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ISSN : 2671-4639(Print)
ISSN : 2671-4663(Online)
Journal of Animal Reproduciton and Biotechnology Vol.27 No.1 pp.21-28

Relationship between Semen Quality Parameters and Field Fertility of Bulls

Anup Kumar Talukder1,2,*, Sushanto Kumar Rabidas1, Md. Golam Shahi Alam1, Farida Yeasmin Bari1
1Department of Surgery and Obstetrics, Faculty of Veterinary Science, Bangladesh Agricultural University
2Department of Medicine and Surgery, Faculty of Animal Science and Veterinary Medicine, Patuakhali Science and Technology University


A study was conducted on four crossbred bulls, used as artificial insemination (AI) sires, to correlate their semenquality with their non return rate (NRR). Semen was collected once a week via an artificial vagina, diluted in eggyolk-citrate and maintained at +7℃ for three days. It was evaluated for sperm motility, viability, morphology immediatelyafter collection and was examined daily for sperm motility, viability and morphology of acrosome, mid pieceand tail for a total of three days. A total of 2016 cows were inseminated by two AI technicians. The proportionsof sperm with normal heads were 83.4% (63.7~91.7%), the proportion of spermatozoa exhibiting normal morphology(acrosome, mid piece and tail), motility and viability were 89.2% (82.3~92.0%), 71.3% (61.7~75.0%) and 76.7%(65.7~85.0%), respectively in fresh ejaculates. Sperm motility and sperm viability was significantly (p<0.05) lowerin Holstein-Friesian × Local bull than in other bulls during all three days of storage. The overall NRR for four bulls was82.7% (72.9-87.5%). Bulls with higher sperm motility, viability and normal morphology of spermatozoa of individualbull had significantly (each p<0.05) higher NRR. The highest (p<0.01) NRR (87.5%) was observed in a Red Chittagongbull whose semen qualities were significantly (p<0.05) higher than Holstein-Friesian × Local bull (NNR 72.9%).The results of the present study concluded that NRR at 56 days post AI is related to parameters of semen quality.Therefore, semen evaluation may allow the discarding of bulls with poor fertility in an AI program.

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 Pregnancy in cows from AI services is the key factor for optimal economic success in dairy farms. AI is the oldest and currently most common assisted reproductive technology and an important tool in animal production (Vishwanath, 2003). Failure of cows to become pregnant and the need for repeated AI are common causes of frustration and economic loss of the dairy farmers (Stevenson et al., 1990). Since the aim of an insemination is to produce pregnancy, the fertilizing capacity of spermatozoa preserved either as chilled or as frozen should never be compromised in an AI program. To make an AI program into an economic success, sires with inadequate fertility(whether in terms in individual poor-quality ejaculates or of a bull whose conception rates are unacceptable) must be identified and removed (Shaha et al., 2008). A national program for crossing local cattle with exotic breeds by AI has been practiced in Bangladesh since 1950 (Ahmed and Islam, 1987). Reproductive efficiency of AI bulls is usually measured by the NRR and it is measured on the cow. It is affected by the fertility qualities of the cow herself and of the bull she is inseminated by, and also by the season and skillness of the technicians who carried out the insemination (Reurink et al., 1990). No other system except NRR to monitor male fertility has been introduced on a large scale (Reurink et al., 1990).

 Semen can be used in AI programs after cryopreservation, or after dilution in either ambient-temperature (e.g. Caprogen) or chilled diluents (Holt, 2000). Semen survives dilution and/ or chilling for a few days, but the motility and morphology of spermatozoa progressively worsen as the period of storage increases (Alam et al., 2005). Moreover, motility can also be affected by the age of the sire and environmental influences(Hallap et al., 2006). However, no information exists on the performance of chilled semen in the national crossbreedingprogram of cattle breeding in Bangladesh, nor has the relationshipbetween semen quality and NRR been evaluated for the bulls in this program. This study was, there fore, performed to identify low fertility bulls, on the basis of their semen qualities(sperm motility, head morphology, morphology of acrosome, mid piece and tail and viability of sperm) and NRR under field condition, so that such bulls can be discarded from a commercial bull AI center.


 The research work was conducted at the University AI Center and Reproduction Laboratory of the Department of Surgery and Obstetrics, Bangladesh Agricultural University, Mymensingh- 2202, Bangladesh.

1. Bulls

 Four healthy vaccinated, dewormed and sexually matured crossbred bulls were selected for this study. The age, body condition score and body weight of the bulls were 3~5.5years, 3.5~4.0 (1 to 5 scales) and 345~561 kg, respectively.

2. Measurement of Scrotal Circumference

 Scrotal circumference was measured in centimeter by using measuring tape (Lane Manufacturing Co., Denver, CO, USA) according to the method described by Foote (1969).

3. Semen Collection and Transportation

 Semen was collected by artificial vagina (AV) once per week, following the method adopted by Islam et al. (1999). The semen was maintained at +37℃ after collection until evaluation and processing.

4. Semen Evaluation

 The routine evaluation of fresh semen was done as describedby Alam et al. (2005) and Shaha et al. (2008). Volume of fresh semen was recorded from the graduated mark of the semen collecting tube. Mass activity was evaluated by placing a drop (25μl) of semen onto a pre-warmed (+37℃) glass slide without cover slip and examined under light microscope at low magnification (100×). The concentration of spermatozoa (million/ml) was determined by using haemocytometer technique as described by Bane (1952).

 Sperm motility was assessed by observing motion under 100× magnifications using 10× optical systems. Sperm head morphology was evaluated in dried semen smears using differential interference contrast (DIC) optics (BX 51, OLYMPUS, Tokyo Japan; 1000× magnification) as previously described by Freneau et al. (2009). Morphology of sperm acrosome, midpiece and tail was examined using samples diluted in buffered formal saline as method described by Barth and Oko (1989). Sperm viability (proportions of live and dead spermatozoa) was estimated by using Eosin-Nigrosin staining (Evans and Maxwell 1990).

5. Semen Processing and Preservation

  Semen was processed in egg-yolk-citrate (EYC) extender (Jha, 2008). After collection, the semen was diluted with EYC to obtain 20×106  progressive motile spermatozoa per ml. Individual insemination doses were transferred into one ml glass vials, which were preserved at +7℃ for three days for artificial insemination.

6. Insemination

  Inseminations of cows were done at 12~18 hours of onset of estrus by two inseminators of University AI center. Cows showing any abnormal genital discharge or having any abnormalities in genital organs on rectal palpation at the time of AI were not considered for this experiment.

7. Data Collection

 Data for individual insemination records were collected from the registers books of University AI center. Data including the bull whose semen used, date of semen collection and insemination, identification of the cow which received insemination, and where applicable, the date on which cows were represented for insemination after return to estrus by 56 days postinsemination were recorded.

8. Calculation of Non Return Rate

 Non return rate was calculated by the formula given below:


9. Statistical Analysis

 Data were entered in a Microsoft excel work book and exported to SPSS (Version 10.0) for analysis of descriptive statistics. Linear regression was performed to figure out the relationship between qualities of fresh semen and NRR of bulls. one way ANOVA was used to see the effects of individual bulls on semen volume, sperm concentration, sperm motility, number of live sperm of fresh semen, and the changes of sperm motility and viability from Day 1 to 3 (on each day) of preservation among the bulls.


 The age, body condition score, body weight and scrotal circumference of the bulls were 3~5.5 years, 3.5~4.0 (1 to 5 scales), 345~561 kg and 26~32 cm, respectively. Details of the bulls used in this study are given in Table 1.

Table 1. Brief description of bulls used at AI center

 Semen quality data for the four bulls are presented in Table 2. The mean volume of fresh semen collected from four crossbred bulls varied from 3.9 ± 0.4 to 7.7 ± 0.3 ml. There were significant differences between bulls in ejaculate volume (p<0.05). The sperm concentrations (× 106/ml) of fresh semen ranged between 1,116.7 ± 28.9 to 1,315.0 ± 13.2 among the bulls. The sperm concentrations are significantly differed among the bulls (p<0.05). The sperm motility (%) of fresh semen varied from 61.7 ± 2.9 to 75.0 ± 5.0. The differences of sperm motility among the bulls are statistically significant (p<0.05). The proportions of live spermatozoa in fresh ejaculates varied from 65.7 ± 4.0 to 85.0 ± 1.0% among the four bulls and this difference is statistically significant (p<0.05).

Table 2. Evaluation of fresh semen (volume of semen, mass activity, motility and viability of sperm) of bulls. Number of ejaculates per bull=3

 Examples of different sperm head abnormalities are shown in Fig 1. The percentages of spermatozoa with normal heads varied from 63.7 ± 3.2 to 91.7 ± 1.6 among the bulls. Significant (p<0.05) differences in sperm head morphology were present between bulls (Table 3). Examples of different abnormalities of sperm acrosome, mid-piece and tail, are shown in Fig. 2. The proportions of spermatozoa with normal acrosomes, mid-pieces and tails were also differed significantly (p<0.05) among the bulls, which varied from 82.3 ± 2.5 to 92.0 ± 1.5% (Table 3).

Fig. 1. Photography of abnormal morphology of bull sperm head observed at DIC optics (1,000× magnification). (a) normal sperm head, (b) small head, (c) pyriform head, (d) detached head, (e) narrow head, (f) abnormal shaped head, (g) pear shaped head, (h) damaged acrosomal membrane.

Fig. 2. Photography of abnormal acrosome, mid-piece and tail of bull sperm observed at 1,000× under DIC optics. (a) detached head, (b) broken of mid-piece, (c) proximal cytoplasmic droplet, (d) cork screw shape tail, (e) broken tail, (f) abaxial tail, (g) simple bent tail, (h) loose acrosome.

Table 3. Evaluation of fresh semen (sperm morphology) of bulls. Number of ejaculates for each bull = 3

 Sperm motility and sperm viability both decreased significantly(p<0.05) as period of storage increases (Table 4). The number of spermatozoa with abnormal acrosomes, mid-pieces and tails were significantly (p<0.05) increased with the advancement of time of preservation. Sperm motility and sperm viability was also significantly (p<0.05) lower in Holstein-Friesian × Local bull than in other bulls on all three days.

Table 4. Changes of semen quality on Days 1 to 3 after preservation at +7℃

 A total of 2,016 cows were inseminated, of which 349 cows were presented for second insemination. Non return rate for each bull is presented in Table 5. The overall NNR is 82.7% (72.9~87.5%) for four bulls. The highest NRR was found in Red Chittagong bull that was significantly (p<0.01) higher with that of Holstein Friesian × Local which achieved 72.9% NNR.

Table 5. Non return rates of bulls

 Bulls with semen of higher sperm motility in fresh ejaculates had significantly (R2 = 0.63, p<0.05) higher NNR (Fig. 3a). Bulls having large number of viable spermatozoa in fresh semen achieved significantly (R2 = 0.73, p<0.05) higher NRR (Fig. 3b). The proportion of sperm with normal head morphology was significantly (p<0.05) correlated to NRR (R2 = 0.90, Fig. 3c) in cows, whilst the number of sperm with normal acrosomes, midpieces and tails was significantly (p<0.05) correlated to NRR (R2 = 0.93, Fig. 3d).

Fig. 3. Relationship between fresh semen quality and NNR of bulls. (a) sperm motility; R2 = 0.63, p<0.05, (b) percentages of live spermatozoa; R2 = 0.73, p<0.05, (c) percentages of sperm with normal head; R2 = 0.90, p<0.05, (d) percentages of sperm with normal acrosome, mid-piece and tail; R2 = 0.93, p<0.05.


 The results of the present study showed that non return rate of bulls was significantly related to their semen quality, such that the bulls which given semen of higher sperm motility and viability and lower proportion of morphologically normal spermatozoa achieved higher NRR.

 The mean volume of fresh semen collected from four crossbred bulls varied from 3.9 ± 0.4 to 7.7 ± 0.3 ml, which is consistent with the other studies of bulls in Bangladesh (Bhuiyan and Shamsuddin, 1998; Jha, 2008). Similarly, Munsi et al.(2007) reported that the fresh semen volume of crossbred bulls ranged between 2.9 ± 0.2 to 4.4 ± 0.5 ml. The sperm concentrations (× 106/ml) of fresh semen varied from 1,116.7 ± 28.9 to 1315.0 ± 13.2, which were consistent with the results of Munsi et al. (2007) and Jha (2008). The sperm motility (%) of fresh semen varied from 61.7 ± 2.9 to 75.0 ± 5.0, which again is in agreement with previous studies in Bangladesh (Sugulle et al., 2006; Munsi et al., 2007; Jha, 2008). Finally, the use of DIC microscopy (range: 63.7 ± 3.2% to 91.7 ± 1.6%) to evaluate sperm head morphology gave results that were comparable with earlier studies (Sugulle et al., 2006; Siddiqui, 2007; Freneau et al.,2009). As previously reported, there is a significant level of variation between bulls and between ejaculates, which has been variously attributed to age, season, technique of semen collection, body weight and scrotal circumference of the bulls (Helbiget al., 2006; Siddiqui et al., 2007), whilst morphology can also vary with processing and storage of semen, diluents and duration of storage (Shamsuddin and Chanda, 1998). Semen is described normal when the frequency of abnormal sperm heads does not exceed 10% and other parameters (acrosome, mid-piece, and tails) exceed 5% each or a total of 10~15% (Rodriguez-Martinez, 2008).

 There was significant difference in the proportion of sperm with abnormal head morphology among the bulls evaluated by DIC microscopy. The process of evaluating head morphology is easier using DIC than using William’s staining technique.According to Freneau et al. (2009), DIC is a more effective technique for visualizing major defects, although bright field, which included stained smear preparations may be preferable for visualizing minor defects.

 In the present study, sperm motility and viability decreased gradually over three days of preservation at 7℃. This trend has been widely reported in the international literature on chilled semen diluents, and is also in agreement with the results of Jha (2008) in Bangladesh. Provided at least 50% of sperm display progressive motility, the semen can be used for AI. Previous work has shown that semen can routinely be maintained at this level of viability in egg yolk-citrate diluent at 7℃ for two days (Alam et al., 2005) in Bangladesh AI programmes.

 Non return rate was significantly related to semen quality,such that the bulls with semen of higher motility and viability and lower proportion of morphologically abnormal sperm had higher NRR. Sarder (2006) similarly reported that the highest NRR (83.1%) was associated with high quality semen and the lowest (70.1%) with poor quality bull semen in the Rajshahi district. The evidence of both the present experiment and the literature is that the variation in NRR between bulls and/or ejaculates is due to differences in the proportion of morphologically normal motile spermatozoa in the insemination dose. Thus, there was a significant (p<0.01) relationship (R2 = 0.63) between sperm motility of fresh semen and NRR; a finding that is in agreement with previous studies. For example, Amann and Hammerstedt (2002) reported that both the number ofmotile sperm per dose as well as the number of viable sperm per dose correlated significantly (p<0.01) with NRR (r = 0.49, and r = 0.83, respectively). Likewise, Bhuiyan et al. (1999) reported that cows inseminated with good quality bull semen (i.e. > 50% motility, > 7.5 × 106 total number of motile spermatozoa and >70% normal sperm) in an AI programme in Bangladesh that used chilled semen, conceived at higher rate (p< 0.001) than did those inseminated with poor bull semen (55.2% vs 37.1%). In the present experiment, a significant (p<0.05) relationship between sperm viability and NRR was observed (R2 = 0.73), which again is in accordance with previous studies, such as that of Decuadro-Hansen et al. (2002) who reported that sperm viability and sperm motility correlates with the fertilizing ability of a particular sample of semen. Similarly, in the present study, the proportion of sperm with normal head morphology was significantly (p<0.05) correlated to NRR (R2 = 0.90) in cows, whilst the number of sperm with normal acrosomes, mid-pieces and tails were significantly (p<0.05) correlated to NRR (R2 = 0.93). Al-Makhzoomi et al.(2008) reported that sires’ having ejaculates containing >10% of morphologically deviating sperm head shapes are associated with poor NRR. Card (2005) suggested differential spermiogram that included less than 30% morphologically normal spermatozoa, more than 10% immature germ cells, more than 30% abnormal sperm heads and/or mid-piece defects, or more than 25% spermatozoa with proximal cytoplasmic droplets with reduced fertility.

 In conclusions, the results of the present study concluded that NRR at 56 days after first insemination is related to parameters of semen quality in the fresh ejaculate. Hence, semen evaluation may allow the discarding of bulls with poor fertility from the AI program. Moreover, evaluation of semen before AI might be a good practice to increase NRR. Further study is needed to investigate the effects of other factors including cows’ age, breed, parity and seasons of insemination performed and skillness of inseminator on NNR.


 We thankfully acknowledge Professor Dr. MAM Yahia Khandoker, Department of Animal Breeding and Genetics and Professor Dr. Mohammed Shamsuddin, Department of Surgery and Obstetrics, Bangladesh Agricultural University, Mymensingh for allowing us to work at the University AI Center and for permitting us to use DIC optic microscope, respectively.


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