E. D. El-Deeb; K. M. El-Sabbagh; G .A. Sosa;  Randa.S. *  And A. I. El-Azab

Dept. Theriogenology, * Dept. Physiology, Fac. Vet. Med., Zagazig Univ. (Benha); P.O. 13736, Moshtohor



In a laten square design, the frozen semen collected from four buffalo bulls (8-10 years old) was subjected to three thawing rates (35C/30 sec, 50C /15 sec and 65C/8 sec). For each bull at each thawing rate, at least three straws were thawed, pooled and incubated in water bath at 37C. The percentage of post-thawed motility was estimated for 4 hours holding time with an hour interval. From the obtained results, it could be concluded that the post thawing motility of buffalo spermatozoa was significantly differed with bull variance, increased by increasing the thawing rate from 35C/ 30 sec to 65C / 8 sec. and decreased by increasing the holding time in water bath at 37C for 3 hours.



One of the main objectives of the worldwide extension of A.I is to improve fertility in cattle. In order to achieve a high conception rate, many investigations have been conducted to maintain the fertilizing capacity of the frozen semen being used. Some of these were directed toward studying the crucial effect of thawing rate on the quality (Sahni and Mohan, 1988; Dhami, et al., 1991and Tyseer, et al., 1996) and the fertilizing capacity (Ahmed, et al., 1988 and Yousef, 1997) of buffalo frozen semen. Thawing of the frozen semen takes place in different forms, e.g. air thawing (Bean, 1972); rolling the straw between hands (Mac Pherson and Penner, 1972) and thawing in warm water (Ahmed, 1984). The duration of thawing varied from seconds to few minutes (Senger, et al., 1976 and Mohan et al.,1992). Not only the rate of thawing, but also time of holding after thawing seems to influence the fertilizing capacity of the frozen semen (Kim and Kim.1978; Morkholm and Filseth, 1988 and Yousef, 1997). Holding semen in warm water up to three hours (Yousef, 1997) showed a decrease in the post thaw sperm motility that postulated to have an association with the fertilizing capacity (Dimitropoulos, 1967). The present study was designed as an attempt to study the effect of thawing rate, holding time, and the bull variance on the post thaw sperm motility of buffalo frozen semen.



Four buffaloes bulls used in the present study (8-10 years old). They kept in Buffalo Frozen Semen Center, Abbasia, Cairo during a period of 6 in the (March 2001 -August, 2001). The buffalo semen was processed by mean of standard French system using milk-based extender (Laciephos) and packaged in 0.50 ml straws. In a laten-square design, the frozen semen collected from each bull was subjected to three thawing rates (35C/30 sec, 50oC/15 sec and 65C /8 sec). At least, three straws were thawed and pooled from each bull at each thawing rate. The pooled semen was incubated for four hours in a thermostatically controlled water bath adjusted at 37oC. The percentage of the post thawing motility of buffalo spermatozoa was estimated at 0, 2 and 4 hours holding time. The obtained data were statistically analyzed, where appropriate, according to Denenberg (1976).



As shown from table (1), regardless of the effect of bull, thawing rate and holding time, the overall mean of percentage of the post thawing motility of buffalo spermatozoa was 38.47 1.09%.


Table (1): Effect of thawing rate, holding time and bull variance on the percentage of post-thawing motility of buffalo Spermatozoa.


No. of samples

Mean S.E.

Bull variance:

. No. (1)

. No. (2)

. No. (3)

. N0. (4)







31.30 1.35(d)

34.26 0.99(c)

41.30 1.12(a)

37.41 1.01(b)

Thawing rates:

. 35C/30 sec

. 50C/15 sec

. 65C/8 sec






33.61 1.03(b)

36.11 1.20(a)

38.47 1.09(a)

Holding times:

. 0 hr

. 2 hr

. 4 hr






40.97 0.86(a)

36.25 0.81(b)

30.97 1.09(c)

Overall mean


38.47 1.09

    Values with different letters within the same factor differed significantly at P < 0.01


A highly significant (P<0.01) difference was observed in percentage of the post thawing motility of spermatozoa between the different bulls. It was 31.301.35, 34.260.99, 41.301.12 and 37.411.01% for the buffalo bulls under investigation (Tables 1 & 2).


Table (2): Analysis of variance showing effects of thawing rates, holding times and bull variance, on percentage of post- thawing motility of buffalo spermatozoa.   

Source of variance





Between bull (B)

Between thawing rates (R)

Between holding times (T)

(B) x (R)

(B) x (T)

( R)x (T)






























     **: P < 0.01


The percentage of post thawing motility of spermatozoa was noticed to increase significantly (P< 0.01) by increasing the rate of thawing. It was 33.611.03, 36.111.20 and 38.471.09% for 35C /30 sec, 50C/15 sec and 65C/sec. Thawing rates, respectively (Tables 1 & 2).

After thawing, when semen was incubated in warm water at 37C for 4 hours, a highly significant (P< 0.01) decrease in percentage of the post thawing motility of spermatozoa was decreased by advancing the time of holding. It was 40.970.86, 36.250.81 and 30.971.09% for 0, 2 and 4 hours holding time, respectively (Tables 1 & 2).

Analysis of variance (Table, 2) did not reveal any significant interaction between the bull variance, thawing rate and holding time in their effects on percentage of the post thawing motility of buffalo spermatozoa.



          The present study revealed a marked difference between buffalo bulls in percentage of post-thawing sperm motility. Individuality between buffalo bulls were highly significant (El-Azab, et al., 1978, Meyer and Barth, 2001).

          From the practical point of view, it has been found that the buffalo semen was very sensitive to freezing (Sahni and Mohan, 1988 and El-Azab, et al., 1977), a finding that explained the lowered percentage of the post-thawing sperm motility in the present study. Not only the difference between buffalo bulls was significant, but also the semen freezability for the same bull differ from time to time as it is more stressful to the change in the environmental conditions (Singh and Singh, 1989 and Yassen, 1995).

          After freezing, different rates of thawing have been practiced to optimize the percentage of the post-thawing motility .The present study showed that the optimal motility was recorded at 65C/8 sec, thawing rate (38.471.o9) compared to other rates (36.111.20 and 33.611,o3) at 50oC/15 sec and 35oC/30 sec respectively. Jainudeen and Dass (1982) recorded that thawing temperature from 5ºC to 50oC resulted in an increase in post thaw motility of buffalo semen. Moreover, Ashamed (1984) found that thawing of straws of buffalo semen in water bath at a temperature of 0ºC, 37ºCor 75C for 2 minutes, 15 seconds and 9 seconds respectively, resulted in average initial post thaw motility of 30.00, 40.00 and 50.00%. The difference between the different rates was significant. Bhalde, et al. (1991) observed that buffalo semen thawed at 30oC for 30 sec and at 5oC for 5 minutes resulted in 57.20 and 55.00% motile sperm, 60.70 and 57.60% live sperm, respectively. It has been noticed that increasing the thawing rate from 5oC/2-4 min. (Ennan, et al. 1976) to 75C/7-12 sec. (Ahmed, 1984;Tysser, et al. 1996) associates with an increase in percentage of the post- thawing sperm motility. These findings disagreed with those obtained by Gilbert (1984) and Dudeja (1990). They reported that the motility of buffalo spermatozoa was maximum on thawing at 35oC for 20 or 30 second respectively. In the same respect, Gilbert (1984) found that bovine semen thawed at 35oC maintained motility for longer time than that thawed at lower temperature. The finding was confirmed by the positive correlation between the sperm motility and fertility (Ahmed, et al., 1988 and Yousef, 1997) however; higher temperature of thawing seems to be harmful on the sperm motility due to any bias in the thawing time. For buffalo frozen semen, the ideal thawing rate was recorded to be from 37oC/30 sec.to 45ºC /15 sec.to obtained reasonable (Sahni and Mohan, 1988 and El- Azab et al.1977) motility.

          After thawing, the present study indicated that holding the thawed frozen semen in a warm water of 35-40oC associated with a decline in the post-thawing sperm motility by increasing the holding time, a finding which came in agreement with some previous studies (Senger, et al., 1976;El-Azab, et al., 1977). This finding could be attributed to the positive equilibration of water temperature toward the ambient temperature during incubation  (Bewn, et al., 1991). Two hours holding time after thawing at 35ºC were suggested to maintain the post-thawing motility of bull spermatozoa (Jondent and Rabodeux, 1977).

          Inscoping on the effect of holding time on conception rate, Rao (1992) and Zair (1997) reported that the C.R was improved (75.86%) by keeping thawed buffalo semen at 37oC for 1/2-1 hour. In the same aspect El Naggar (1999) found a marked improvement of fertility during the green season of the year (69.23%) in buffaloes inseminated after 30 minutes of thawing compared to 40.00% for animal inseminated after 30second of thawing.          


          From the present study, it could be concluded that the percentage of pos-thawing motility of buffalo spermatozoa is differed significantly by the bull variance, increased by increasing the thawing rate from 35ºC/30 sec. to 65ºC /8 sec. and decreased by increasing the holding time up to 3 hours incubation water in bath at 37oC.



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