Semen Characteristics and Artificial Insemination in the Buffalo

Buffalo bull semen has been collected and cryopreserved [1,2] and has been widely used for artificial insemination [3-5] basically employing procedures similar to cattle. In India (between 2002-2007), 350,000 doses of frozen semen were available from buffalo bulls under progeny testing of Murrah and other breeds and artificial insemination was introduced in Bhadawari, Pandharpuri and swamp buffalo breeds [6]. Specific characteristics of buffalo bull semen include their smaller volume [2,7-10], typically white color [9], lower buffering capacity with a slightly acidic pH [10], presence of static ejaculates that regain motility on dilution [11-14] and higher contents of calcium and phosphatases and low contents of citric acid, hyaluronidase and fructose [10]. Buffalo bulls exhibit a profound effect of season on the semen quality [15-21]. Semen quality is better in swamp buffalo [22]. These and other factors render the freezability and fertility of buffalo semen poor compared to cattle bull semen [23-29]. Approaches to circumvent some of these problems in the recent past have led to experimentation with the use of various dilutors, cryoprotectants and cryoprotectant protocols [2]. When fully optimized, buffalo bull semen can be cryopreserved for prolonged periods with optimal fertility and can then be widely disseminated [2]. Poor estrus expression and variable time of ovulation in female buffaloes [30-34] is another obstacle as to the proper time of insemination in buffaloes. This has led to the use of timed inseminations procedures at many locations, yet the overall coverage of buffaloes with artificial insemination continues to be low. Based on a large number of studies we are critically mentioning the semen characteristics and artificial insemination in the buffalo.

1. Sexual Maturity

Sexual maturity is attained in buffalo bulls at a higher age than cattle bulls [10,35]. Prepubertal development of the male reproductive organs in Nilli Ravi [35], Egyptian [36-38], Surti [39] and Murrah [40,41] buffalo calves revealed that the reproductive organs develop in buffalo calves by 6-12 months of age. Studies also revealed that the seminiferous epithelial cycle required 16 days in buffalo [42,43] and the duration of spermatogenesis in buffalo bulls was estimated to be 75 days [44,45]. The complete establishment of spermatogenesis occurs by 24 months in buffalo bulls [10] in contrast to 4-6 months in cattle bulls [46]. In a study on buffalo calves, spermatozoa were present in the epididymis at the age of 90 weeks [47]. The time lag between testosterone secretion (12 months) to support spermatogenesis (16 months) and finally achievement of puberty (2.5-4 years) is long [48] in buffalo. The average age at first service was 3.7 years in one study on Egyptian buffaloes [49] whereas in Murrah buffaloes the age was 27.2±1.37 months [50] and in Italian buffaloes the age was 2 years [51]. In Brazil, male Murrah buffaloes when reared in controlled management, with proper nutrition and subjected to a schedule of prophylactic health and weaning, reached puberty between 15-18 months [9,52].

An important parameter for evaluating male buffalo breeding capacity with little citation in the literature is the relation to the scrotal circumference (SC) [53,54]. Bhosrekar [55], recorded that the scrotal circumference for adult buffalo in India ranges from 30-34 cm. The relationship of scrotal circumference with age revealed that the scrotal circumference in buffalo bulls increases with age (Table 1). In studies on Jaffarabadi buffalo bulls the mean scrotal circumference in normal bulls was 33.89±1.19 cm [56] and the mean scrotal length and width was 15.20±0.14 cm and 12.39±0.51 cm respectively. A recent study found that Murrah buffalo bulls between the age of 25-36 months had a mean SC of 26.9 cm, the estimated testis weight was 261 g, semen volume was 2.5 mL and sperm concentration was 1677 x 106 /mL whereas the same parameters at the age of 60 months were 32.4 cm, 652 g, 3.6 mL and 1666 x 106 /mL respectively [57]. The mean trans-scrotal diameter in Surti buffalo bulls was 36.28 cm (29-45 cm) with high correlation of testis size with semen characteristics [58]. Testosterone levels in buffalo bulls has been observed to be nearly half compared to that observed in cattle [10] with testosterone levels being 1.58±0.32 ng/mL in young bulls [59] and 0.69 ± 0.12 ng/mL in aged buffalo bulls [53]. The lower level of testosterone may be a cause of poor libido and shorter breeding life of buffalo bulls. Initiation of sexual function is influenced by season, independent of age [60].

2. Selection and Training Male Buffalo for Semen Collection

Similar to cattle, buffalo bulls to be used as semen donors are selected on the basis of pedigree or progeny records. It has been mentioned that milk yielding traits and birth weights are highly heritable, whereas the reproductive traits have a low inheritance in the buffalo [61] and thus care should be taken in the selection. Traits of economic importance that need attention during selection of buffalo bulls include age at first calving, calving interval and age at first collection of semen [62]. Methods of selection should be based on clear breeding goals, aimed to increase milk or beef and improved productivity. Sexual behaviour index has been suggested for selection of buffalo bulls [63]. A complete breeding soundness evaluation as described for cattle bulls [64] should be performed before selecting a buffalo bull as a semen donor. This includes a physical examination, evaluation of scrotal circumference and evaluation of semen samples. A good buffalo bull should possess an appropriate body conformation (Fig. 1) and both the testes should be uniform (Fig. 2). The scrotal circumference is evaluated using a scrotal tape (Fig. 3) and the optimum scrotal dimensions in buffalo bulls of different age have been mentioned (Table 1). Adult buffalo bulls evidence good elasticity of the scrotal skin (Fig. 4). Buffalo bulls to be maintained as semen donors should also be tested for diseases such as Brucellosis, Campylobacteriosis, Bovine Virus Diarrhea, Leptospirosis, Tuberculosis or any other disease pertaining to the area where they are kept. Buffalo bulls with reproductive or systemic diseases described previously [52] should be excluded from being used as semen donor.

Figure 1. Selected semen donor of Murrah buffalo bull, 22 months age.

Figure 2. Good scrotum conformation in a sexually mature Murrah buffalo, 24 months age.

Figure 3. Scrotal circumference measurement in a young Murrah buffalo bull using a scrotal tape.

Figure 4. Good scrotum conformation in an adult Murrah buffalo, 43 months of age. Observe the elasticity and capacity of contraction of the scrotum.

The minimum standards for production of frozen semen had been laid down by the central monitoring unit, department of animal husbandry and dairying, Government of India in 2005 and similar standards are operative at other countries as well. The male buffalo is perhaps the easiest domestic species to be trained on female or even male to ejaculate into an artificial vagina [2,9,30] and generally it will ejaculate into the artificial vagina at the first attempt. Young buffalo males mounting other males at puberty can be easily trained to donate semen. Young males at puberty start to mount other males and flehmen response is observed when the bull sniffs the other herd mates male genitalia and urine, extending its chin and inhales with a slightly open mouth [1,9,65]. The artificial vagina is generally accepted as the preferred method of semen collection in order to ensure optimal semen quantity, quality, hygiene, and thermal protection. Once selected as semen donor, the male should be sent to an Artificial Insemination Center, specialized in deep freezing semen which usually occurs when buffalo bulls attain the age of 16-18 months.

Moreover, once arrived at the AI Centre, the animal must complete a normal period of quarantine throughout which he will receive special attention regarding his management and nutrition. It is important that young buffalo males after arrival at the AI Center for semen collection be maintained alone in a barn, however, it is recommended to put it together once or twice a day with other young buffalo males so that they start to acquire homosexual behavior, a very important aspect for further breeding management involving semen collection [1,9,65,66].

3. Management

3.1. Housing

In the tropical and subtropical countries with warm and humid temperatures, housing may be open, semi-open and rarely closed. Moreover, buffalo bulls raised in tropical and subtropical conditions require protection from heat and adequate ventilation because thermal stress is known to reduce sperm quality [21,67]. Shade trees, shade cloth and thatch are effective. Fine water sprays with fans or air conditioning stall can be used to cool buffalo bulls under extremely hot conditions. Buffaloes should be housed securely so there is no chance of escape and interaction with other males, staff and the general public. The use of electrical fences separating the stalls and pastures is recommended.

3.2. Feeding

A balanced protein supplement with 16% crude protein should be fed. Care should be taken not to overfeed bulls as fat deposition in the inguinal canal negatively affects fertility. Condition score is an important guide to nutritional requirements. Working bulls should have a score of 3 to 3.5 on a scale of 1-5.

3.3. Male Management (Handling)

Buffalo bulls should be provided regular exercise as this improves the seminal quality [68,69]. The establishment of a firm relationship between the personnel involved in the management, mainly the handler, and the buffalo is essential and cannot be over emphasized. The buffalo should be at ease when handled and the handler should not be afraid. The application of a buffalo nose ring is rarely required; this fact seems to be important. Usually for old buffalo bulls at semen collection centers it is not recommended to use the nose ring. Our experience has demonstrated the use of nose ring sometimes is a disaster and no semen can be collected from some old buffalo bulls. The buffalo should be handled by a strong halter with a metal chain that once pulled and forced, causes the bull some discomfort. The use of small sugar tablets, good care and comfort, cause a positive impact in the buffalo male's behavior. Buffaloes should always be handled optimizing semen production. This includes taking note of all aspects of the physiology of male sexual behavior. Negative stimuli such as noise, low hygienic conditions, stress of any origin, should be avoided as much as possible in the collection area [1,9,65,66].

4. Sexual Behaviour

Buffalo bulls are considered sluggish breeders [1,9,70,71] with lower libido and lower ejaculatory thrust. The libido (interest of male towards a female) appears to be suppressed during the hotter months and the hotter times of the day [21,72,73]. Buffalo bulls lose their libido in a short time when let loose in buffalo herds [9]. Buffalo bulls evidence a less forceful thrust at services compared to cattle bulls [74,75]. Courtship behaviour in buffalo bulls is evidenced by licking of the vulva by the male, flehmen and nudging behaviour which appears to be of lower intensity [76-78], however, a few studies with Brazilian Murrah buffaloes observed a more intense presence of flehmen activity [1,9]. The service (copulatory) behaviour is manifested by mounting, intromission, ejaculation and dismounting [76]. Parameters to evaluate libido include reaction time (time taken by the male in mounting when brought near a female in estrus), flehmen reaction (curling of the lip after sniffing the vulva of the female), mounting stimulus required, ejaculatory thrust, and dismounting time. The overall mean libido score, reaction time and service behavior score was 72.22±0.07 sec, 59.13±2.98 sec and 56.6±0.20 percent in Murrah buffalo bulls [78]. Reaction time and ejaculation time were significantly higher in swamp buffaloes as compared to river and river x swamp buffaloes [22]. The poor libido does not seem to improve by changing the sexual stimuli at the time of semen collection. In a study on 182 buffalo bulls aged between 1 and >8 years, neither age of the bull nor the physical state of the teaser (male, estrus- or non-estrus female) exerted any significant effect on the degree of sexual interest expressed by bulls [76]. However, each of the degrees of erection, holding, and ejaculatory thrust was significantly influenced by age of the bull but not by type of the teaser. Judged by the reaction time, some authors such as Oloufa [79] reported improvement of libido in winter (43.5 sec) and autumn (42.6 sec) compared to spring (49.1 sec) and summer (56.2 sec). The libido improves in spring and this is attributed to the supply of highly nutritive green fodder [72]. In another report [80], the reaction time was significantly lower in autumn (67.7 sec) compared to that observed in spring (165.0 sec), winter (87.7 sec) and summer (85.2 sec).The mean reaction time can be as high as 245.56±30.37 sec in Jaffarabadi buffalo bulls [56]. In addition to the significant differences between bulls, and probably other factors, feeding systems as well as interactions of feeding system with season contribute significantly to the variations in reaction time. Buffalo bulls in regular use as semen donors at A.I centers can be ejaculated thrice in rapid succession, two times a week without serious effect on their semen quality, apart from a slight decrease in ejaculate volume from the first to the third consecutive ejaculate [70]. 0n the contrary, other semen quality parameters (mass activity, individual motility, live sperm percent, sperm abnormalities, and methylene blue reduction time) appear to improve with sequence of ejaculation. It was also noted that 2 successive ejaculates every 3-4 days from young bulls produced significantly greater ejaculate volume and sperm output per week [71].

Poor libido does not seem to improve by changing the sexual stimuli at the time of semen collection [76]. Some studies depict that the libido is known to be affected by the season [72,79,80] with high libido during the cooler months of the year.

5. Semen Characteristics

5.1. Semen Collection

Semen is routinely collected from trained buffalo bulls by using an artificial vagina (20-35 cm in length) (Fig. 5) maintained at temperature between 44-46°C [1,2,9,10,81] although semen can also be collected by electroejaculation [82].

Figure 5. Components of an artificial vagina (Minitüb model) used for buffalo semen collection.

It is important to use an artificial vagina with a temperature higher than that used in bovine. Semen is usually collected early in the morning, before feeding, and each collection consists of two ejaculates taken within a short interval of 30 minutes [2,52]. Similar to cattle bulls, the erect penis of the buffalo bull is deflected into an AV when he mounts another male (Fig. 6). One to four collections per week can be taken from a buffalo bull [10,83] without affecting fertility. In extreme winter, the collection tube fitted with AV needs to be protected from the chilling environmental temperature to avoid cold shock to spermatozoa. Immediately after collection the semen should be placed in a water bath at 37°C and evaluated for its quality [9,66].

Figure 6. Semen collection from a buffalo bull (Photo courtesy Dr R.K. Sharma Central Institute for Research on buffaloes, Hissar, Haryana, India).

5.2. Physico-Morphological Characteristics

The physical morphological characteristics of buffalo bull semen include color, volume, pH, mass activity, sperm motility, sperm concentration, live sperms, sperm morphology and semen biochemical properties.

5.2.1. Colour and Density

The colour of buffalo semen varies from a milky white to creamy colour, with a light tinge of blue [9,84]. The density of the semen depends upon the spermatozoa concentration, age of male and season of collection.

5.2.2. Volume

There is significant variation in the volume of buffalo bull semen (1.3-4.5 ml) with young bulls producing lower quantity (1-3 mL) and adult bulls producing up to 8 mL (Fig. 7) [9]. The volume of semen recorded in different studies (Table 2) reveals marginal differences between the breeds. Several factors like age, breed, and season influence the ejaculate volume [10,73]. Usually the volume of a second ejaculate collected in quick succession is higher than the first ejaculate. The rainy season is considered to be the optimum season when buffalo bulls ejaculate the maximum volume [10,85]. Cooling arrangements during hot summer months can thus increase semen volume. The transrectal massage method is not a satisfactory technique for semen collection from buffalo bulls as it results in poor volume, low sperm concentration and a high pH [52,75,86].

Figure 7. Buffalo ejaculates; excellent quality volume - 5.5 and 6.0 mL respectively.

5.2.3. pH

The pH of semen is generally evaluated by pH strips or by using a pH meter. Freshly ejaculated buffalo semen has a slightly acidic pH, with a range reported from 6.2-7.0 [19,87-94]. The buffering capacity of buffalo bull semen is much lower compared to cattle bull semen probably because of lower concentration of protein, citrate and carbonate ions present in the seminal plasma [10]. Therefore, semen extenders should have sufficient buffering capacity.

5.2.4. Mass Activity

The mass motility of freshly collected buffalo bull ejaculates evaluated under a microscope is known to be low compared to cattle bull semen. The wave motion observed in buffalo bull semen is slow compared to cattle bull semen. On a 0 to 5 point scale, the mass motility of buffalo bull semen varies between + 2.4 to + 4.0 (Table 2), with repeatability of 0.40 [10,95] and seasonal variations in the mass activity. A lower mass activity in the buffalo bull ejaculate is considered to be due to lower sperm concentration and poor individual motility [52].

5.2.5. Individual Sperm Motility (Initial Sperm Motility)

Individual sperm motility is assessed in extended semen samples under a microscope and is a visual estimate of the percentage of motile cells. A small drop of semen is placed on a dry slide (Fig. 8) maintained at 37°C covered with a cover slip and examined at a magnification of 40-100 fold. The proportion of spermatozoa with a progressive forward motion is counted. Estimates of initial sperm motility vary widely from 57.37±1.06% to 82.0±5.0% [9,12,18,50,96,97] and are affected by the extender used, season, breed, age of buffalo bull and the error in laboratory processing [10,123]. Vale [9] considers that ejaculates with over 70% of sperm cells with progressive forward movement are the minimum standard for a good semen sample suitable for cryopreservation (Table 2). During the last decade the introduction of computer assisted semen analysis (CASA) has enabled those working in this field to use new parameters in assessing sperm motility [124,125]. The CASA systems can evaluate parameters like speed, direction and the beat cross frequency of the sperm cells [2].

Figure 8. Placement of a small drop of semen for evaluation of individual motility.

5.2.6. Vigor (Individual Motility)

Vigor or individual motility is evaluated using microscopy looking at the highest number of motile sperm cells possible. An individual sperm cell must be followed until it disappears from the microscopic field. This operation must be repeated many times. A good ejaculate must have a vigor of >3 in a scale of 1-5, that means more than 60% individual motility [9,66].

5.2.7. Sperm Concentration

The sperm concentration in buffalo bulls is known to vary between 534 and 1400 million per ml [9,12,18,30,87,89,92,94,120,126,127] (Table 2). Variation in the spermatozoa concentration per unit volume occurs due to breed, age, climate, restraints, number of false mounts and the frequency of ejaculations [10]. Sperm concentration determines the dilution rate of semen at semen collection and processing centers. Sperm concentration is determined by counting the cells in a Neubauer chamber of a hemocytometer. With the aid of an automatic micropipette 20 μL of fresh semen is diluted in a graduated tube containing 4 ml of a buffered formol-saline, to give a final dilution of 1:200 (Fig. 9). The sample is carefully homogenized and the hemocytometer is charged with the diluted semen (Fig. 10). Sperm cells are counted under an optical microscope 40X objective, performing the sum of at least five large squares (compounds 16 small squares). The result is multiplied by the dilution factor of 10,000 to obtain sperm concentration per ml of ejaculate. More recent studies suggest that the concentration of spermatozoa in ejaculates can be satisfactorily evaluated using a spectrophotometer [2]. Woelders [128] suggest that the fluorometric measurement of the amount of DNA might give a reliable estimate of the concentration of spermatozoa. Commercially available instruments (Fig. 11) for evaluation of sperm concentration should be calibrated by evaluation of sperm concentration using a hemocytometer.

Figure 9. Buffalo sperm cells diluted in a buffered saline-formalin solution.

Figure 10. Illustrating scheme showing procedure to determine the sperm concentration by using a hematometric chamber model Neubauer.

Figure 11. A commercially available automatic machine for the evaluation of sperm concentration.

5.2.8. Live Sperm Cells (Viability)

The percentage of live spermatozoa determines the quality of the ejaculate [2]. Since a positive correlation between conception rate and viability of spermatozoa does exist, the importance of live spermatozoa in a semen sample is great [10]. In freshly collected semen from buffalo bull the percent live spermatozoa vary from 60 to 95% [10,79,96,129-131]. Semen samples with more than 30% initial dead spermatozoa may not be suitable for storage and freezing [2] (Table 3). Differential staining techniques have been used for differentiation of live and dead spermatozoa [11,97,126,132-134]. The staining solution is prepared by dissolving 1.67 g of eosin yellow and 10 g of nigrosin in distilled water to make 100 ml. The solution is boiled for 20 min and then cooled, filtered and stored at refrigeration temperature. One part of semen is mixed with two part of the stain and smears are prepared on a slide. The smears are then air dried and examined under microscope. The head of the dead sperm typically stain pink due to changes in membrane permeability whereas live sperm continue to be colourless (Fig. 12). A total of 100 sperms should be counted to evaluate the percent of live/dead spermatozoa. The libido of the bull significantly influences the proportion of live spermatozoa in an ejaculate with greater viability of sperms in ejaculates from buffalo bulls with higher sex drive [10]. It has been mentioned that triple staining (TriSt) techniques are better for evaluating the viability of frozen thawed sperms compared to eosine nigrosine stains as this stain overestimate the number of live sperms in cryopreserved samples [135].

Figure 12. Supravital stain eosine. Top: Live sperm (colorless); Bottom: Dead sperm (pink).

5.2.9. Abnormal Spermatozoa

The evaluation of sperm morphology is usually performed through dried semen smears in slides and stained with carbo-fucsine-eosin stain method according to Williams or Red Congo-Cerovsky (Fig. 13). The various types of structural abnormalities associated with head, middle piece, and tail are recorded. The abnormalities may either be hereditary or arise because of defects caused by infectious diseases or environmental factors. They can be classified as in the bovine according to their source and nature. Stained smears are examined under oil immersion (X1000) and 200 sperm cells are counted. An alternative approach is to examine a wet preparation of sperm fixed in formalin buffered saline solution, mounted with a cover slip and examined on a phase or interference contrast microscope, counting 200 sperm cells. Two hundred sperms are visualized to calculate the proportion of abnormal spermatozoa [9,127]. The average proportion of abnormal spermatozoa varies from 3 to 20% [12,15,89,94,129,130,136-138]. The abnormal spermatozoa are detected by staining and are usually classified as head, middle piece and tail abnormalities [12,130]. Most abnormalities were found on the sperm head (2 to 5%) [12,88,94,138], while middle piece abnormalities were less than 1% and abnormal tail (coiled tails, bent tails, protoplasmic droplets and tailless) varied from 2 to 9% [12,138]. The proportion of abnormal spermatozoa is influenced by breed, season, and frequency of use and sex drive [10].

Figure 13. Abnormal forms of sperm morphology. Cerovsky stain.

5.2.10. Acrosomal and Membrane Integrity

The abnormal acrosome percentage varies from 5 to 18% in the freshly collected buffalo bull semen [10]. The detachment of the acrosome results in a decrease in ATP and loss of intracellular proteins responsible for fertilization [139]. No significant difference in the acrosome structure of cattle and buffalo spermatozoa exists; however, certain enzymes responsible for fertilization like acrosin and hyaluronidase are known to be low in buffalo semen [10].

5.2.11. Static Ejaculates

Freshly collected buffalo semen sometimes does not show any mass activity but the majority of these ejaculates regain motility when mixed with an extender. Such ejaculates are called static ejaculates [10]. The occurrences of static ejaculates are more frequent during extreme climatic conditions and vary from 25 to 66% [11,12,14,134]. Significant differences between the percent of motile sperms and percent live sperms were recorded between static and normal semen samples [11,134] and the freezability of static ejaculates was low with a high discard rate [14]. A significant variation in cytomorphological and biochemical characteristics between static and normal ejaculates was observed [12,13]. Possible reasons for static ejaculates are the presence of a spermiostatic factor, motility inhibitory factor or the absence of forward motility proteins [10].

5.2.12. Morphology of Spermatozoa

Buffalo bull spermatozoa are more rectangular as compared to cow bull spermatozoa. The head of the buffalo bull spermatozoa resembles that of the ram but is shorter and narrower [136,140]. It is narrowest at the base and widest at a point slightly behind the anterior end [141]. Spermatozoa measurements reveal that compared to cattle bull, spermatozoa of buffalo bulls are smaller (Table 4).

5.2.13. Semen Biochemical Characteristics

The earliest reports on biochemical studies on buffalo semen appear to be those of Pal [143] and Sayed and Oloufa [98]. Buffalo semen is characterized by high contents of calcium, chloride, acid soluble phosphorus and, acid and alkaline phosphatises [10]. The concentration of alkaline phosphatase could be a factor adversely affecting the viability of buffalo spermatozoa during in vitro storage. The total electrolyte contents in buffalo semen has been found lower compared to cattle bull semen [10]. The altered citrate: chloride ratio might be responsible for poor buffering capacity of buffalo bull semen. Significantly lower levels of ascorbic acid [144] in buffalo semen may be responsible for its poor preservability. Seminal protein and total cholesterol are also lower in buffalo bull semen compared to cattle bull semen [10,145]. Buffalo spermatozoa are rich in all lipid components but in seminal plasma the situation is reversed [10,146]. Triglycerides are the major lipids in buffalo semen. High levels of alkaline phosphatase coupled with high concentrations of phosphatase esters tend to suppress the spermatozoa's oxidative metabolism and result in poor in vitro survival. Buffalo spermatozoa have intrinsically poor oxygen uptake capacity and aerobic fructolysis index, which results in a decrease in sperm motility more rapidly under aerobic than under anaerobic conditions. The lower initial content of fructose and citric acid adds to the poor preservability of buffalo spermatozoa [10,144]. The fructolysis index of buffalo spermatozoa is low [147]. A critical factor governing buffalo semen preservation is the cold susceptibility especially damage to the acrosome and spermatozoa membrane, and leakage of acrosomal enzymes [10].

In some studies it has been found that buffalo semen contains higher fructose [148,149], acid and alkaline phosphatase activity [144,150,151] and inorganic phosphorus [151-153], but lower ascorbic acid concentration [151,154] compared to cattle bull semen [29]. Acid phosphatase shows significant direct correlation with sperm concentration, percentages of initially motile and live spermatozoa, dehydrogenase activity and rate of fructose utilization. On the other hand, and in contrast to cattle bull semen, higher alkaline phosphatase in buffalo semen is concomitant with decreased motility; per cent live cells, depressed dehydrogenase activity and a slight decrease in fructolytic rate [151]. It could be inferred that higher concentration of alkaline phosphatase in buffalo semen adversely influences the viability of spermatozoa during in vitro storage. Significantly lower levels of ascorbic acid in buffalo semen [155] may be responsible for poor in vitro preservability of buffalo spermatozoa since it has been demonstrated that buffalo spermatozoa are more susceptible to oxidative damage during storage and preserve better under an environment with low oxygen [156]. Both acetylcholinesterase [157] and amylase [158] are significantly lower in buffalo bull than in cattle bull semen. Buffalo semen has markedly lower cholesterol content [153]. Buffalo semen contains characteristically low levels of potassium [153,159,160] compared to sodium. The sodium: potassium ratio in buffalo seminal plasma is 1: 3.7 on the average with well over 60% of the ejaculates having a ratio of 1: 2.86 [160]. The chloride content of buffalo semen is considerably higher [153]. The total protein content in buffalo semen (2.27 g/100 ml) is also significantly less compared to that observed in cattle bull (5.43 g/100 ml) semen [161,162].

As in bulls, glutamic acid is most predominant in seminal plasma but arginine is predominant in spermatozoa. The DNA / arginine ratio is significantly higher in buffalo (1.08) than in bull (0.93) spermatozoa. There are also considerable qualitative differences in buffalo seminal protein fractions separated by starch gel electrophoresis. In a few studies the fructose utilization rates and fructolytic indexes were significantly higher in semen of buffalo bulls compared to that of Bos taurus bulls [149,]. Partial correlation studies revealed that fructose consumption in buffalo semen is primarily influenced by sperm cell concentration [153,165]. Initial fructose level does influence fructose utilization acting as a rate-limiting factor. Moreover, the live sperm percent in buffalo semen independent of initial fructose level, sperm concentration and initial motility influences considerably the rate of fructolysis, whereas initial motility independent of other factors does not correlate with the amounts of fructose utilized. Buffalo sperm acrosome is rich in hydrolytic enzymes, as alkaline phosphatase [166] and beta-glucuronidase [167], but acid phosphatase is localized mainly in the post acrosomal segment [168]. There are many other differences in the various biochemical constituents of buffalo semen which have been recently described in detail [29].

5.2.14. Semen Microbiology

Many previous [169,170] and recent [171-173] studies evaluated the presence of microbes in freshly ejaculated and extended buffalo semen. The organisms isolated include Staphylococcus, Bacillus, E. Coli, Alcaligenes faecalis, Proteus, Citrobacter and Pseudomonas sp. Many of these organisms are environmental and fecal contaminants. Thus semen collection and preservation should be performed under aseptic conditions as far as possible. The use of amikacin and enrofloxacin has been suggested as additives in semen due to their high efficacy against the microbes compared to the routinely used streptomycin and penicillin [172,174].

6. Preservation of Buffalo Semen

Preservation of buffalo bull semen appears to be a little difficult compared to bull semen due to many factors including lack of proper extenders, difference in freezability of semen and difference in the semen quality during different seasons.

6.1. Liquid Storage of Semen

Buffalo semen can be stored at 5°C for up to 72 h without significant decrease in motility, if it is extended with media that have the same composition as those used for cryopreservation [175]. However, the use of liquid semen for artificial insemination in buffalo has not been readily accepted, natural mating is preferred by some breeders and AI is not available at many locations. Tris citrate based extenders are considered the optimum for liquid storage of semen [175]. The collected semen is first examined and then extended with suitable extender at similar temperatures. The extended semen is then slowly cooled to 5°C and then stored at this temperature. Reports on the fertility with liquid buffalo semen are scarce.

6.2. Cryopreservation of Buffalo Semen

Buffalo semen can be cryopreserved by extension with suitable extenders, with addition of cryoprotectants, additives and antibiotics, filled in semen straws and then frozen by different procedures. Many procedures mentioned for bull semen are used for cryopreservation of buffalo semen; however, there are subtle differences in the extenders, cryoprotectant proportions and processing which are mentioned.

6.3. Freezability of Buffalo Semen

Many studies in different countries have demonstrated differences in the freezability of buffalo semen collected during different seasons with cooler months and shorter days yielding better freezability compared to the hot summer months [121,176,181]. It has also been mentioned that semen from different individuals differs greatly in the ability to withstand freezing [181,182]. Freezability of buffalo semen, on the other hand, seems not to be influenced by the bull or the extender used (bull x extender interaction) [182] but addition of fructose affected the freezability [113].

Equilibration nullifies the effect of season but freezing and thawing operations exert definite effects on post-thawing sperm recovery. The size of the straw is considered important in the cryopreservation of buffalo semen [183] and likewise the extenders and additives exert a difference in the freezability of buffalo semen [93,175,184-187]. Sephadex filtration of semen appeared to improve the freezability of buffalo semen [188], however, this was not validated in clinical studies. Monthly variations in the freezability of buffalo semen have been mentioned [85,97]. Few scattered reports are available that describe the differences in chemical composition of buffalo seminal plasma and spermatozoa under different climatic conditions [189-191]. However, the information provided in these studies is insufficient to explain the variation in freezability of buffalo spermatozoa during the different seasons. Thus more studies are required to explain these differences in the freezability of buffalo semen during different seasons.

6.4. Selection of Freezable Ejaculates

Since the discovery that buffalo spermatozoa could be preserved at subzero temperatures, rigorous efforts have been made to predict the freezability of buffalo spermatozoa on physical, morphological, biological and biochemical basis. The tests used with buffalo semen are essentially those recommended for usage with bull semen. These include mainly detection of the susceptibility of spermatozoa to cold effects by determining the percentage of motile spermatozoa, monitoring some valuable parameters as freeze-injury of acrosomal membranes, leakage of acrosomal enzymes as acrosin, hyaluronidase and transaminases. It has recently been reported [181] that cold shock inflicted damages on buffalo spermatozoa resulting in changes in the percentage of individually motile spermatozoa, percentage of damaged acrosomes, amount of Glutamic-Oxaloacetic Transaminase (GOT) released extracellularly comparable to those which take place upon freezing and thawing. However, in view of the extremely low associated variance calculated between various semen parameters (7.29-9.61%), the use of cold shock test to predict freezability of buffalo semen continues to be questionable.

6.5. Extenders

A large number of extenders have been tested for cryopreservation of buffalo bull semen including coconut water [66,127], citrate based diluters [192-194], lactose-egg yolk diluents [160], milk diluents [1,18,93,195-196], soya milk [197,198], Tes [199-201], citrate based media [25,175,202-204], commercially available Laiciphos and Biochiphos [167,182,205-207] and Tris based extenders [175,200,201,208-219]. Andrabi [29] concluded from analysis of a large number of studies in buffalo that Tris-citric acid provides the most satisfactory buffering system to improve the freezability and post-thaw motility of buffalo semen and Agarwal and Tomer [10] also expressed similar views previously with comments that this is the most commonly used extender for buffalo semen. The composition of commonly used Tris buffer utilizes the addition of egg yolk, citric acid and fructose [220] in the extender (Table 5). The composition of Tes Tris, Lactose Tris, skimmed milk and ringer lactate extenders (table 6, Table 7, Table 8 and ) all utilize 7% glycerol.

6.6. Cryoprotectants and Additives

The permeable and non-permeable cryoprotectants commonly added to buffalo semen are described separately although they are used in combination. Egg yolk, glycerol and sugars are commonly used in combination along with antibiotics and other agents.

6.6.1 Permeable Cryoprotectants

Glycerol is the common permeable cryoprotectant added to extenders for cryopreservation of buffalo semen. The most acceptable proportion of glycerol according to many studies varies from 5% to 6% [175,186,214,221-225] with single step addition being better over two step addition [226]. Other permeable cryoprotectants tested for buffalo semen include propylene glycol, ethylene glycol [227-229] and dimethyl sulfoxide [230] with little improvement over glycerol. Thus glycerol continues to be the preferred permeable cryoprotectant suggested to be included in extenders for cryopreservation of buffalo bull semen.

6.6.2. Non-permeable Cryoprotectants

Egg yolk is used as a cryoprotectant for buffalo bull semen. The lecithin and low density lipoprotein contents in egg yolk contribute to the preservation of the lipoprotein sheath of the sperm cell [187]. However, in addition to its protective action against cold shock, the egg yolk also stimulates the enzyme system of spermatozoa. This result in deamination of certain specific amino acids normally present in the dialysable fraction of egg yolk and yields hydrogen peroxide, which is toxic to spermatozoa during storage under aerobic conditions [231]. For this reason, the egg yolk may be dialysed before addition to the extender. Little attention has been paid to the level of egg yolk necessary for freezing buffalo semen, and the majority of workers are using concentrations around 20% [2,232]. It should be noted, however, that as the yolk concentration is increased in the diluent, the pH of the medium decreases and tends towards the acidic side. This may also be the reason for the depressing effect of higher amounts of yolk on motility of thawed spermatozoa. The toxic effect of egg yolk may be combined with toxicity of dead spermatozoa [233].

One study examined various levels of egg yolk in a tris-glycerol-based freezing diluent and found that egg yolk beyond 5% did not show any significant improvement in post-thaw motility [231]. This indicated the scope of reducing the yolk level from 20% to 5% without adversely affecting the freezability of buffalo semen. In the absence of egg yolk, none of the diluents used (tris-, milk- and citrate-based) gave protection to spermatozoa during freeze-thawing. After addition of egg yolk, post-thaw motility improved significantly, but there was no further increase for concentrations higher than 5% in the three buffers [93]. A few studies also examined the value of duck egg yolk, guinea fowl egg yolk and Indian hen egg yolk in place of normally used hen egg yolk with results of improvement in freezability of buffalo bull spermatozoa [232], however, the difficulty in obtaining such yolks limit their wide scale use.

The cryoprotective effects of six sugars - glucose, xylose, raffinose, fructose, sucrose and cheeni (a high molecular weight sugar containing 99.5% sucrose) each at 1%, 1.5% and 2% concentrations, were examined by various workers [187,234-236]. They found that the protective effect of sugars depended on the type of diluent used for freezing. Sugars create an osmotic pressure, including cell dehydration and therefore, a lower incidence of intracellular ice formation.

6.6.3. Other Additives in Semen during Cryopreservation

Keeping in view the poor freezability of bubaline semen, attempts have been made to improve the basic buffers developed to minimize the deleterious effects of cryogenic procedures. There are few scattered studies that have used additives such as antioxidants, chelating agents, metabolic stimulants, detergents, etc. for improvement in post-thaw quality of buffalo spermatozoa. In this regard, some studies [236,237] examined the effect of addition of caffeine or triethanolamine lauryl sulphate to Tris-citric acid-based extender. The addition of the detergent improved post-thaw spermatozoa motility. However, addition of caffeine to the extender did not made any improvement in motility. The addition of caffeine, cAMP and cattle seminal plasma improved the freezability of buffalo semen in one study [238]. It is believed that the protective effect of detergents may be exerted directly on the sperm membrane or is mediated through a change in the extending medium such as emulsifying the egg yolk lipids to make them more readily available to the plasmalemma during cryopreservation [239-241]. On the other hand, the failure of caffeine to produce any improvement is not understood. The effect of 0.1%cysteine or 0.1% EDTA (sperm membrane stabilizer and capacitation inhibitor) in semen diluents (Tris-fructose-yolk- glycerol, egg yolk-citrate-glycerol or lactose-egg yolk-glycerol) on enzyme leakage (lactate dehydrogenase) from buffalo spermatozoa during freezing was also examined [236]. The addition of cysteine or EDTA to the experimental extenders did not improve the post-thaw quality of spermatozoa in terms of release of lactate dehydrogenase. Similarly the inclusion of ascorbic acid (2.5 mM) in the semen extender yielded non-significant higher post-thaw motility and survivability [238,242]. The antioxidant effect of ascorbate is related to direct vitamin E regeneration by reducing the tocopheroxyl radical in the one-electron redox cycle [243,244]. The effects of vitamin A, D and E in extender on motility, survivability and acrosomal integrity of cryopreserved buffalo bull spermatozoa evidenced marginal improvements with addition of vitamin E at 0.3 mg/ml only [171,245]. Similarly the addition of ascorbic acid (0.02%) improved the semen quality and fertility of buffalo semen [246]. The effects of addition of amino acids (glutamine, glycine, alanine and cysteine) enhanced the post-thaw motility and acrosomal integrity [247]. It is well known that α-tocopherol inhibits lipid peroxidation (LPO) in biological membranes, acting as a scavenger of lipid peroxyl and alkoxyl radicals, thus preventing oxidative damage in cryopreserved bovine semen [248]. Sodium pyruvate (1.25 mM) addition to the extender resulted in significantly better post-thaw progressive motility and viability [249]. The beneficial effect of pyruvate and α-ketoacids is attributed to its antioxidant property. The addition of oviductal proteins obtained from various stages of the oestrous cycle to Tris-based extenders on spermatozoa characteristics in buffaloes revealed marginal improvements (Kumaresan et al., 2006). A recent study [250] attempted to improve buffalo semen cryopreservation with the incorporation of bradykinin (0.5, 1.0 and 2.0 ng/ml) in a routinely used extender. It was found that incorporation of bradykinin (2 ng/ml) in Tris-based extender might be useful in improving the quality of frozen-thawed bubaline spermatozoa as determined by live percentage, motility and plasma membrane integrity, however, the mechanism of this improvement was not elucidated. It appears that there are some additives, which have some useful effects in terms of improvement in the quality of frozen-thawed buffalo spermatozoa; however, their routine use cannot currently be justified.

6.6.4. Antibiotics

It is important to efficiently control the population of microorganisms in the semen. Conventionally, benzyl penicillin 1000 IU/ml and streptomycin sulphate 1000 μg /ml alone or in combination is commonly added to the freezing diluents of buffalo bull semen [2,251]. Some workers found that control of bacteriospermia in buffalo bull semen with streptomycin and penicillin (SP) is not an effective combination [252-256]. More recently, a few studies [174,257] found that bacteria isolated from buffalo bull semen were resistant to penicillin and gentamicin (500 μg/ml), amikacin (500 μg/ml) or norfloxacin (200 μg/ml) were the antibiotics of choice to be added in the extender for efficient preservation of buffalo spermatozoa. Also SP was deleterious to the post-thaw quality of spermatozoa. A few other studies [251,258,259] investigated the effects of a relatively new antibiotic combination (gentamicin tylosin and linco-spectin, GTLS) in extender and concluded that GTLS is more capable than SP for bacterial control of buffalo bull semen as judged by total aerobic bacterial count and/or in vitro antibiotic sensitivity. Moreover, GTLS is not detrimental to spermatozoal viability of buffalo bull.

7. Semen Processing for Cryopreservation

It is generally accepted that the cryopreservation process itself reduces more than 50% of the sperm viability [260]. During this process, the spermatozoa are subjected to chemical/toxic, osmotic, thermal, and mechanical stresses, which are conspicuous at dilution, cooling, equilibration, or freezing and thawing stage [29]. The various procedures for processing of buffalo bull semen are mentioned.

7.1 Transport of Ejaculates

When the collection farm is away from the processing laboratory, ejaculates need to be transported as early as possible. No change was found in the subsequent quality of semen when it was processed within 1 h of its collection [261]. It was also suggested to add the diluents and cool the semen to 5°C when processing of semen will not occur shortly after collection [65,262]. It is recommended to locate the donor buffalo bulls in the vicinity of the processing laboratory.

7.2. Methods of Dilution and Dilution Rates

Dilution rates of 1: 1 to 1: 12 have been successfully used for buffalo semen. Perhaps dilution rates for semen are based on the sperm concentration [263]. The dilution is carried out either in one or two steps to a final concentration of about 50-150 x 106 sperms per mL. Reports on diluted buffalo semen with acceptable fertility described sperm concentrations ranging from 30 x106 to 120 x 106 cells/ml [264,265]. When the two-step method is adopted, the second diluents portion has a higher cryoprotectant concentration than the first portion [2]. The addition of glycerol 1 h before freezing is the best approach when a two-step freezing is used [2].

7.3. Initial Cooling

After dilution, the semen is cooled to a temperature close to 4°C or 5°C. Cooling is a period of adaptation of spermatozoa to reduced metabolism. Extended semen is cooled slowly to avoid potential cold shock [29]. Cold shock is believed to impair function of membrane proteins that are necessary for structural integrity or ion metabolism [266]. Major changes in bovine spermatozoa during this phase occur near 15 to 5°C, and do not happen below 0°C [266]. It has been empirically determined that cooling cattle bull spermatozoa from body temperature to 5°C performed at a rate of 10°C/h has minimum deleterious effects [267]. In this regard, Dhami et al., [268] studied the effect of cooling rates (5, 30, 60 and 120 min from 10 to 5°C vs 120 min from 28 to 5°C) on deep freezing of buffalo semen diluted in Tris-based extender. Their results suggest that buffalo semen can be frozen successfully after cooling for 30 min at 10°C as judged by motility and survivability.

7.4. Equilibration

The time period between addition of glycerol and the initiation of freezing is known as equilibration (glycerol equilibration period). It has been demonstrated that a minimum period of equilibration is required for obtaining optimum post-thaw motility and fertility in semen. A wide range of equilibration period from 2 to 18 h has been reported. In Tris-based buffers, 4 h equilibration time gave better post-thaw motility compared to 2 or 6 h [269,271]. In other reports, a 2 h equilibration period was considered optimum [10]. The added glycerol penetrates into the sperm cell to establish a balanced intracellular and extracellular concentration. It should not be overlooked that the equilibration includes the concentration balance not only of glycerol, but also of the other osmotically active extender components [272]. Therefore, this phenomenon interacts with the type of extender (buffer and cryoprotectant) used and could easily interact with other cryogenic procedures.

7.5. Freezing of Semen

Earliest work on freezing of buffalo semen appears to have been initiated in the 1950s [273,274] using solid CO2 and alcohol mixture. Subsequent studies cryopreserved buffalo semen using dry ice coolant and different extender formulations for cooling with inconsistent results. Many of the earlier studies utilized glass ampoules for cryopreservation and cooling rates from 5°C to -15° C and from - 15° C to - 79°C were manually regulated by adding dry ice to the alcohol bath. The results of such studies yielded inconsistent post-thaw recovery of spermatozoa. Studies 20 years later started utilizing vapor freezing techniques for cryopreservation of buffalo semen [196,275] and because of its simplicity, convenience and better post-thaw recovery, these techniques have become popular with later improvements in extenders, programmable freezing apparatus and cryopreservation protocols. Currently vapor freezing techniques employing liquid nitrogen are in use at most buffalo semen freezing centers.

After equilibration, the diluted cooled semen (+4°C) is packaged in pre-sterilized empty semen medium (0.5 mL) or mini (0.25 mL) straws either manually or by using automatic instrumentation available commercially (Fig. 14). The straws should be printed before filling with semen (using straw printing equipment) with the bull number, breed and, date of collection. The open end of the straws is sealed with PVC powder in manual packaging. To maintain the temperature at 4°C all these procedures are carried out in a cold handling cabinet (Fig. 15). Mini straws are currently used for semen packaging and freezing due to their cost effectiveness and saving of storage space [276]. An improvement in conception rates were observed with the use of mini straws compared to the medium straws [183,277]. Freezing in liquid nitrogen vapor is practical and can be done by using an isotherm box. The straws are placed in horizontal racks placed in the isotherm box 1-4 cm above the liquid nitrogen for 10-20 min, after which they are directly immersed into the liquid nitrogen container (-196°C) in goblets [2]. Many freezing equipments (programmable freezers) are commercially available which control the drop in temperature of semen.

Figure 14. Commercially available automatic semen filling and sealing equipment.

Figure 15. Commercially available cold handling cabinet for semen processing.

Various approaches of temperature decline have been suggested while using these instruments. The temperature drop from +4°C to - 40°C have been elucidated to be the most important with temperature drop of 20°C per min being the optimum approach [127,278,279], the temperature drop then being 15°C per min up to -100°C [249]. Semen is then held at this temperature for 5-15 min before being immersed in liquid nitrogen containers. Based on wide analysis of literature, the cryogenic procedures for buffalo semen were outlined as: cooling from 37° or 39° to 4°C at the rate of 0.2-0.4°C per min, equilibration time of at least 2-4 h at 4°C, freezing of straws approximately 4 cm above liquid nitrogen for 10-20 min, or by the fast freezing rates (programmable freezing), and thawing at 45-60°C for at least 15 sec.

7.6. Thawing of Semen

The thawing of frozen semen (bringing of frozen semen into liquid state) is as important for sperm survival as is the freezing procedure [29]. In order to avoid recrystallization, rapid warming rates (placing semen straws in a water bath with temperature of 40°C for 30 sec) are commonly suggested for buffalo semen [127,268,280,281]. Somewhat slower thawing rates (35° to 39°C for 30 sec) have also been suggested [93,216,261,282,283]. Although the benefits of slow thawing were mentioned to be better post-thaw motility and acrosomal integrity, no differences between thawing at 35°C for 30 sec or at 50°C for 15 sec were observed by some workers [268,280].

7.7. Effect of Freezing on Sperm Motility and Morphology

The freezing procedure results in significant reduction of sperm motility and in sperm morphology, especially the sperm head length, maximum breadth, base width, and acrosome length and head area. The bulk of evidence indicates that changes of sperm head measurements (except acrosome length) are primarily due to the combined effect of dilution, cooling, glycerolization and equilibration prior to the freezing process [167,284]. Buffalo spermatozoa are believed to be inherently more fragile than bull spermatozoa, and upon freezing and thawing they are subjected to ultra-structural damage and subsequent detrimental chemical changes in the molecular organization of their membranes with leakage of vital material important for fertilization [184]. Electron microscopic investigations indicate that ultra-structural changes start at the very initial steps of processing, mainly after the addition of glycerol and increase progressively with each further steps of processing [2]. However, the sequential increase of ultra-structural damage after dilution, cooling, glycerolization and cold equilibration does not correlate in any way with the little change of sperm motility observed after these processing steps. The first changes due to freeze processing are relevant mainly to the acrosomal region of the sperm that shows distension and loosening of the peri-acrosomal plasmalemma followed at late steps by ruffling and swelling. Plasmalemma overlying the post-acrosomal sheath is rather resistant. Drastic alteration of sperm ultrastructure, particularly of sperm nuclei and mitochondrial sheath, become evident mainly after freezing and thawing (Fig. 16).

Figure 16. (A). Acrosome membrane normal, (B). Acrosome membrane amended, (C). Early detachment and (D). Total acrosome membrane separated. (Courtesy of Prof. Dr. K.F Weitze, Hannover-Germany).

Several parameters are used to evaluate the morphological and physiological state of spermatozoa after freeze-thawing, particularly motility [10,285-287], forward motility and membrane integrity monitored by many approaches.

7.7.1. Post-thaw Motility

The post-thaw motility is the most common parameter to assess the effects of freezing on spermatozoa and also as an indicator governing the use or discard of semen for artificial insemination. Post-thaw motility rates vary from 30%-70% (Table 10) and are known to be influenced by a number of factors such as cryoprotectants, freezing procedures, age of the bull and season of semen collection.

7.7.2. Acrosomal Membrane Integrity

Approaches to monitor acrosomal membrane integrity utilize the fixation of a small quantity (250 μL) of semen in 25 μL of 1% formal citrate (99 mL sodium citrate dehydrate 2.9% and 1 mL formaldehyde 37%) and counting of 200 spermatozoa under a phase contrast microscope (100 X) [292,293]. The post-thaw proportion of sperms with intact acrosomes varied from 51.30±0.56 to 64.10±0.52 in one study [293] whereas in another study the proportion of spermatozoa with intact acrosomes varied from 76.9 to 84.0% immediately after thawing [232].

7.7.3. Plasma Membrane Integrity

The hypo-osmotic swelling test (HOST) also known as osmotic resistance test is the usual test to evaluate the plasma membrane integrity of sperm cells [19,113,114,121,123,200,292,294-296]. The test is usually performed by incubating 10 μL of semen in 100 μL of 100 mOsm/k hypo-osmotic solution (0.735 g sodium citrate and 1.351 g fructose at 37°C for 45 min [293]. After incubation the sample is gently mixed and spread (0.1 mL) with a cover slip on a warm slide. Sperms (200) with swollen or coiled tails are recorded [297] and reflect the proportion of sperms with changes in the membrane permeability. The integrity of plasma membrane appears important and has been evaluated by lectins and sperm oocyte interactions [125].

7.7.4. Sperm DNA Integrity

Bull sperm DNA integrity has been determined by acridine orange staining [298]. Similar tests have been performed on buffalo semen to evaluate the sperm DNA integrity [293]. Air dried slides of semen are fixed in freshly prepared Carnoy's solution (three parts methanol and one part glacial acetic acid) and allowed to dry. Dried slides are stained for 3 min with acridine orange and evaluated immediately under fluorescence microscope with 490 nm excitation light and 530 nm barrier filter. Normal DNA shows green fluorescence over the head region while sperms with DNA abnormalities show varying fluorescence from yellow-green to red [293]. The proportions of sperms with abnormalities are derived by counting at least 100 sperms.

7.7.5. Metabolic Tests on Semen

Tests like methylene blue reduction and resazurin dye tests have been used in the past to evaluate bull semen [299] and a short resazurin reduction test was related with high fertility. However, such tests are uncommon in buffalo bull semen. Tests like fructolysis index (mg of fructose utilized by 109 sperms in 60 min) were calculated for buffalo semen and the index was found to be higher for semen diluted in citrate buffer compared to phosphate buffer [163,300]. An assay of maximal activities of 11 glycolytic enzymes in cell-free buffalo sperm extracts showed that hexokinase, phosphofructo-kinase and glyceraldehydes-3-phosphate dehydrogenase had the lowest activity suggesting regulation of fructolysis at steps catalysed by these enzymes [301].

7.7.6. Viability Tests

In vitro testing of fertility of buffalo bull spermatozoa for use in IVF has utilized a combination of the above tests and the trypan blue / giemsa staining techniques [302]. The test is considered suitable for evaluating the membrane status of sperm cell subdomains (head, acrosome, tail) through differential staining of the viable and non-viable segments of the sperm cell [303]. Semen traws are thawed and 1 part of semen is diluted with 9 parts of 0.9% NaCl. One drop of diluted semen is mixed with one drop of 0.27% trypan blue on a slide and thin smears are prepared by spreading on another slide and air dried. The slides are then kept in a fixative (86 mL of 1N HCL and 14 mL of 37% formaldehyde and 0.2 g neutral red) for 2 min and rinsed with distilled water. Slides are then kept in jars containing Giemsa (7.5% in distilled water) solution and kept overnight (16-20 h) at room temperature. The slides are then rinsed with distilled water and air dried [302]. Sperm cells displaying a viable head and acrosome but trypan blue stained tails are deemed non-viable [303,304]. The technique was successful in evaluating viability and acrosome status of water buffalo spermatozoa [303] and in selecting acrosome intact live spermatozoa for IVF [302].

Tests like cervical mucus penetration assay in the buffalo have yielded few conclusions [286]. An MTT assay has been recently mentioned to be more efficient compared to HOST and eosine staining to determine viability of buffalo sperms [305]. Similarly a recent report mentioned flow cytometric evaluations as a useful tool for a quick multiparametric evaluation of buffalo sperm quality [306].

7.7.7. Enzyme Leakage

Another method for evaluation of frozen-thawed semen is the analysis of enzymes which may be related to fertility. A significant increase in the hyaluronidase activity in frozen thawed semen was observed [202]. The activities of aspartate aminotransferase (AAT), hyaluronidase aminotransferase (HAT), lactic dehydrogenase (LDH) and acid phosphatase (APH) were found to be increased in extracellular medium after freeze thawing indicating leakage of enzymes from sperm cells [278]. The release of HAT and AAT showed a highly significant negative correlation with motility and acrosomal integrity, while LDH and APH had a positive correlation with motility and acrosomal integrity. The transaminases in spermatozoa are intrinsically associated with their metabolic activity and function as a reservoir of energy. A few studies [23,167] stressed the importance of levels of GOT/GPT, LDH, ACP and AKP enzymes in the seminal plasma as an indicator of the quality of frozen-thawed semen. Since the GOT/GPT, LDH, ACP and AKP release is influenced by factors such as cold shock, glycerol concentration, cooling and freezing rates [211,280,295,307], their levels can be used to assess the quality of spermatozoa after freeze-thawing. Fertility rates showed a highly negative correlation with the release of all five enzymes which indicates that enzyme leakage may be a good marker for the assessment of freezability and fertility and also for sperm damage after freezing. The questions of how far the antigenic properties of spermatozoa are affected by the customary methods of cryopreservation has been proposed by Bratanov [308] and indicate that the various steps of the freezing procedure significantly lower the antigenic activity localized on the surface of the buffalo sperm acrosome as visualized by indirect immunofluorescence.

8. Artificial Insemination (AI)

Artificial insemination in buffaloes has been reported to be initiated in 1954, Shafi and Wierzobowski, 1979 [309], however, the first reports of pregnancy in buffaloes with frozen semen appears to be those of Basirov [310]. Systematic buffalo semen freezing, distribution and insemination appear to have been initiated in 1973 in Pakistan [309] and in 1976 in India [311]. Currently artificial insemination procedures in buffaloes are carried out by using procedures similar to that in cattle [10]. The minimum number of spermatozoa required in a frozen semen straw for optimum conception in the buffalo has not yet been determined, however, the usual dose used is 30 million spermatozoa at the time of freezing [10,312]. The recto-vaginal approach used in cattle is also used in buffalo and two inseminations (12 h apart) starting from 8-12 h of estrus onset are suggested for the buffalo [10]. The major difficulty for AI in buffalo is the lack of appropriate methods for estrus detection as a high proportion of buffaloes exhibit poor estrus expression. Therefore, it becomes difficult to time insemination in the buffalo [31,127]. The fertile life of frozen semen in the female genital tract of buffalo is also shorter compared to fresh semen [313,314]. Deep intrauterine AI has been suggested using the Ghent device and one fourth number of sperms in buffalo without any significant decrease in conception rates [27].

Figure 17. Artificial insemination with frozen semen being performed in a buffalo.

8.1. Conception Rates for Artificial Insemination

Conception rates to artificial insemination in buffaloes appear to be low and highly variable due to many confounding variables including season, nutrition, body condition score, exposure to vasectomized bulls, technicians, difficulty in proper estrus detection and time of insemination [9,33,315-321]. Conception rates with frozen semen in earlier reports varied from 16% to 28% [322,323]. Conception rates varied from 40% to 50% for 13,659 inseminations at Bangalore (India) [311] and Pakistan [315]. Similarly for 35,292 inseminations in buffaloes at a semen station (Bangalore, India) that were followed for pregnancy diagnosis between 1977-1982, the conception rates varied from 40% to 50% [3]. First service conception rates for Egyptian buffaloes varied from 36% to 46% [324]. Conception rates varying from 38.0% to 43.8% were observed for AI with frozen thawed semen from Surti [328], Mehsana [325], Murrah [326,327] and Nili Ravi [110] buffaloes. The first service conception rates for 1,500 inseminations with frozen semen in 808 Surti buffaloes (1984-1985) were 30.88% and overall conception rates were 69.69% [328]. Similarly for 3,682 inseminations using semen from Egyptian buffalo bulls (5-10 years) and stored for 1 to 3 years, the conception rates varied from 60.08±1.50 to 63.30±0.87% [329]. Pregnancy rates in spontaneous estrus Italian buffaloes that were exposed to a vasectomized buffalo bull were 42.5% [33]. Trials on insemination of Italian buffaloes with frozen semen at three farms resulted in conception rates varying from 30.5% to 57.1% [320] whereas in other studies on Mediterranean Italian buffaloes conception rates varied from 40.4% to 60.6% [33,331]. Conception rates in Brazil for buffaloes inseminated during natural estrus were higher: 43.3%-81.4% (Table 9), however, the data involved only 724 buffaloes over a period of 10 years. Similarly high conception rates were recorded for buffaloes inseminated in Venezuela during natural estrus (Table 10). In Argentina, conception rates with frozen semen during natural estrus in buffaloes were 48.9% (Crudelli personal communication). In one recent study the conception rates were 25.52% for 1,943 buffaloes inseminated with frozen semen [332]. Similarly at NDRI, Karnal (India) analysis of AI records of 138 sires and 1,239 Murrah buffalo dams, for a period of 25 years, revealed an overall conception rate of 33.19% [317]. For the year 2012-2013 the conception rates at Central Institute for Research on Buffaloes (Hissar, India) were 45.75% and 40% for the Murrah and Nili Ravi buffalo herds respectively.

The coverage of animals with artificial insemination in different Asian countries varies from 20% to 90% and need substantial improvement [333]. In Sri Lanka only 2% of AI performed annually is on buffalo [334]. In Italy also the use of AI is limited to 10% of the buffaloes enrolled to the herd book [335] due to a low efficiency.

8.2. Conception Rates with Timed Inseminations

Estrus synchronization programs and timed AI at induced estrus has been used in buffaloes in India, Pakistan, Egypt, Argentina and Brazil [336-343] and are popular in Italy and South America [317,320,331,341,344-346]. These programs utilize a combination of prostaglandins, GnRH and steroids [331,341,346-348] for estrus synchronization and timed insemination with frozen semen. Conception rates in most of these protocols for timed insemination in buffaloes vary from 33% to 57% [344,345,349-352] with decreasing conception rates when such approaches are used during periods of increasing day length [317,353]. Crudelli et al., [341], however, recorded pregnancy rates up to 78%.

The use of a new protocol - CLsynch - which is based on the detection of a CL (by ultrasound) on the ovary of a postpartum female buffalo followed by the use of PGF2α and GnRH, has demonstrated to be very effective with pregnancy rates of 36.68% in one study [354] and high pregnancy rates in another study (Vale et al., non-published data) (Table 13) that compared conception rates at fixed time with Ov Synch and CL Synch protocols and two group of control buffaloes (1a and 2a) that were inseminated at natural estrus without any treatment. The overall conception rates for treated buffaloes were 68.9% whereas the overall conception rates for untreated control were 54.5% (Table 13).

8.3. AI with Sexed Semen

With the first reports of pregnancies and live births in buffaloes using sex selected semen [355-356], more reports have become available recently [357-360]. Sex selection of spermatozoa is performed by flow cytometric approaches used for cattle [355,356,361] and sex sorted semen filled in 0.25 mL straws with 4 million spermatozoa are usually deposited at the utero tubal junction [355,360]. The combined use of IVF and sexed semen has proven successful in some studies in buffaloes [362,363]. Conception rates vary from 38.8% to 49.8% [355,356,359,360].

8.4. Improvement Programs for AI

Efforts to improve the overall coverage of buffaloes with artificial insemination have been initiated in many countries. In India, a National Project on Cattle and Buffalo Breeding (NPCBB) was initiated in 2000 with the objective of improving AI services in cattle and buffaloes and supply of frozen semen and bulls. During a period of 10 years, semen production increased from 22 to 63 million doses and the number of AI increased from 21.8 to 52 millions with overall increase in conception rates from 20% to 35%. In the same way, in countries like Pakistan programs for progeny testing and semen dissemination are carried out [364]. Systematic breeding programs have also been initiated in Sri Lanka [334] and Bangladesh [365]. In Indonesia (between 1998-1999) 7,958 frozen semen doses of Murrah buffalo were produced [366]. In Italy it has been mentioned that many millions of semen doses from buffalo bulls of proven high genetic merit are available for AI throughout the world [367]. In Thailand approaches to improve genetic performance of Thai swamp buffalo have been initiated by using frozen semen from river buffalo [368-370]. Similarly, in Philippines frozen semen of Murrah breed was introduced in 1980 to crossbreed Philippine Carabao [319,371]. Improvement programs for buffalo were also evaluated in Australia using AI with semen from Italy [352]. The Bulgarian National Association for development of buffalo breeding initiated steps to cross Bulgarian buffalo with the Murrah buffalo from India for fast genetic progress [372].

In Brazil some attempts have been done in order to improve the genetic potential of milk buffaloes mainly using the Murrah breed. AI in buffaloes started in Brazil at 80' decade of the past century and then was disseminated to other countries of the continent. In Brazil there are many farms using AI as a routine since a long time ago. There are a couple of University laboratories providing facilities for deep freezing semen as the Centre for Animal Reproductive Biotechnology-CEBRAN at Castanhal county, Pará State, the pioneer centre in buffalo reproduction in Brazil. One of the most important programs within the Murrah breed was established in the São Paulo State, which originated the famous commercial trademark known as WB-Genetics. This program was extended to other farms in 1992, the Fazenda Laguna, at Ceará state, northwest of Brazil, and both together have supplied improved males and females for other farms in Brazil and other countries of the world. The project on buffalo improvement was accomplished together in 1992 throughout the increasing of the daughter's number per sire and increasing the intensity of selection with the emphasis for milk production through a careful selection of buffalo males avoiding inbreeding. Till date a total of more than 100 males were selected including 12 top levels males for deep freezing program where more than 40 thousand semen doses were produced and exported for different states of Brazil and other countries. Indeed such program was one the most important highlights on buffalo genetic improvement in Americas. Furthermore, it has produced one of the most important and legendary buffalo male in the Americas, named Guatambú. In this program the top level selected buffalo dams reached a milk production of 6000 kg per 300 days of lactation and a herd average of 3000 kg per 300 days lactation.

8.5. Crossbreeding of River and Swamp Buffaloes with the Use of AI

Crossbreeding among the various river buffalo breeds is not popular due to lower differences in milk production across the different breeds, and distinct features of various breeds suited to different agro-climatic conditions. The major use of river buffalo is for milk production and many buffalo breeds such as Mehsana, Bulgarian, and Mediterranean have originated by crossing with the Indian Murrah buffalo [373,374]. It has been mentioned that countries such as India, Malaysia and Philippines export large quantities of buffalo meat to countries such as China and Pakistan; however, efforts for systematic development of beef buffalo breeds are scarce [375]. The introduction of riverine buffalo genetic materials into distinctly swamp buffalo populated countries of China and South East Asia started as early as 1917 in the form of both live animals and frozen semen [373] (Table 12). Most of the breeds infused were Murrah Buffalo from India and Nili-Ravi from Pakistan. Attempts to improve Anatolian buffaloes of Turkey using proven river buffalo semen from Italy resulted in 105 crossbred buffaloes up to March 2008 [376]. Chinese indigenous buffaloes are swamp type with smaller body-size and lower milk and meat production when compared with river type animals [377]. To improve the production performance of Chinese indigenous buffaloes and to increase the economic benefits for buffalo rearing, Murrah and Nili-Ravi breeds were imported from India and Pakistan in 1957 and 1974, respectively to cross with the indigenous buffaloes. Such approaches have led to increase in milk production varying from 13.5% to 30.3% [378,379].

The early introduction of riverine breeds resulted in crossbreds, either by way of natural mating between the introduced breed and the indigenous swamp buffaloes or by AI in the late 60's or early 70's. In the Philippines, many buffaloes were imported from the United States and Bulgaria (1994-1999) [380] (Table 13). It has also been mentioned that many doses of Italian buffalo frozen semen were imported to China from Italy [379]. Current collated data on global transport of semen is largely unavailable and only scarce information (Table 14) is described mentioning the dissemination of semen.

China and the Philippines are working on the improvement of genetic potential of milk buffaloes, including the crossing of swamp females with progeny tested river buffalo bulls with the aim of increasing milk production in these countries. In the Philippines, the Philippine Carabao Center was established in 1993 for this purpose with 13 regional centers [319]. There are compelling social and economic reasons for the decision to pursue wide-scale crossbreeding and continuous backcrossing of swamp buffaloes with the riverine buffaloes in countries such as China and the Philippines [381].