation, the use of body reserves cannot satisfy the production needs of buffaloes, as the low carbohydrate-protein ratio of the pastures creates a negative production factor (Fig. 16) .
Fodder availability seems to be very important for buffaloes in the tropical areas of South America. Forage availability enhances the reproductive performance in buffaloes raised in range conditions. In Fig. 17a and Fig. 17b it can be seen how food plays an important role in the BCS of buffaloes.
Figure 16. Effect of the dry season with low pasture supply on body condition score in female buffaloes raised in an extensive management system in tropical areas.
Figure 17a. Effect of bad management and poor nutrition on buffaloes. Too many animals placed on a very poor pasture.
Figure 17b. Buffaloes on the poor pasture in A where a buffalo cow with a poor BCS can be seen. The cow was reported in anestrus since a long time, and its calf is more than 15 month old.
To the south of the equator, due to a natural characteristic of sensitivity to positive photoperiod-conception takes place between December and February. In those regions in which the daylight relationship increases or decreases, the calving season occurs when there is little forage. In some areas of Argentina and southern Brazil, it is necessary to give a timely mineral supplement to eliminate uterine and vaginal prolapse. This pathology is frequently found in buffaloes north of the equator . The birth period  is concentrated in different months (Fig. 19 and Fig. 20), from January to July around the Amazon River, and the year round with a higher concentration in November and December on the Jarí River, from April to October on the Marajó island and in the Amazon River Delta.
Thus, in two of the three regions considered, the calving period follows the wet and rainy season (November-May) and therefore there is a high availability of forage. This situation allows energy storage for an increase in body weight during the dry period. However, in flat flood areas the percentage of births is evenly distributed throughout the year and in those regions where photoperiod has no influence on the equatorial region, the effect of food availability on pregnancy rates is quite evident .
On the other hand, transferring species that originated south of the equator results in a reproductive activity that is conditioned more by light stimulus than by satisfying nutritional requirements. The same phenomenon occurs in the Southern Hemisphere when animals that originate in regions around the tropic of Capricorn are transferred to regions near the Equator. In Brazil, the case of the Laguna Farm located in the northwestern part of Brazil, (Ceará state, latitude south 3° 28' west longitude 39° 3' minutes) is well known. A herd of Murrah buffaloes were transferred in 1992 from a region in the Capricorn tropic in Sao Paulo. In this new environment the animals showed an atypical seasonal pattern in their first and second year calving females with births concentrated in April and June and no calves were born between September and November. The farm had excellent management, artificial insemination, and veterinary services. After six years the situation changed to a pattern where 60.9% of births occurred between January to July and 39% between August to December (Vale, unpublished data). Thus, it was possible to establish a calving schedule in which parturition occurred in every month of the year.
These findings have demonstrated that:
- Although the domestic buffalo shows calving patterns throughout the year there is a great tendency to concentrate this physiological phenomenon in the months in which the day light decreases (September in the North of Equator) or increases (April in the South of Equator) compared to the hours of darkness.
- Where light/dark relationship is constant throughout the year, even if the tendency to seasonality does not exist, the calving concentration varies from area to area and from year to year. It would appear that in practice the onset of the reproductive cycle requires particular environmental conditions, either individual or group related to sexual behavior.
- This last aspect that has been noticed in the areas of the Amazon and has also been demonstrated in Italy, where alternating phases of intense sexual activity independent of season, results in periods of high concentrations of births followed by periods of sexual stillness, showing a tendency in certain species to group sexual activity. Such phenomenon is influenced by environmental conditions.
Buffaloes are extremely sensitive to variations in climatic conditions [7,8,64] and this is confirmed by an advance or delay in the time of calving and also by the resumption of ovarian activity after parturition.
This has been noted in numerous farms where mating outside the season is practiced in those places that have the same macroclimate but which suggest better methods of handling nutrition, hygiene and veterinary practices.
It should not be considered strange if up to now the importance (it deserves) of nutritional factors in the fertility of buffaloes has not been emphasized. Buffaloes do not seem to have the same physiological functions as high-yielding dairy cows that use their reserves to compensate for deficiencies of energy and protein during the early stages of lactation. If this is the case, only females that produce more than 3000 kg of milk per lactation would have a catabolic pattern, which translates into 8% of the buffaloes registered in the Italian buffalo production association and around 30% buffaloes of all the farms.
As a matter of fact, buffaloes use their reserves within reasonable limits, to assure their reproduction, however, this does not appear to be detrimental to their production. Thus, the reproductive seasonality of the species represents a physiological characteristic that has remarkable economic implications. In the south of the equator in areas that are far from cities, extensive buffalo farming for beef production has been specialized. In such cases due to its reproductive seasonality, calving and weaning coincide with an increase in forage availability which allows buffaloes to mate and be more profitable than cows. This fact seems to be responsible for increasing the number of buffaloes in South America. In some tropical countries to the north of the Ecuador, the environmental conditions favor bovines because of religious considerations.
In this case, buffaloes are used to produce milk and their seasonality is useful since it provides an alternative to fresh bovine milk in periods of food scarcity [3,20]. Therefore, buffalo milk production is more economically viable if it coincides with the availability of fodder. In South America the opposite has been found. The manufacture of specialized milk products requires a constant supply of fresh milk, which is hampered by the reproductive seasonality of the species.
In Italy the buffalo breeding is specially directed to produce a type of mozzarella cheese. The demand for mozzarella is mainly concentrated in spring and summer, which coincides with the periods with the lowest incidence of births and consequently with low milk production . In order to cover the milk demand, during the last 20 years some Italian breeders have tried to modify the natural calving calendar separating the females from the males between October and February when conceptions are not desired (also known as the out-of-cycle mating scheme). This modification of the natural calving period is easily achievable over time due to the high reproductive efficiency of some farms and the laborious work of many years. This can be achieved by differences in the seasonal sensitivity of animals. However, in some farms where the out-of-cycle mating scheme has not been successfully used, the same goal has been achieved by culling females that are not pregnant at the time of separation from the male and that become pregnant only a few months after re-introduction. The use of these two reproductive strategies to meet market demands is expanding more and more among buffalo breeders as well as in countries such as Argentina, Brazil and Venezuela. This is also due to the differential price of buffalo milk during the spring and summer, and autumn and winter seasons. Based on findings from these studies, it is expected that the low sensitivity to the characteristic seasonal effect will increase in farms where the scheme is properly used and this situation is expected to continue. Calvings between September and December decreased from 23 to 16%, between 1983 and 1995 on controlled farms. Actually, this system is largely used in Italy.
2. Anestrus - Off-Season Breeding System
Anestrus can be defined as the non-resumption of the estrous cycle after calving, or as previously mentioned as the interruption of cyclic ovary activity for several reasons. The latter is preceded by normal estrus followed by inadequate luteal secretion. This phenomenon has been found by the appearance of seasonal anestrus in sheep, in the first postpartum ovulation and during the prepubertal phase in sheep and cows. These reproductive disorders can be divided into two main causes first: a short luteal phase and second: a normal luteal phase with low levels of progesterone production.
Anestrus is usually attributed to nutritional factors in cattle. In buffaloes, nutrition is only one of the causes, other factors such as photoperiod sensitivity, environmental factors and uterine inflammatory processes (endometritis/pyometra) if parturient hygiene is poor, play an important role on the reproductive pathology of buffaloes [33,34,65,66].
This can get worse over time, especially during the negative photoperiod months. The phenomenon is particularly evident when animals are subjected to off-season mating and affect buffaloes born in the first two months of the year or in the first five months of low temperatures.
To the south of Italy, 70.3% of primiparous and 24.0 % of pluriparous buffaloes have been found to be acyclic, and buffaloes that are not pregnant before 70 days postpartum enter into deep anestrus that ends after 200 days when they are exposed to at least 2 months of the short photoperiod .
Changes in the endocrine balance may be responsible for resumption of delayed ovarian activity and may have a negative influence even when buffaloes become cyclic, especially on long days. It can be assumed that this behavior peculiarity could depend on an endocrine disruption that is related to the photoperiod. Buffaloes found to be acyclic at 70 days postpartum subsequently become cyclical and have a 16% pregnancy loss.
This phenomenon is partly seen in the results of progesterone levels in milk for the diagnosis of gestation. It is less accurate in spring than in the fall, 64. 3% vs 93. 6% respectively. The difference may be due to a large number of animals found in anestrus or embryonic mortality during the spring season .
It is possible to differentiate a temporal anestrus from a deep anestrus according to the length of anestrus. Temporal anestrus is considered less than 150 days, and deep anestrus is considered longer than 150 days.
Taking into account some hormonal factors that are the basis of anestrus, and according to other studies, the buffalo has a low hypothalamic-pituitary activity that consequently causes a decrease in ovarian activity . A low pulsatile release of LH after an estrous cycle causes the buffalo to become acyclic and it fails to conceive. The acyclic buffalo shows low levels of progesterone, FSH and estradiol-17β [48,68,69]. High levels of prolactin with low levels of thyroid hormone are found during the spring months when the incidence of acyclicity is greater . The causes of anestrus in Italy are extremely different compared to other countries that raise buffaloes.
Market demands cause an increase in the number of farms that change the calving calendar (approximately 60% of farms follow this practice). In developing countries, with some exceptions, reproduction is naturally seasonal and the reasons for anestrus are almost always attributed to nutritional factors. In these countries, there are no long intervals between calving in buffaloes that have offspring. In Italy there are long intervals between calvings for all pluriparous females that have an offspring.
Buffalo heifers are more frequently affected with anestrus. The birth-conception interval is generally longer (60 days) than for pluriparous, old buffaloes (which solve their breeding problems in the fall) and also those that have recently calved. The trend towards seasonality in the herd is, however, influenced by the amount of animals in the herd, and heifers have trends related to photoperiod .
Old buffaloes (older than 10 years) and buffaloes having had more than five calves are the ones that most frequently have anestrus especially during spring, representing 20 and 55% of animals, respectively. These groups resolve their breeding problems in the fall as well. Even so, animals older than 10 years remain in optimal health because their reproductive function only takes place in the fall. During this period, ovarian activity resumes, the uterine tone improves and usually favors the spontaneous solution of moderate uterine inflammatory processes .
This marked seasonality is not found in heifers in which fertility is not affected by the breeding period. These primiparous animals are not influenced by the season in which they calve, but are affected by the stress of the first lactation, management conditions in the first months of gestation and the first postpartum period (PPP) or puerperium. Postponement of the resumption of ovarian activity easily occurs in PPP, which is occasionally disrupted, and pregnancy may not occur.
In some well managed farms this does not happen due to the different weaning techniques used, strategies for special care of growing animals, and to supplementation of growing animals during the puerperium.
It is important to take into account the age, the stratification of the animals and the tendency to seasonality that is related to the age of the herd.
First it is necessary to consider that several authors consistently found little sensitivity of the CL to the action of PGF2α. This was not related to differences in CL size, progesterone production, number of LH receptors, or number of cells when comparing large-related/ small abnormal luteal bodies to normal CLs .
The second phenomenon is due to inadequate follicular development during the preovulatory period, because of low luteal support or because of premature PGF2α production . Inadequate LH secretion due to a short luteal phase, or a long luteal phase with low progesterone has also been reported by several authors [58,71]. These factors that have not been identified and confirmed in relation to the poor response to PGF2α in the bubaline species. Buffaloes frequently have an inadequate luteal activity after induced estrus. The causes of low fertility in cows are sporadically found in buffaloes, however, they play a different clinical role, for example in buffaloes, retained placenta is rare because the caruncles are less convex than in cattle  creating fewer junctions in the cotyledon and facilitating placenta expulsion.
In some farms in Italy an increased incidence of retained placenta has been reported, however, the real cause has not yet been identified. This phenomenon causes a delay in conception of up to two months. Uterine diseases are rare in Italy, probably because of better management. In other management systems, it is common for endometritis to appear with thickening of the uterus affecting fertility .
In relation to ovarian pathology, it can be said that the persistence of the CL can be seen but ovarian cysts are extremely rare in buffaloes and usually resolve spontaneously . As mentioned before, Italian breeders allow suckling of buffaloes during the months of March and September with the aim of obtaining births beginning at the end of January until the first days of August. Usually breeders who use this system for the first time, keep buffalo bulls away from females in October and December during the first year and between October and March during the third year. In this way there is a gradual change in the patterns of births and a profit in production is quickly observed. In the first year, the results obtained depend on the degree of seasonality, genital problems and reproductive efficiency that existed at the time of standardizing the off-season breeding method . The anestrus that characterizes all the primiparas and all the old females can be attenuated with hormonal treatment, a practice that nevertheless has very little benefit to solve the real problems of the farm. The economic gain is due to the low percentage of non-pregnant females discarded at the end of lactation (18. 1%) compared to controls (31. 2%). Breeders who have been using this system for quite some time, keep male buffaloes with milking buffaloes from March to September to obtain births from June to July. These males are always together with dry buffaloes. Some sporadic cases of pluriparous buffaloes may appear in the autumn-winter period. Buffalo heifers reach reproductive age at 2 years but puberty is reached during the first seven months of life. Most heifers are born between June and July, however, pregnancy usually does not occur until late July even if they have been maintained with the male. A study revealed after analyzing more than 7000 births that the effect of day length on the reproductive efficiency of buffaloes depends on the values existing before introducing the herd to off-season breeding systems. A shortening of the interval between births is observed in those farms that have used these systems for many years (over 15 years) compared to others that have used this system for less than 5 years, probably because of the high culling level animals that are more sensitive to photoperiod .
The specific fertility of a pluriparous female adjusted for the interval between births  was 74% after using out-of-season mating strategy whereas before it was 81%. Other farms that have used this strategy for many years show a specific fertility rate of 79%. In the first years of use of the out-of-season mating strategy, the birth rate drops by about 7% but after some years it returns to the previous value.
Changes in the proportion of births that can be obtained through the out-of-season mating strategy can be made by selecting the buffalo less sensitive to photoperiod and increasing the use of artificial insemination, however, it is important to note that these strategies should be used continuously to avoid the return of the seasonal births within a few years.
Vale (unpublished data) set up a calendar of births for pluriparous buffaloes on a farm where technical assistance began with an out-of-season breeding program since 1994. The first results came about in 1995. Between 1992 to 1994, there was a drop in birth efficiency between February and April. When conception took place between April and June coinciding with the period of increase in the relation light/darkness. Thus, after 1995, parity declined dramatically between September and November and was concentrated especially on the first eight months of the year. The minimum parity was recorded in the months of February and April proving that herd fertility is always negatively influenced by sexual activity in the months in which the light-darkness relationship increases.
This technique does not influence the physiological characteristics of the female buffalo, but it prevents calving concentration in certain months (July and December), allows for the postponement of births in the first months of the year and consequently conceptions occurs in the spring months at the time when suckling begins to decline.
It is of the outmost importance to continue using this strategy for five or six consecutive years to avoid having buffaloes breeding in the fall-winter period. This happens in buffaloes that are raised on farms where the use of out-of-season breeding is disrupted. In such a case, buffaloes born in the spring-summer period, lengthen their calving interval by one month each year in conditions of natural mating. Then 5 or 6 years later, the birth calendar becomes seasonal. Finally, in mature heifers that are less sensitive to photoperiod, it is possible to avoid having any calvings between February and April. To achieve this goal, male buffaloes must be introduced into the female group at the end of the year. On the other hand, in primiparous females since August 15 (with removal of the male since October 1st) has resulted in a monthly reduction in the amount of milk produced by 15% between September to January, a stabilization from February to April and an increase between May and August that is coincident with the demands of the market.
The adoption of this technique implies a high reproductive efficiency, as well as forage conservation like silage and hay production. The increase in the costs of the farm is offset by the value of the milk used in the production of certain dairy specialties.
3. Effect on Semen Production
In the male buffalo, environment plays an important role on the reproductive behavior which can affect semen production . Oloufa  reported that libido in autumn and winter is more pronounced when compared to spring and summer. The reaction time was shown to be significantly lower in autumn and winter when compared to spring and summer. Some studies depict that libido is known to be affected by the season with high libido during the cooler months of the year. Another negative effect caused by the environment is the thermal stress. In the tropical areas and during the hot summer in temperate areas of the world, the heat stress results in reduced seminal quality and lower sperm viability [11,12]. Therefore, in most of the Brazilian territory, especially in the humid tropical climate of the Amazon, environmental management practices associated with animal management are fundamental for the sustainability of the buffalo semen production system. Furthermore, considering that in some areas buffalo species present reproductive seasonality, the variation in seminal quality may occur as a function of the season, with a longer reproductive activity in the autumn/winter seasons, which represent the short days (hour x light) [73,75,76]. However, under the humid Amazon rainforest conditions, with climate characterized by rainy and non-rainy periods, where temperature and relative humidity remain very high throughout the year in certain places, it causes discomfort and thermal stress in farm animals, especially in buffaloes . In these areas, management must be improved in order to enhance semen production in the male buffalo [75-79].
1. Bronson FH. Climate change and seasonal reproduction in mammals. Philos Trans R Soc Lond, (B) Biol Sci 2009; 364(1534):3331–3340.
2. Jöchle W, Lamnod DR. Control of reproductive functions in domestic animals. VEB Gustav Fischer Verlag Jena, 1980.
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Affiliation of the authors at the time of publication
1Faculty of Veterinary Medicine, Post-Graduate Programme of Veterinary Sciences (PPGCV), Ceará State University (UECE), Fortaleza, Ceará, Brazil. 2Laboratory of Biotechnology and Reproduction, Institute for Biodiversity and Forrest, Universidade Federal do Oeste do Pará UFOPA, Santarém, Para, Brazil. 3 Facoltá de Medicina Veterinaria e Produzioni Animali, Centralino, Napoli, Italy.