BUFFALO MALE REPRODUCTIVE BIOLOGY
and
SEMEN PRESERVATION
(A Review Article)
M.S.S. ABDOU
Dept. of Theriogenology, Faculty of Veterinary Medicine,
Cairo University
Buffaloes constitute a significant part of the domestic stock in Egypt. The increasing importance of buffaloes in the dairy industry of Egypt, has made artificial insemination technology a requisite to improve productivity of this animal species, which would require full exploitation of the reproductive potential of buffalo bulls. Until recently, Obtaining better semen from outstanding males has, along with a multiplicity of other factors, been one of the major constraints that stood against wide spread of artificial insemination. This has instigated the interest of research workers allover the world to study the reproductive profile and breeding capacity of buffalo bulls and the characteristics of their semen.
During the last 25 years, considerable data has been obtained on various aspects of reproductive capacity of buffalo bulls including sexual behaviour and semen production as well as specificities of buffalo semen and spermatozoa and frozen semen technology.
PUBERTY AND SEXUAL MATURITY
The age of puberty and first ejaculation in Egyptian buffalo bulls (14.2 month) is reasonably early (Ali et al, 1981). Indian workers believe that initiation of sexual function is influenced by season, independent of age (Narasimha Rao and Kotoyya, 1979). However, in spite of early puberty, bulls in Egypt are put to service at about 3 - 3.5 years of age 8=](El-Itriby and Asker , 1957) and so are the bulls in India (Majeed et al , 1961). Appropriate feeding and management of prepubertal buffalo bulls is thought to be of value in enhancing puberty , since first signs of sexual interest and meiotic divisions of spermatogonial cells were found to occur as early as 9 month (Ali et al, 1981). During the first postpubertal year, a marked increase in the sperm producing capacity has been reported (Sayed, 1958).
SPERM PRODUCING POTENTIAL
Testicular weight , a reliable index of semen producing ability, has been shown to increase between 2.5 - 3.0 (68.5 g), 3.5 - 4.0 (96.2 g) and 4.5 - 5.0 (114.2 g) years of age(El-Azab et al, 1978). Essentially similar results were obtained by Ibrahim (1985) upon investigating the testes size of 3 groups of buffalo bulls aged 2-3, 3-4 and over 4 years, although other research workers (Abdou et al, 1982) failed to record any significant increase in the average weights of testes, testicular parenchyma and epididymides between 2.5 and 5 years of age. Variations in the testes weight are markedly greater between younger bulls and decrease with advancement of age. Paired testes weight correlates highly significantly with body weight. There is evidence that
the larger, faster gaining bulls would have larger testes than smaller bulls of
comparable age.
As with other animal species, gonadal sperm numbers are positively and significantly correlated with the weight of testicular parenchyma (Abdou et al., 1982). Moreover, the sperm numbers per gram of testicular parenchyma are much higher (P <0.01) at 3.5-4 years (85.8 million) and 4.5-5 years (75.7 million) than at 2.5-3 years (50.7 million). We estimated that, on the average, a buffalo bull produces 2.74 billion sperm daily and that the daily sperm production per gram of testicular parenchyma is about 13.74 million which is much less than the value of 32 million reported by Indian workers (Verma et al., 1965). In another report (Ibrahim,1985), we have found that although the gonadal sperm number per testis are significantly lower in males aged 2-3 years (7.81 billion) than in those aged 3-4 years (10.97 billion ) or those over 4 years of age (13.70 billion) -average 10.79 billions-, the parenchymal sperm concentration per unit weight do not vary appreciably between ages (93.97, 99.62 and 99.34 millions) indicating that 2 - 3 year- old bulls have already attained the mature rate of spermatogenesis. Nevertheless, among-bulls variations appeared to decrease with advancement of age. Daily sperm production increased markedly with age from 2.39 to 3.35 and 4.19 billions (average 3.30 billions), whereas daily sperm production per gram of testicular parenchyma did not (28.82, 30.46 and 30.38 millions, average: 29.93 millions), for bulls of the three mentioned ages, respectively. Indian workers reported 4 billions as the daily sperm production of a typical buffalo bull as revealed from quantitative testicular histology studies (Sharma and Gupta, 1979 and Sengupta and Bhela, 1988).
The duration of seminiferous epithelium cycle was studied by Sharma (1987) using tritiated thymidine and autoradiographic techniques. The duration of one cycle was 16 days, whereas the approximate duration of spermatogenesis was 75 days. It is thought that such long spermatogenic cycle may be in some way behind the low rate of daily sperm production in this species. Moreover, as found by Abdel-Rahman (1980) the frequency of stage 8 of the seminiferous epithelial cycle averaged 8.9 - 10.5% which does not vary from that reported for cattle. The recorded frequency appears not to vary with age.
Concerning seasonal effects, neither testicular weight, parenchyma weight nor the number of sperm produced by testis were season -dependent (Ibrahim, 1985).
Epididymal sperm reserves per bull are in the range of 3.9 - 8.51 billions (EL-Azab et al., 1978), 8.23-10.17 billions (Abdou et al., 1982) or 6.23- 10.98 billions (Ibrahim, 1985). Sharma and Gupta (1978,1979) reported 36.2 billions for the total epididymal sperm reserves per bull. In adult bulls, the relative distribution of sperm in the three major segments of the epididymis is about 30.4-33.3% in the caput, 8.79-20.5% in the corpus and 49.1-55.5% in the cauda.
The epididymis of the buffalo is a rather intricately convoluted duct with complicated structure. It is much shorter than that of the bull. Histological assessment of the epididymal duct (Abdou et al., 1985) revealed eight histologically distinct regions, which could be differentiated according to tubular and luminal diameters, cell types and regional differences in their distribution, distinctive features of their principal cells, changes in epithelial height as well as on the height of stereocilia. Our observations do indicate that the caput and proximal corpus are the most active sites at which maturation of spermatozoa is likely to occur. Various maturational biophysical, biochemical and morphological changes have been recorded. Passage of spermatozoa from caput to corpus and then to cauda epididymidis is associated with significant diminution of sperm head length, breadth and area with the head appearing more elongated ( Abdel-Rahman, 1980 and Abdou et al., 1983 a).
ACCESSORY GENITAL GLANDS
The gross appearance of the vesicular glands resembles that of the bull except for the pattern of lobulation and the shape of urethral end. These glands are markedly smaller than in Bos taurus bulls of comparable age as regards their size and weight. As observed by Osman (1965), these glands show rapid growth during the first 1.5 years of age but growth rate slows down till the age of 3.5 years when it appears to cease completely. There is an indication, however, that the glandular epithelium is significantly higher at 3-4 years than at 2-3 years of age which could entail not only higher secretory activity but also age differences in the response of glandular epithelium to male sex hormone (Eissa, 1980).The length of the ampullae of the ductuli deferentis testis does not vary significantly with age although their diameters do. External prostate of the male buffalo is ill-developed and resembles that of Bos taurus bulls in histological features. The pars interna is well developed and can be easily peeled off the surrounding urethral muscle (length= 6.7 cm, proximal width= 3.9cm, distal width= 2.9cm, thickness = 2.4cm, weight= 46.7 g). In this portion of prostate gland, two types of secretory units (arbitrarily termed A and B) are present. These are different in their location, frequency, size height of epithelium, cytoplasmic affinity to staining and nature of secretory material present therein. The height of epithelium lining the more frequent type B units does not show marked change with advancement of age. Bulbo-urethral glands do not vary significantly in weight or dimensions in bulls aged 2-4 or more years.
On the other hand it has been found that neither the general chemical composition (moisture content, percent organic and inorganic matter) nor the secretory products (fructose and citric acid ) of any of the accessory glands did vary significantly with age. However, accessory organs of adult buffalo bulls were found to be the subject of considerable seasonal variation in weight as well as functional activity. All glands revealed marked decrease in weight in summer and autumn. During these seasons, the weights of the vesicular glands, internal prostate, and bulbo-urethral glands constituted almost 86%, 80% and 72% of the weights recorded during winter and spring, indicating that the vesicular glands were least affected by seasons. Vesicular glands of the male buffalo were found to contain fructose (267.0 mg/ 100g)) in amounts much higher than those contained in internal prostate (21.3 mg/ 100 g) or bulbourethral glands (52.3 mg/ 100g). Citric acid changes with seasons were even more prominent than those of fructose. Accessory glands exhibited markedly higher citric acid levels during winter.
Bulk and trace elements concentrations in the accessory glands have recently been reported (Eissa et al., 1992). Seminal vesicles contain higher calcium and copper than other glands. Prostate gland is relatively more rich in zinc. Higher concentrations of sodium, potassium, magnesium and inorganic phosphorus are found in the bulbo-urethral glands. Copper is the only element that decreases with age. Season exerts significant effect on the levels of potassium, calcium and zinc.
SEXUAL BEHAVIOUR
Buffalo bulls are sluggish breeders. The poor libido does not seem to improve by changing the sexual stimuli at the time of semen collection. From a study on 182 buffalo bulls aged between 1 and >8 years , Badawy et al (1972) observed that neither age of the bull nor the physical state of the teaser (male, estrous- or non-estrous female) exerted any significant effect on the degree of interest of bulls. However, each of the degree 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 (Oloufa et al., 1959) 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). El-Sawaf et al. (1971) found that the libido improves in spring and attributed this to the supply of highly nutritive green fodder. In another report ( El-Azab et al., 1980), the reaction time was significantly lower in autumn (67.7 sec) than in spring (165.0 sec), winter (87.7 sec) and summer (85.2sec). In addition to the significant differences between bulls, and probably other factors, feeding system as well as interaction of feeding x season contribute significantly to the variations in reaction time.
As for ejaculation frequency, El-Menoufy (1974) found that buffalo bulls in regular use as semen donors in A.I.centres can be ejaculated thrice in rapid succession , two times a week without serious effect on their semen quality, apart from slight decrease in ejaculate volume from the first to the third consecutive ejaculate. 0n the contrary, other semen quality parameters (mass activity, individual motility, live sperm percent, sperm abnormalities , methylene blue reduction time) appear to improve with sequence of ejaculation. Yassen et al. (1985) found that 2 successive ejaculates every 3-4 days from young bulls produced significantly greater ejaculate volume and sperm output per week schedule.
SEMEN CHARACTERISTICS
The literature is full of information on various aspects of semen quality viz., sperm concentration , sperm motility, incidence of live and abnormal cells.. Buffalo semen is milky white; never yellow. Its consistency depends on its content of spermatozoa and is affected, among other factors, by frequency of ejaculation. Sperm concentration is about 800 million /ml on the average . Values as high as 1500-2000 millions sperm/ml and as low as 200 million sperm /ml have been recorded. First ejaculates contain higher number of spermatozoa per ml compared to second ones. According to several reports, sperm concentration is higher in summer (Oloufa et al., 1959 and Sayed et al., 1962, Abdou et al., 1978 b), or spring (El-Hariri, 1973). Initial motility and live sperm percent are also optimal during summer (Sayed et al., 1962 and El-Hariri, 1973). Improvement of semen quality during summer and /or spring is consistent with higher conception rates of buffaloes observed during these seasons (El-Wishy, 1971). The percentage of abnormal spermatozoa in buffalo semen varies between 3 and 26% (El-Menoufy, 1974) being less frequent in summer (Oloufa et al., 1959). Compared to Egyptian buffalo bulls, the quality of the Indian Murrah semen is optimal in spring (Sengupta et al., 1963).
Buffalo spermatozoa have distinct morphological features. A typical spermatozoon is shorter than that of Bos taurus bulls and measures 62 microns, on the average. The mean head length, maximum head breadth and breadth at the base of the head are reported to be 7.20, 4.45, 2.40 microns , respectively. Head area is between 24.3 and 26.6 sq.micron and head shape index 1.62. Various mensuration characteristics of buffalo spermatozoa are affected by seasons, feeding regime and sequence of ejaculation (El-Azab, 1980).
BIOCHEMICAL CHARACTERISTICS.
Striking compositional differences from semen of Bos taurus bulls have been reported. Buffalo semen contains higher fructose (Abdou et al., 1977 a), acid and alkaline phosphatase activity (Abdou et al., 1974; 1978 a) and inorganic phosphorus (Abdou et al., 1977/78 a) , but lower ascorbic acid concentration (Abdou et al., 1977/78 b) than bull semen. 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 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 (Abdou et al., 1978 a). It could be inferred that higher concentration of alkaline phosphatase in buffalo semen influences adversely the viability of spermatozoa during in vitro storage.
Significantly lower levels of ascorbic acid in buffalo semen 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 a reduced environment (Sengupta et al., 1969).
Both acetylcholinesterase (Abdou et al., 1977c) and amylase (El-Guindi and Abdou, 1978) are significantly lower than in bull semen.
Buffalo semen contains characteristically low levels of potassium (El-Chahidi et al, 1985 and Nour, 1985) 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 (Nour, 1985).
A highly significant difference exists between the nucleic acid contents of bull (3.52 pg /sperm) and buffalo bull (3.25 pg /sperm) spermatozoa (El-Guindi et al., 1975).
Total protein content in buffalo (2.27 g /100ml) is also significantly less than in bull (5.43 g /100 ml) semen (Khilo, 1986). 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.
Fructose utilization rates and fructolytic indexes are significantly higher in semen of buffalo bulls compared to that of Bos taurus bulls (Abdou et al., 1977 b). Partial correlation studies revealed that fructose consumption in buffalo semen is primarily influenced by sperm cell concentration. Initial fructose level does influence fructose utilization acting as a rate-limiting factor . Moreover, the live sperm per cent 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 (Abdou et al, (1984 ) and beta-glucuronidase (Abdel-Rahman, 1988 ), but acid phosphatase is localized mainly in the post- acrosomal segment ( Abdou et al., 1983 b).
PRESERVATION OF BUFFALO SEMEN
The conventional egg-yolk citrate extender originally developed for bull semen appears not so suitable for cold preservation of buffalo semen at 4 - 5 oC compared to skim-milk diluent and particularly egg-yolk glucose bicarbonate (El-Hariri, 1973). In these diluents, summer and spring semen preserves better than semen collected during autumn or winter. Although the conception rate resulting from semen stored in any of the the three mentioned diluents does not vary significantly because of the diluent factor, yet there is a noticeable tendency for better fertility from semen preserved in milk diluent. Besides, the average number of inseminations per conception is always significantly lower (1.77) for semen diluted in milk than in egg yolk glucose bicarbonate (1.97)or egg yolk citrate (2.05). Nour (1985) reported a new diluent composed of 2.0 g lactose (20 ml of a 10% solution), 0.6 g sodium citrate dihydrate (24 ml of a 2.5% solution), 0.576 g anhydrous sodium phosphate (36 ml of a 1.6% solution ) and 20 ml egg yolk per 100 ml of distilled water. This is probably one of the very few extenders designed specially for buffalo semen. It is able to support sperm viability and mensuration characteristics than do other commonly used diluents.
FREEZING OF BUFFALO SEMEN
1- Freezability of buffalo semen:
Valuable data on the freezability of buffalo semen have been reported by Tayel et al.(1988) from analyses of the data accumulated over two years of work in the governmental Centre for Frozen Semen belonging to the General Organization for Veterinary Services, Egypt. For 1183 ejaculates obtained from 8 bulls, the prefreezing and post-thawing motilities differed only slightly with sperm losses due to procedural treatments of about 16%. Some frozen ejaculates showed much better resistance in withstanding low temperature effects and displayed better survival rates at thawing; higher than 65% with sperm losses lower than 5%. Semen from different individuals differ greatly in the ability to withstand the rigour of freezing (El-Sheltawi, 1989, Ziada 1994 and Ziada et al., 1995 b). Freezability of buffalo semen, on the other hand seems not to be influenced by bull x extender interaction (Ziada, 1989, 1994 and Ziada et al., 1995 b).
Season affects buffalo semen freezability. Analysis of the kept / collected ratio for 1620 ejaculates (Tayel et al., 1988) indicated that good quality semen which could be successfully frozen was that obtained over April to July (74.4 - 82.2%) as well as in November (75.5%). According to Ziada (1994), season exerted significant effect on preefreeze sperm motility after primary dilution as well as glycerolation. At these steps of processing, a significant season x bull interaction existed, indicating that not all bulls react similarly towards the effect of season. Equilibration nullifies the effect of season but freezing and thawing operations per se exert definite effects on post-thawing sperm recovery. Comparatively better post-thawing motility has been observed for winter as well as summer collections. Autumn semen is relatively less freezable. Post-thawing acrosomal maintenance, on the other hand, is optimal in winter.
2- 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, biochemical bases. 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 (El-Sheltawi, 1995) that cold shock inflicted upon buffalo spermatozoa causes changes in the percentage of individually motile spermatozoa, percentage of damaged acrosomes, amount of G0T released extracellularly comparable to those which take place upon freezing and thawing. However, in view of the extremely low associated variance (r2%) calculated between various semen parameters (7.29 - 9.61%), the use of cold shock test to predict freezability of buffalo semen becomes questionable.
3- Diluents
A wide array of extenders originally evolved for bull semen was, and still being, used in “hit- and- miss“ empirical fashion. To our knowledge, there is no diluent which has been composed specially for buffalo semen freezing that has taken into consideration the peculiarities of semen of this animal species. Because of this and of other associated factors like cryoprotectant level, equilibration period, freezing rate, thawing regime and differences in freezability of semen from individual bulls, reported cryopreservation results have always shown great variation and have often been contradictory. As with chilled semen, milk has been found better than egg yolk citrate even after 8 month of storage in liquid nitrogen. (El-Kafrawi and Barrada, 1974). In a comparison between Tris -citric acid -fructose-yolk (TCFY), egg yolk citrate (EYC), egg yolk- glucose- bicarbonate (EYGB), egg yolk- skim milk (EYSM) and Laiciphos-271 (Nour, 1980), EYSM and Laiciphos yielded higher post-thaw motility than other diluents. Sperm recovery in skim milk diluents has even been better than in Triladyl- a commercial Tris-based extender (Abdel-Rahman, 1988). According to Abdel-Malak et al., 1993), post-thawing motility in Tris buffer, has been better at pH 7.0- 7.5 than at 6.5 or 8.0, but Tris molarities between 0.20 and 0.35 did not vary significantly. In a recent report (Ziada et al., 1995 a), commercial diluents Triladyl and Laiciphos resulted in comparable post-thawing motility.
3- Cryoprotectants:
As with bull semen, glycerol concentration in the semen diluent has been shown to play a decisive role in post-thawing sperm recovery and viability. Concentrations of 6.4% in Tris, or 7.0% in skim milk appear better than lower or apparently higher concentrations. Glycerol x thawing rate interaction is a factor of extreme importance (Ziada et al., 1992). For pellet freezing of buffalo semen in lactose diluent 2 - 3% glycerol results in better sperm revival (Ismail, 1990). Although glycerolation at 4-5 oC is a common practice, satisfactory post-thawing motility and least morphological and biological damage have been recorded after room temperature glycerolartion of buffalo semen processed in Tris (Fath -El-Bab et al., 1985).
To our knowledge, no studies have hitherto been conducted to clarify the proper freezing rate of buffalo semen but rapid freezing in a way similar to that adopted for bull semen is generally used.
4- Thawing rate:
According to Ziada et al. (1995b), thawing of buffalo semen at relatively fast rates (50 oC for 15 sec or 35 oC for 30 sec) was found superior to thawing at slow rates (20 oC for 1 min or 5 oC for 2 min).
5- Packages of frozen semen
No particular difference in post-thawing revival has been noted between freezing in ministraws or in minitubs as packages for frozen semen in spite of the differences between the two packages in the ratio of their surface are to volume.(El-Sheltawi et al., 1991a ).
6- Effect of freezing on sperm morphology:
Freezing procedure results in significant reduction of sperm head length, maximum breadth, base width, 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, glycerolation and equilibration prior to freezing process (Abdel-Rahman, 1988 and El-Sheltawi et al., 1991b).
Buffalo spermatozoa have been believed to be inherently more fragile than bull spermatozoa that upon freezing and thawing they are subjected to ultrastructural damage and subsequent detrimental chemical changes in the molecular organization of their membranes with leakage of vital material important for fertilization. Electron microscopic investigations indicate that ultrastructural changes start at the very initial steps of processing, mainly after the addition of glycerol and increase progressively with each further steps of processing (Ziada, 1994). However, the sequential increase of ultrastructural damage after dilution, cooling, glycerolation and cold equilibration does not correlate in any way with the little change of sperm motility observed after these steps of processing. 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.
7- Enzyme leakage:
Various procedural treatments included in the technique of freezing contribute to the leakage of vital material as GOT, GPT, beta-glucuronidase and lactic dehydrogenase to the extracellular medium(Abdel-Rahman et al., 1989). As reported, the Tris-based extender-Triladyl affords better protection to sperm membrane integrity than reconstituted skim milk diluent as monitored by GOT and GPT release.
8- Acrosomal antigenicity and integrity:
The question of how far the antigenic properties of spermatozoa are affected by the customary methods of cryopreservation has been posed by Bratanov (1979). However, since then no significant studies appear to have been made and the available literature is virtually devoid of results of research on this topic. Our studies (Ziada, 1994) indicate that the various steps of freezing procedure lower significantly the antigenic activity localized on the surface of the buffalo sperm acrosome as visualized by indirect immunofluorescence. The diluent composition has seemingly no obvious effect on acrosomal antigenicity. There is reason to speculate that the loss of acrosomal surface antigens due to freeze processing is associated with evident morphological damage in the acrosomal region of the sperm head. A highly significant negative correlation has been observed between antigenic and acrosomal scores.
Because of the well known importance of the acrosome in fertilization and the possible significance of sperm surface antigens, evaluation of acrosomal status using a fluorescent probe could be more valuable, than other acrosome staining methods ,for simultaneous determination of both integrity and antigenicity of this sperm organelle.
9- Assessment of the quality of frozen semen:
Visual estimation of sperm motility is in most cases used for appraisal of frozen semen quality. A number of reports (El-Sheltawi, 1989 and El-Sheltawi et al, 1993 a, b, c) recorded significant positive correlations between post-thawing motility on one hand and each of viability index, cervical mucus penetrability, percent live sperm that passed through a column of sephadex and percent sperm displaying gelatinolytic activity. Negative correlations were also found between motility and the percent morphologically damaged acrosomes, bent tails and amount of GOT released from the spermatozoa in the extracellular media. In spite of being statistically significant, the obtained correlation coefficients were not high enough to be useful in predicting semen quality from motility estimates.
Dark field microscopy can be used efficiently for clearly viewing sperm acrosome morphology in wet mounts. This method has decided advantages over other methods Abdou and Fattouh, 1990).
10- Fertility results from frozen buffalo semen:
Fertility results from frozen buffalo semen vary significantly due to a number of factors. The same factors that control the fertility results of frozen bull semen seem to be involved also here. The individual variation in freezability of semen appears to be of prime importance. El-Sheltawi (1989) recorded significant differences of 42.33 - 70.68% among bulls indicating that the choice of buffalo bulls whose semen can withstand the hazards of freezing and thawing can increase the net fertility rate from frozen semen.
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